TWI296285B - Promoter sequences from wssv immediate early genes and their uses in recombinant dna techniques - Google Patents

Description

1296285 IX. INSTRUCTIONS: L P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P 5 (immediate early genes), and they have promoter activity to drive transcription of a target gene in a non-native host cell, thus having great potential for use in the field of biotechnology. [m sleeve] (Description of related techniques) • The production of recombinant peptides/proteins is a very important genetic engineering technique in the field of biotechnology. Its basic principles involve the ability to express a desired gene product [eg, ribozymes and RNA transcripts, industrial and agricultural enzymes, therapeutic proteins, interferons, inter The target genes of interleukins, hormones, growth hormones, antigenic polypeptides, antibodies, and analogs are selected into a suitable vector, and subsequently The recombinant vector formed is transferred into a competent host cell. The recombinant host cell thus formed can be cultured in a suitable medium under suitable culture conditions, and Table 2 of the target gene can be induced at an appropriate timing to achieve mass production. The purpose of the desired gene product. According to current knowledge and technology in the field of genetic engineering, E. coli (heart cells are the most widely used and most effective host cells, and many types of plastid vectors have been developed for retention in these 1,296,285 bacterial species. The plastid vectors are typically constructed to have an inducible artificial promoter that is selected upstream of a target gene to control the performance of the target gene. The most commonly used artificial start The sub-promoter includes /ac, noisy, _, ire, araBAZ), children?/^ and butyl 7 5 promoters, and these promoters can be obtained by isopropyl-/5 thiogalactophosphate (isopropyl-million- D-thiogalactopyranoside, IPTG), lactose, arabinose, temperature change or the like is induced (SC Makrides ei αΖ· (1996), jRev., 60: 512-538). 10 On the other hand, if a selected target gene contains a constitutive promoter, it can be directly colonized into a vector. When using this target gene to construct a recombinant vector, it is usually not necessary to induce the production of recombinant polypeptide/protein by induction. Other 15 prokaryotic cells that can be used for vector transformation include, but are not limited to, cells derived from: other bacterial species/genus [such as Bacillus subtilis (jgac/ZZws) , Serratia marcescens, Lactobacillus species (L like π ·), Streptomyces species (still (7) m; yCa π ·) and Salmonella typhimurium 0 ^ / η ·)], blue green swing 20 Actinomycetes (AcizViomycWM) and so on. However, prokaryotic cells do not, and yeast only performs limited post-translational modifications of the expressed polypeptide/protein, whereas higher eukaryotes are capable of performing the complexities normally required for proper functioning of the protein. Protein modification. Thus, if translation 6 1296285 post-modification is required for recombinant polypeptide/protein, it may be more desirable to use eukaryotic cells to produce the recombinant polypeptide/protein. Eukaryotic cells suitable for use in vector transformation include, for example, fungal cells, protozoan cells, plant cells, insect cells, animal cells, and human cells. Examples of suitable fungal cells are: yeast cells, such as baker's yeast (Sacc/mramyca or methanolic yeast CP/c/n_(2) and Kluyveromyces [such as K. lactis (K Mci (4) and Marks Rude yeast ([ cells. Suitable plant cells are those derived from gynosperms or angiosperms, preferably monocots and dicots, especially crops). (crops), and is derived from the roots, stems, leaves or meristems of these plants, and is cultured in the form of protoplasts or callus. Examples of suitable insect cells are fruit. Fly S2 15 cells and Sf21 cells derived from S. cerevisiae/rwg/penia), Sf9 cells, etc. Suitable animal cells may be cultured cells or cells in vivo 'preferably derived from vertebrates, more preferably Derived from mammals' and is an organ or tissue derived from these animals (such as kidney, liver, lung, ovary, breast, skin, bone, blood). 20 representative samples of animal cells : CHO, COS, BHK, HEK-293, HeLa, NIH3T3, VERO, MDCK, MOLT-4, Jurkat, K562, HepG2, etc. Carriers for the transformation of the host cells shown above include those commonly used in In genetic engineering techniques, for example: phage, such as phage; plastids, such as from Escherichia coli (including plastids (including 7 1296285 pBR322, pBR325, pUC12, pUC13, pQE-30, ρΕΤ12, ΡΕΤ30, etc.), Plastids from Bacillus subtilis (including pUBllO, pTP5, pci94, etc.), shuttle vectors for Escherichia coli and Bacillus subtilis (eg pHY300PLK), and plastids from yeast (including pSH19, PSH15 5, etc.), sticky The plastid, the virus 'including · insect viruses, such as baculoviruses; animal viruses, such as vaccinia viruses, cytomegalovirus (CMV), retroviruses, etc. 0 ^ In order to achieve an effective expression of a selected target gene, it is desirable to construct a vector containing a functional promoter/regulatory sequence, and the functional promoter/tune The sequence is operably linked to a target gene carried by the vector. The (etc.) promoter/regulatory sequence can be derived from any of the following: viruses, bacterial cells, yeast cells, fungal cells, algal cells (algal cells) ), plant cells, insect cells, animal cells, and human fine cells. For example, a promoter that can be used in E. coli (five (7)/〇 cells includes, but is not limited to, such as c promoter, T5 promoter, T7 promoter, T7 A1 _ actor, lac neutron, trp Therefore, the mover, trc loser, recA, mover,

Zpp promoter, arajBAD promoter and; I promoter. A promoter useful for cells of the genus Bacillus includes, but is not limited to, SP01 promoter 20, SP0 promoter, penP promoter, and the like. A promoter useful for yeast cells includes, but is not limited to, a PH05 promoter, a PGK promoter, a GAP promoter, an ADH promoter, and the like. A promoter useful for insect cells includes, but is not limited to, a polyhedrin promoter, a p10 promoter, a 〇pIE2 (OpMNPV promoter, etc.) a promoter that can be used to mobilize cells of 8 1296285, but Not limited to: SRα promoter, SV40 early promoter, RSV-promoter, HIV-LTR promoter, HSV-TK promoter, CMV promoter, CMV-HSV thymidine kinase promoter, etc. One is available Promoters for plant cells include, for example, a 35S CaMV 5 promoter, an actin promoter, a ubiquitin promoter, etc. Suitable for supplying regulatory elements for sputum animal cells (regUlat〇ry) Elements) include: CMV-HSV thymidine kinase promoter, SV40 early promoter, RSV-promoter, CMV enhancer, and SV40 enhancer. In addition, representative RNA polymerase promoters can be classified into the following 10 types. : an inducible promoter, a constitutive promoter, a tissue-specific promoter, and a synthetic promoter. Representative inducible promoters are: a heat-inducible Hsp70 promoter, a metallothionein (me Taii〇thi〇nein) promoter, an alcohol dehydrogenase promoter and a galactose promoter. A representative constitutive promoter is Rous sarcoma virus (RSV). LTR promoter, human cytomegalovirus (CMV) major very early gene promoter and Sv4〇 early promoter; and ® representative tissue-specific promoter is an alpha globm promoter and a beta The betaglobin promoter. The synthetic promoter can be produced by chemically or recombinantly modifying the native promoter. In addition to the lack of post-translational modification, there are other and expressed in prokaryotic cells. Some proteins have associated problems. For example, some heterologous proteins (heterolog〇Us pr〇teins) are expressed as insoluble inclusion bodies are stored in prokaryotic cells, which makes the recovery of such proteins sleepy. 1296285 Difficulties. Many of the difficulties associated with prokaryotic expression systems can be produced by translating mammalian cell culture systems. Post-translationally processed proteins are overcome. However, mammalian cell cultures may be relatively less effective because they are slower to grow and more difficult to maintain and costly. Advances, as well as the development of baculovirus-based expression systems, have promoted the expression of heterologous proteins by transduced insect cell lines (Lwcbw Swmmers (7988), iB/cvTec/i·, (5, 47) -55·, Miller (1988), Annu· Rev·Microbiol·, 42, 177-199). In the case of transgenic insect cell lines, the heterologous protein is mainly derived from the bacillus virus californica multicapsid nucleopolyhedrosis virus (AcMNPM) (Luckow and Summers (1988), as described above The Miller (79SSJ, eg 2遂) vector is completed. 15 Baculoviruses are double-stranded DNA viruses that kill infected by lysis (lySis) after a typical infection cycle. Insect cells. A variety of different baculoviruses are known, each of which is unique to a particular arthropod species. It is not known whether baculovirus can replicate in animals other than arthropods. Gene expression during an insect's natural baculovirus infection

It is controlled by Southern 0 weeks and has a sequence of cascades (JerecJ cascacje). Viral genes can be classified into four different categories based on their position within the cascade of genes. Very early (IE), late onset (DE), late, and very late. Early gene expression occurred before the onset of viral dnA 1296285 replication and appeared to be necessary for induction of late viral gene expression (Blissczrd and Rohnnann (1990) ), Amm· Rev· Entomol 35: 127-155·, Guarino and Summers (1988), J. Virol 62 * 463-471·, Miller et al. (1983), Virology, 126·376-380. The experimental 5 evidence shows that in the absence of other viral factors, the baculovirus gene is transcribed by the host RNA RNA peak. Therefore, it is known that the baculovirus gene has a promoter that can be recognized by the transcription mechanism of the host cell. The above description of the genital pathogen and its genes is quoted from us 20020116723 A1, which discloses the use of a promoter derived from a very baculovirus very early 10 promoter to control a selectable marker. Due to the performance, the marker gene provides resistance to one of the families of bleomycin/phleomycin-type antibiotics. In particular, US 20020116723 A1 discloses: derived from the genus of cedar moth Polyhedrosis virus (0 </ br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> The transcription of the marker gene can be selected, and the selectable marker gene can be a Shigella bentgrass (10)//such as a Me gene that provides Zeocin resistance to insect cells. 2. Describe the viral vector and/or contain Patents for the construction of vectors for viral promoters and published patent applications include, but are not limited to, US 5,077,214, US 5,162,222, US 5,168,062, US 5,385,839, US 20020116723A, US 20030108524A1, US 20030108863

Al, US 20030229046 Al, US 20040082531 Al, US 11 1296285 20040161841 A1, US 20040197313 A1, WO 99/61636 A1 and WO 0105992 A1. Literature references related to the identification of viral promoters and/or regulatory sequences 5 for constructing vectors for transformation and/or transfection applicable to host cells include, but are not limited to: r〇 m A.

Et al. (1997), Gene, 188, 183-190·, Steven S. Pullen and Paul D. Friesen, June 1995, 69 (6), 3575-3583·, Fan Xiu Zhu et al·, J· Virol” July 1999, 73 (7), 5556-5567·, RL· Harrison and BC· Bonning (2003), J. Gen·Virol·, 84 (Pt 7), 1827-1842·, 10 Ε·Β· Carstens et al ·, Virus research (2002), 83, 13-30\Μ·Κ· Barnhart et al·, J. Virol·, Jan·1997, 71 (1), 337-344\LA· Guarino and MD· Summers, J · Virol” Feb· 1986, 57 (2), 563-571\ VA Olson et al, J. Viroly May 2003, 77 (10), 5668-5677·, Ε·Α·van Strien et al·, Arch Virol· (2000), 145, 15 2115-2133, VA· Olson et al” J. Virol” Sep. 2002, 76 (18), 9505-9515·, Andrew K. Cheung (1999), Nucleic Acid Research, 17 (12 ), 4637-4646; Alexandra Garcia-Maruniak et al., J. Virol, July 2004, 78 (13), 7036-7051·, K. Kojima et al. (2001), Arch Virol·, 146, 1407- 1414 〇20 Although as mentioned above, the researchers in this art are still committed to exploring any potential Promoter and / or regulatory sequences, they may be used for constructing recombinant can be used in the production of a recombinant polypeptide / protein expression vector. White spot syndrome virus (WSSV) or white spot bacilliform virus (WSBV), an elliptical large double-stranded DNA virus with 12 1296285 coat, is a cultured shrimp in the world. One of the most toxic and dangerous viral pathogens ([/nowK by αΖ·, Fish Pathol· (1994), 29:149-158 recognizes S^Fish Pathol· (1996), 31: 39-45\ Nakano Et al., Fish Pathol· (1994), 29 (2): 135-139\ 5 Takahashi et al” Fish Pathol· (1994), 29 (2): 121-125', Η·-Υ·Chou et al · (1995), Dis· Aquat· Org·, 23: 165-173·, J. Huang et aLy Marine Fish Res. (1995), 16:1-10 and Marine Fish Res. (1995), 16:11- 23; C.-F· Lo, et al·, Dis· Aquat· Org· (1996), 27, 215-225 to anti-J·Fish·Soc, Taiwan (2003), 30, 1-13·, C, H. 10 Wang et al. (1995), Dis Aquat. Org., 23:239-242; C. Wongteerasupaya et al. (1995), Dis· Aquat· Org·, 21:69-77·, TW· Flegel (1997), World J. Microbiol. Biotech·, 13, 433-442·, Y·Lu et al· (1997), J. Gen· Virol” 84: 1517-1523, it also attacks many other crustaceans, such as crabs and crayfishes. In addition, due to the uniqueness of WSSV, it is necessary to directly apply infections of other viruses. Modes to explain WSSV infection strategies are difficult. As a result, WSSV infection strategies may have to be studied from scratch. Morphologically, WSSV virions are about 275x120 20 nm and have a squint-to-rod shape. Non-embedded has nonoccluded, enveloped particles and has a nucleus shell with periodic fringes perpendicular to the long axis (3〇〇X7〇nm) (d in aL (J995), Dis Aquat Org·, 23: 239-242·, C.

Wongteerasupaya et al (1995), Dis· Aquat· Org,, 21: 13 1296285 still-77). The most striking feature of WSSV is the presence of a caudal extension at one end of the virion (intestinal fistula (7995), such as 2遂; S· Durand et al. (1997), Dis. Aquat· Org·, 29:205 -211) 〇 complete genome sequencing has been performed on 3 WSSV sub- 5 isolates (for Taiwan isolate WSSV Tl, see NCBI accession number AF440570; for Thai isolates, see NCBI accession number AF369029; and For Chinese isolates, see NCBI Accession No. AF332093). The WSSV genome (~300 kb) is smaller than the 335,593 bp genome of the long-sac water cloud virus [five cioarp(10)hZ/cw/omy virus, EsV-1, algae deoxyribonucleic acid 10 (PA; ycW(10)v/r/also ^)] ~30 kb, long-sac water cloud virus is the largest viral genome that has been sequenced to date (/.L. van Etten et al. (2002), Arch Virol" 147, 1479-516. Previous studies on individual genes and complete Analysis of the genomic sequence suggests that WSSV does not belong to any known virus (1)·ei β/., 15 Virology (2000), 277, 92-99 and Virology (2000), 277:100-110; WJ· Liu et Al" (2001), Virology 289: 362-377·, Feng Yang et al., J. Virol" Dec. 2001, 75 (23): 11811-11820·, CW Marielle et aL, Virology. July 20, 2001, 286 (l): 7-22\ L.-L. Chen et al. (2002), Virology 301: 136-147·, H. Marks et 20 al· (2003), J Gen Viwl 84:1517-1523) Recently, WSSV has been proposed to be a type species belonging to the genus Plasmodium (Μπ·v/rws) of the genus Line Virus (M.i4·Mfl;yo (2002J, Arc/t·Vz&gt;&lt;&gt; 9/·, 147, 1655-1663). In Shen In an earlier genomic analysis of human microarray technology using the microarray technique (microarray 14 1296285 technique), this isolate was identified as having a total of 532 putative open reading frames (〇RF), which are An ATG start codon begins and may encode a polypeptide of at least 60 amino acids long. Of these 〇RFs, 39 have been identified as WSSV structural genes and less than 12 are non-structural In addition, for ~90% of these ORFs have been detected transcripts ^.-C. Wang, et aL UDNA microarrays of the white spot syndrome virus genome: genes expressed in the gidls of infected shrimp," Marine Biotechnology, Fu Tezhong) 〇男10's According to the homology searches for the NCBInr database, most of the ORFs published for this Taiwan isolate showed no significant difference with other known proteins. Similarity. Similar results have been reported for other rain species isolates (Μαη·έ7&amp; C.W·van with αΖ·,

Virology· July 20, 2001, 286 (l): 7-22; Feng Yang et al” J. 15 Viwl” Dec· 2001, 75 (23)·· 11811-11820, however, although the temporary performance of the WSSV gene Already by individual genetic studies (L, L. Chen et al. (2002), Virology, 301, 136-147·, J.-H. Leu, et al. (2005), J. Virol” Jan. 2005 , 79 (1), 140, 149., W.-J. Liu, et al. (2001), Virology 289, 362-377·, M.-F· Tsai et al·, 20 Virology (2000), 277 , 92-99 and Virology 277 (2000), Sichuan and by total analysis (from the above to (200 sentences, 乂Virol 78, 11360-11370·, H, C· Wang et al, “DNA Microarrays of the white spot syndrome virus genome: genes expressed in the gills of infected shrimp," Marine Biotechnology, P. chinensis, 15 1296285 was explored, and so far, no WSSV very early (IE) genes have been identified. As a result, the expression of the viral IE gene will depend on the host cell mechanism and does not involve any virus a novo protein synthesis, which means that the IE group It is particularly important to determine the host range (PD·Friesen (1997), “heart suppression/如·(10)&lt; Baculovirus early gene expression^ In: Miller, LK, (Ed.), The baculoviruses. Plenum Press, New York and London, pp. 141-170). These IE gene products, once expressed, can produce functions such as regulatory trans-acting 10 factors during infection and can be used to initiate Replication events of viruses. In the cascade of regulatory events of the virus, the successive stages of viral replication rely on the proper expression of genes located in the previous phase, for example, in baculoviruses and herpesviruses. During the infection period caused by large DNA viruses, gene expression is regulated such that the very early (IE or α) 15 genes are first transcribed, followed by early (Ε or /3) and late (L or τΟ gene expression (GW) Mouth 99 (5J, C; yi (9iec/m (9/(9g; y, 2 (9, 73-93; GW· Blissard and GF· Rohrmann (1990), Annu· Rev· Entomol·, 35, 127- 155. PD·Friesen and LK Miller (1986), C Urr. Top. Microbiol Immunol. 131 f 31-49; R.W. Honess and 20 B. Roizman (1974), J. Virol, 14, 8-19. To investigate the transcription of the viral IE gene, viral infection is induced in the presence of a protein synthesis inhibitor [usually cycloheximide (CHX)] that prevents de novo protein synthesis by preventing translation. If the translation of the IE gene (rather than transcription) is blocked, the viral infection cycle will also be repressed by the IE phase in 16 1296285. Thus, in the constant presence of CHX, the presence of RNA transcripts detected during viral infection is a good evidence for identifying positive viral genes. For the first time, despite the lack of any recognized immortal shrimp cell line and the difficulty of using CHX in vivo, Applicants successfully used CHX as an inhibitor to deter virus de novo protein synthesis. A total analysis of microarray technology and reverse transcriptase polymerase chain reaction (RT-PCR) was subsequently used to determine the transcription pattern of WSSV, and the results obtained from this were three "very early (10) genes. The 10th donor was identified and named as 2, and 3. In addition, the promoter-regulated region cloned from the wgjsv gene was confirmed in a non-native host cell (ie, Sf9). The insect cell has promoter activity and thus has great potential for application in the field of recombinant DNA technology. C SUMMARY OF THE INVENTION J 15 SUMMARY OF THE INVENTION [In accordance with the first aspect, the present invention provides an isolated WSSV very early a promoter/regulatory region consisting essentially of a nucleotide sequence selected from the group consisting of: (1) a nuclear g acid sequence of sequence identification number: 29; (11) a sylvestre acid of (1) Sequence 5, short-cut fragment (5,-truncated fragment), [with cut-off number: continent, at least 92 nucleotide residues from the end; (10) nuclear sequence, which is used by - White spot virus (WSSV) genomic DNA The plate and the polymerase chain reaction having a cis- forward primer and a primer 17 1296285 to the primer are amplified, and the forward primer is substantially selected from a sequence identification number : 15 is composed of a nucleotide sequence among nucleotide sequences indicated by SEQ ID NO: π, and the 5 reverse primer is basically composed of a nucleotide acid sequence of sequence identification number: 16;

10 15

(iv) - a nucieic acid analogue of the nucleotide sequence of (1) having at least about 6% sequence identity to the nucleotide sequence of (1) and capable of driving an operably linked ( The expression of the target gene operatively connected to the nucleic acid analog; (v) a nucleic acid analog of the 5'-truncated fragment of (ii) having at least about a 5'-truncated fragment of (1)) 60% sequence identity and can drive the expression of a target gene operably linked to the nucleic acid analog; (vi) - a variant of the nucleotide sequence of (1), which contains at least one conserved species Sexual substitution (conservative subs_ti〇n), and can drive the expression of a target gene operably linked to the variant; and (VII) - a variant of the 5'-truncated fragment of (ii), Containing at least one constant substitution, and can drive the expression of a target gene operably linked to the variant. In the above-mentioned WSSV very early promoter, the nodal region can trigger the expression of a heterologous t-base in the non-small 彳 main cell, and thus can be used to construct 18 1296285 to construct various recombinant expression vectors for use. In the transformation - a wide range of primary cells. Thus, according to a second aspect, the vectors are constructed to contain *, and the wssv very early promoter regulatory region is operably linked to the target gene. According to a third aspect, the present invention provides a recombinant host cell which is produced by transforming a host cell with the above recombinant expression vector. 10 15 It is contemplated that the practice of the invention is not limited by the use of particular host cells. In fact, the invention can be applied to a wide variety of prokaryotic and eukaryotic host cells 'including bacterial cells, yeast cells, fungal cells, plant cells, insect cells, mammalian cells, etc., and can be used to produce Useful ribozymes and RNA transcripts, as well as different types of proteins, including: white shellfish present in the cytoplasm (4) 〇plasm) or pedpiasmic space, proteins or cells present on the cell membrane (ceu membrane) Proteins, as well as enzymes for use in the health, agriculture, food industry, brother industry, aquaculture and animal husbandry, especially pharmaceutical proteins and peptides such as interferons, human and animal hormones, immune antigens And antibodies. According to a fourth aspect, the present invention provides a primer set for the selection of a 4-week region of a WSSV very early 20 promoter, comprising a forward primer and a reverse primer, the forward primer being substantially One selected from the group consisting of sequence identification number 15 (i.e., the primer 126-lk-F shown in Table 2 described in the examples) and the sequence identification number: 17 (i.e., as described in the examples). The primer sequence i26-2k-F in Table 2 is composed of the nucleotide sequence of the nucleotide sequence 19 1296285, * the reverse primer is basically the phase identification number: 16 (also shown in The nucleotide sequence of the scorpion 126_R) in Table 2 described in the examples was constructed. BRIEF DESCRIPTION OF THE DRAWINGS Other features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments and the accompanying drawings, in which: FIG. In a virus challenge trial with different doses of CHX, 532 on the microarray under conditions of WSSV infection (vertical axis) versus mock infection (horizontal axis) A scatter plot of normalized Cy3 florescence (ie, performance level) of 10 WSSV open reading architectures (〇RFs), group A: 12·5 mg/kg CHX treatment; group B : 62·5 mg/kg CHX treatment; and Group C: 250 mg/kg CHX treatment;

Figure 2 shows the results of agarose gel electrophoresis RT-PCR of ORFs (ORF126, 15 ORF242, ORF418) of three WSSV IE gene candidates and WSSV DNA polymerase gene (such as 叩(6) (positive control group). , where Μ : 100 bp DNA ladder (Lambda Biotech Inc., Taiwan); diameter 1: 250 mg/kg RT-PCR product of CHX-pretreatment group; diameter 2: vehicle-pretreatment group (only 20% ethanol) RT-PCR product; 20 PATH 3: 250 mg/kg PCR product of the CHX-pretreatment group; and PATH 4: PCR product amplified from WSSV genomic DNA [amplicon size reference]; Figure 3 shows the promoter activity of WSSV IE gene candidates in Sf9 insect cells after 72 h of plastid transfection as shown, wherein these 20 1296285 cells were in bright field (BF group) and dark field (EGFP). Groups, to observe the presence/absence of green fluorescence, were examined; scale = 1 〇〇 μιη; Figure 4 shows SDS-PAGE-separated cell lysis from the same transfected sf9 insect cells of Figure 3. Western blotting results of SDS_pAGE separated cell 5 lysates (Western blotting re Sults), wherein the total protein (bl〇tted t〇tal proteins) of the cell lysate is detected using anti-EGFP or anti-no-actin (control) antibody, and Imaging by an ECL chemiluminescence system; 10 Figure 5 shows the 5, and 3, end of the WSSV 7 transcript, which was used for 5' RACE and 3' RACE primers (126SP1) 126SP2, 126SP3, and 128SP1) are underlined, and the shaded area between -92 and -43 nt in front of the translation start point (transiati〇I1 start) shows possible basic promoter elements (as predicted by the NNPP program) The curved arrow indicates the transcriptional start sites, which are indicated by sequencing 7 randomly selected 5' RACE strains; TATA and polyadenylation signals The polyadenylation signal (AATAAA) is framed and bolded separately, and the poly (A) addition site [p〇ly(A) addition site] is indicated by an arrow; 20 Figure 6 shows WSSV DNA 5'-untranslated of polymerase, RIU, RR2 and IE1 genes Region (5' UTR), where the starting start address deduction (10) "also (10) start) is located at ~26 nt downstream of the TATA box; these TATA boxes are shaded and the 5 ' RACE The identified transcription initiation site is indicated by a curved arrow; 21 1296285 Figure 7 shows the results of a transient transcriptional analysis of WS SV by RT-PCR using ki-specific primer 126F/126SP1 (Α group), such as 特异性 specific primer (group B) and (C) vp2S-specific primer (group C), while shrimp cold-actin-specific primer (group D) and intergenic (intergenic) The primer 5 Ι(^Ρ2/Κ&gt;Ϊ13 (Ε group) was used as the internal control group separately; the diameter]v [is a 100 bp DNA ladder (Lambda Biotech Inc., Taiwan), and was marked above other diameters) The number represents the transfection time expressed as hours p0St infection (hpi); and Figure 8 shows the 10 predicted regulatory elements in the 2 kbp WSSV 7 promoter/enhancer region (regulatory The motif and location of the DNA sequence. [Embodiment 3] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For clarity, the following definitions are used herein. As used herein, the term "promoter sequence" refers to a DNA sequence which is usually located upstream of a gene present in a DNA polymer and which provides a gene for initiating the gene. Turn 20 to generate the address of 1111^^^. Promoter sequences suitable for use in the practice of the invention may be derived from viruses, bacteriophages, prokaryotic cells or eukaryotic cells, and may be a constitutive promoter or an inducible Promoter (in(jucible promoter). When a promoter is not directly adjacent to (ie, covalently linked to) 22 1296285 ^ is a contiguous coding within the naturally occurring genome of an organism derived from 匕In the case of sequence, the promoter is isolated. By "separated, it refers to an isolated substance that has been associated with it under other conditions [eg, 'in vivo (Z&gt; 2 V/V0)). Other components (such as biological components), scallops or mites are purified, and the isolated material itself can be manipulated or processed. The term "isolated" thus includes: by standard purification methods And the purified material, the substance produced by a recombinant DNA technique in a host, and the chemically synthesized substance. Therefore, it is expected that the isolated WSSV very early promoter-adjusted according to the present invention Domain 10 can be obtained by isolation, chemical synthesis, and recombinant DNA techniques from natural sources. 15 20 Regulatory sequences for transcription and translation are DNA regulatory sequences, such as promoters, enhancers, and poly-tartification signals. (p〇lyadenylati〇n signal), terminator (r) and analogs, which provide a coding sequence for expression in a host cell. A "cis-element, a chlorophyll sequence" Incremental regulation (uPre§ula(6) or downregulation of protein expression in specific gene loci (gene 1〇cus). Certain DNA sequences, which are usually located in a gene in a _DNA polymer, And providing a site for initiating transcription of the gene to become mRNA, referred to as "promoter, sequence. Other face or visceral sequences" but "needs to be located "upstream" of a structural gene , , in combination with those proteins that determine the frequency or rate at which transcription and/or translation initiation begins. These other sequences, including attenuator, enhancer, and operator (operato) r) and analogues are spears as regulators. Sequences 23 1296285

Thus, the sequence that will be manipulated to determine whether the transcription and final expression of the gene are to be generated is also referred to as "promoter/regulator, DNA sequence. X-DNA'' coding sequence, is - double strand The sequence, when placed under the control of an appropriate regulatory sequence, is transcribed into RNA in vivo, and 5 the RNA is transduced into a polypeptide. The boundary of the coding sequence is at one position at 5' (amine) The start codon at the basal end is determined by a stop codon at position 3, (silk). A coding sequence can include, but is not limited to, a sequence of a prokaryote, a virus from a prokaryotic or eukaryotic organism. The sequence of the genome, the cDNA from eukaryotic cDNA, the genomic DNA sequence of DNA from eukaryotes (eg, 10 mammals), and even the synthetic DNA sequence. - A polyadenylation signal and transcription termination sequence are usually Is located downstream of the coding sequence. A "cDna" is defined as a copy-DnA (copy DNA) or a complementary-DNA (complementary DNA) and is a reverse transcription reaction from an mRNA transcript (reverse transcripti a product of 〇n 15 reaction. A "message sequence" can also be included within the coding sequence and encode a message peptide at the N-terminus of the polypeptide that communicates with the host cell and Directing the polypeptide to the appropriate cellular location. The message sequence can be found to associate with various proteins that are naturally possessed by prokaryotes and eukaryotes. The terms "nucleic acid" and "nucleic acid sequence," , refers to a single or double-stranded form of deoxyribonucleotide or ribonucleotide sequence containing naturally occurring and known nucleotides or aritificial chemical mimics . As used herein, "nucleic acid" is used interchangeably with the terms "gene,", "cDNA,," "mRNA,", "nucleotide, and 'polynucleotide" unless It is also indicated that a nucleic acid sequence encompasses complementary sequences, in addition to the specific sequences disclosed herein, 5 and their conservative analogs, associated naturally occurring structural variants. And/or synthetic non-naturally occurring analogs, such as homologous sequences with degenerative codon substitutions. In particular, constitutive substitutions can be made, for example, by A nucleotide residue at a 10-nucleotide residue of a particular sequence disclosed is substituted without affecting the promoter activity of the sequence. "Recombinant DNA technology" means for combining two heterogeneities The technique of DNA molecules is usually a result of in vitro ligation of DNAs from different organisms. Recombinant DNA molecules are usually experiments by genetic engineering. Generics are produced by 15. Synonymous terms include "gene splicing," "molecular cloning," and "genetic engineering." The products of these operations form a "recombinant" or "recombinant" Molecules. Techniques for manipulating nucleic acids, such as those used to generate mutations in a sequence, subcloning, labelling, probing, sequencing, Hybridization, etc., are described in detail in scientific journals and patent documents. See, for example:

Sambrook J, Russell DW (2001) Molecular Cloning: a Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York; Current Protocols in Molecular Biology, 25 1296285

Ausubel ed., John Wiley &amp; Sons, Inc., New York (1997); Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I, Theory and Nucleic Acid Preparation, Tijssen ed., Elsevier, 5 Ν ·Υ· (1993). The sequence identity of the two polynucleotides can be determined by a number of different methods known to those skilled in the art, including, but not limited to, the BLAST program of Altschul et al. Mo/. Price, 215, 403-410, 1990) 〇10 To define the present invention, a promoter region is defined as having a transcription start site at its 3' end, and upstream (non-transcribed strand) The 5, direction) extends to contain the minimum number of nucleotides required to initiate transcription at a detectable level above the background value. In addition, a sequence of promoter regions can be extended upstream (in the 5, direction of the non-transcribed strand) to contain all of the nucleotides that will affect the operation and/or efficiency of the promoter region qualitatively or quantitatively. Within the sequence of promoter regions, a transcription initiation site&apos; and a domain of protein binding responsible for the binding of RNA polymerase will be found. The promoter of eukaryotes usually, but not always, contains a "TATA" box and "CAT," box 0. The term "operably connected" is used herein to mean that a first sequence is Arranged in close proximity to a second sequence such that the first sequence can affect the second sequence or a region under the control of the second sequence. For example, a promoter sequence can be operably linked to a gene a sequence, and usually at the 5, terminus 26 1296285 of the gene sequence such that the expression of the gene sequence is under the control of the promoter sequence. Additionally, a regulatory sequence can be operably linked to a promoter. a subsequence, 俾 to enhance the ability of the promoter sequence to initiate transcription. In this case, the regulatory sequence is typically located at the 5' end of the promoter sequence. 5 The term "expression vector, as used herein, is Refers to any recombinant expression system that can be selected in vitro (/n or in vivo (ίη νζ·νο), in any species of host cell, c〇nstitutively or inducibly Nucleic acid sequence. The expression vector can be a display system in a linear or circular form and encompasses a system of expression that maintains the episomal form 1 〇 or is integrated into the genome of the host cell. Or does not have the ability to self-replicate, and it may only drive transient expression in a host cell. According to the invention 'when the term 'transformi〇n'' refers to an exogenous nucleic acid When a molecule is introduced into a selected host cell, the term can be used interchangeably with the I5 term 'transfection.' A nucleic acid molecule (eg, a recombinant DNA construct, according to techniques known in the art) Or a recombinant vector) can be introduced into a selected host cell by a variety of different techniques, such as calcium phosphate- or calcium chloride-mediated transfection &gt; Transfection by 20-well electroporation, microinjection, particle bombardment, liposome mediation Transduction with (liposome-mediated transfection), transfection with sputum, retrovirus or other viruses [such as baccinvirus of vaccinia virus or insect cells] 1296285 Transduction, protoplast fusion, Agrafeacia/wm-mediated transformation or other methods. The terms "cell", "host cell", "transformed host cell" and "recombinant host cell" as used herein are used interchangeably and refer not only to Specific individual cells also include sub-cultured offsprings or their potential offsprings. Subcultured progeny formed in subsequent generations may undergo specific genetic modification due to mutation or environmental impact, and therefore, the subcultured progeny may not actually be identical to the derivative. The mother cells of the subcultured progeny. However, subcultured cells still fall within the scope of the terminology used herein. The terms "polypeptide", "peptide" and "protein" as used herein may be used interchangeably, and 1 refers to a polymer composed of amino acid residues in which one or more amino acid residues are present. The base is a naturally occurring amino acid or an artificial chemical analog. In an earlier study by the applicant, a WSSV virus isolate, also known as the grass shrimp 1994 Taiwan isolate (WSSV 20 T-1), was isolated from grass shrimp (Pen like w m (10), according to the Budapest Treaty (Budapest) Treaty) was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Lushan, Wuhan, Hubei, 430072, People's Republic of China on January 11, 1996, and It is given the registration number 28 1296285 CCTCC-V96001 (see also granting Guo Guangxiong et al. still 5,824, 53^us 6,190,862 and L.-L·C7z(9) from (2002), 3〇D6-W7). This Taiwan isolate The complete genome was then sequenced and logged into the NCBI Data Library 5 via direct delivery under accession number AF440570. To the best of the Applicant's knowledge, none of the reports have identified the WSSV Very Early (IE) gene. In the present invention, Applicants used oxycodone (CHX) as an inhibitor to repress the virus from WSSV-infected shrimps from the primordial white matter synthesis, and then by using 532 0 RFS 10 derived from WSSVT-1 according to use. The WSSv DNA (0RF/gene) microarray designed by the WSSV PCR product of the specific primer (see Table 1 in the following examples) was used to examine the RNA transcript of the very early viral gene. It may be very early for WSSV. The three 〇RFs of the gene candidate, namely ORF126, ORF242 and ORF418, were successfully monitored. A genomic sequence alignment of the three previously reported WSSV 15 isolates showed: None of the three 〇RFs in the known WSSV isolates have been deleted. ORF126, 〇RF242 ® and 〇1^418 were therefore named WSSV 分别 (very early genes, ie2 and ie3) were briefly reported. A transient reporter assay is then performed by 20 to explore that the promoter regions of the three WSSVIE gene candidates trigger a heterologous gene in a non-native host cell (eg, Sf9 insect cells) [eg, enhanced The expression of the green fluorescent protein (EGFP) gene] is also used as a reporter gene in this analysis. The primer used to construct the transient expression vectors was Table 2 is described in the Examples in Example 2. Surprisingly, the 1 kbp and 2 kbp promoter regions of WSSV W selected according to the present invention are functional in WSSV non-native host cells, suggesting The promoter region of the WSSV id has great potential for supplying a variety of recombinant expression vectors for constructing a wide range of host cells for transformation. WSSVization/further analysis was performed using the 5,/3, RACE analysis of the primers listed in Table 3 in the examples. The results obtained show that -52 nt G relative to the ATG translation start point represents the major transcriptional initiation point of WSSV7. In addition, a putative TATA box (TATAA) was found upstream (-26 nt) from the start of transcription (ie, at _82 nt to -78 nt relative to the start of the ATG translation). This tata motif, together with the transcriptional initiator, is considered to be an essential element of the RNA polymerase II promoter. The NNPP of the upstream sequence of the putative transcription start site of WSSv w [Neural Network for promoter prediction] analysis 15 identifies a high probability that the predicted basic promoter region is a bit Between -92 nt and -43 nt in front of the tweet start codon. Furthermore, the experimental results obtained by promoter activity analysis suggest that transcription of wssv is not restricted to its native host RNA polymerase II. In addition, according to the nucleotide sequence analysis, the WSSVid 5, UTR (region not translated) was found to contain several sequences that are consistent with the GATA element (A/T) GATA (G/A) consensus sequence (consensus seqUences) ). This may be important because the GATA element is identified as a binding site for the transcription factor, for example in the promoter of the baculovirus 0pMNPV IE gene, 卯64, (Ρ·Η·Kogan and GW Blissard (1994), j· virol·, 68 30 1296285 S73-S22). There are also several other possible regulatory factors, including: 2 direct repeats 歹U (CACACACA and CTCTCTCTCT), 2#1 short-order 歹ij repeats (TTTCTGG and CCAGAAA), baculovirus early gene promoter elements ( Upstream Regulatory Factors) (CGTGC) (/?.Z&gt;. and Bc 5 jBcmmTig (2003J, /· S岑 (Pi 7 human 7S27-7842), baculovirus late promoter promoter (TTAAG) (LA Gwar/ (10) and M.叱

Smith (1990), Virology, 179, 1-8\ T.D. Morris and L.K·

Mi...r (7994), 7祁, 7” 53), and a palindromic seqnence that produces a function as a transcriptional enhancer (C. Γ. 10 McMurray et al. (1991), Proc · Natl·Acad·Sci·USA·, 88, 666-670·, C. Rasmussen et al. (1996), Virology, 224, 235-245) In addition to the above, the sequence of the selected 3' RACE product Analysis showed that poly(A) was added at a 17 nt address downstream of 15 of the AATAAA polyadenylation signal (see Figure 5).

BLAST analysis of the GenBank/EMBL, SWISSPROT, and PIR databases predicted that the WSSV id coding region contained the Cys2/His2-type zinc finger motif. This element has a role in DNA binding, and its presence suggests that the WSSV may produce a function as a 20 transcription factor. The 2 kbp promoter region of WSSV W was cloned [it was amplified by PCR using primer 126-2k-F (SEQ ID NO: 17) and primer 126-R (SEQ ID NO: 16) The nucleotide sequence of [exit] is shown in Sequence Identification Number: 29. The nucleotide sequence of the WSSV coding region and the putative amino acid sequence encoded by 31 1296285 are shown in Sequence Identification Number: 30 and Sequence Identification Number: 31, respectively. In accordance with the above, the present invention provides an isolated Wssv very early promoter-regulatory region consisting essentially of a nuclear nucleoside sequence selected from the group consisting of: (1) a sequence identification number: a nucleotide sequence of 29; (ii) a 5, truncated fragment of the nucleotide sequence of (1) (5, seven unsecated fragments) 'It has at least 3 of the sequence identification number: 29 92 nucleotide residues; 10 (丨丨丨) a nucleic acid sequence which is composed of a white spot virus (WSSV) genomic DNA as a template and a primer set having a forward primer and a reverse primer. Amplification is carried out by a polymerase chain reaction (P〇lymeraSe chain reaction), which is basically composed of a nucleotide sequence selected from the group consisting of sequence identification number: 15 and sequence identification number: π 15 a nucleotide sequence consisting essentially of a nucleoside acid sequence of sequence identification number 16; (iv) - a nucleic acid analog of the nucleotide acid sequence of (1) Nucleic acid analogue), which has the nucleotide sequence of (1) to Approximately 60% less sequence identity and can drive the expression of a target gene operably linked to the nucleic acid analog; (v) - a 5'-truncated fragment of (ii) a nucleic acid analog having at least about 60% sequence identity to the 5'-truncated fragment of exemplified, and 32 I296285 and which can drive the expression of a target gene operably linked to the nucleic acid analog; (vi) - a variant of the nucleotide sequence of (1) which contains at least one conservative substitution and which drives the expression of a target gene operably linked to the variant; (vii) - a variant of the 5'-truncated fragment of (8) which contains at least _ conservation substitutions and which drives the expression of a target gene operably linked to the variant. 10 15 20 According to the invention, when the isolated WSSV very early promoter-regulatory region consists essentially of a 5,_ truncated fragment of the nucleotide sequence of (1), it preferably has a sequence Identify at least 160 nucleotide residues from the 3' end of the number. More preferably, the 5,~ truncated fragment of the nucleotide sequence of (1) has at least 25 桉4 acid residues from the 3' end of the sequence number:29. More preferably, the 5th truncated fragment of the _nuclear (four) sequence has at least 5_nuclear acid base from the end of the column identification number: 29. Preferably, the truncated fragment of the (1) p-acid sequence has at least 1 nucleus: acid residue from the 3' end of 29. According to the present invention, the isolated very early initiation of the amplification reaction is carried out; the introduction of the scorpion and a reverse primer. Cr column identification number: the order of the shirt = the primer is basically composed of - selected from the nuclear singularity shown in the sequence: 17, and the reverse is basically identified by the _ sequence 33 1296285 column The nucleotide sequence of number: 16 is composed. In a preferred embodiment of the present invention, the DNA template is extracted from the Taiwan isolate WSSVT-1, and the sample of the isolate is deposited with the China National Culture Collection under the registration number CCTCC-V96001. 5 According to the present invention, the isolated WSSV very early promoter/peripheral region may also comprise a conservation variant of the nucleotide sequence of sequence identification number: 29 and synthetic analogues [ Such as sequence deletion (deieti〇n), insertion (inserti〇n), inversion (inverison), substitution or addition, but there are 10 pieces of such variants and the like can be similarly The transcription of the sequence at which the trigger bit is downstream and operably linked. In various embodiments, the variants and analogs may be substantially homologous to the term used in the preceding text, or greater than 60% when measured using the algorithm described in the foregoing. , 7〇% to 1〇〇%, at least 1580%, at least 9%, or at least 95% identity. According to the present invention, the isolated WSSV very early promoter-regulatory region can be carried in a performance sputum. As used herein, the term &apos;sees &quot;a synthetically or recombinantly produced nucleic acid construct that carries a series of nucleic acid elements to permit transcription and translation of a particular nucleic acid within a target cell. A variety of different strategies can be used to supply fragments for combining or combining DNA, and depending on the nature of the terminal of the eight segments, a suitable strategy will be immediately apparent to those skilled in the art. The above-mentioned WSSV very early promoter-regulatory region is capable of driving a heterologous 34 1296285 (translation termination site), an insertion cloning location, an enhancer sequence, and a polyadenylation site. (polyadenylation site), a regulatory sequence, and the like. In addition, the vector of the present invention may comprise a nucleotide sequence relating to one or more restriction endonuclease recognition sites. These sequences are well known to those skilled in the art. Marker genes suitable for use in the present invention include, for example, a dihydrofolate reductase gene for eukaryotic cell culture and a G418 or neomycin resistance gene, Zeocin resistant group 10 for insect cell culture. And, for ampicillin, streptomycine, tetracycline or kanamycine resistance genes for E. coli and other bacterial cultures. In a preferred embodiment of the invention, the recombinant expression vector further comprises a second target gene located downstream of 15 of the other promoter sequence and operably linked to the other promoter sequence, the A promoter sequence may or may not be a wssv very early promoter-regulatory region as disclosed herein. According to the present invention, the first and second target genes independently encode a gene product selected from the group consisting of an enzyme, a therapeutic poly 20 peptide, an epitope, and an antibody. In a preferred embodiment of the invention, the recombinant expression vector is pIZME/WSSV126-2k/V5-EGFP-His. In another preferred embodiment of the invention, the recombinant expression vector is pIZAIE/WSSV126-lk/V5-EGFP-His. 36 1296285, ,, and the type of performance carrier can be used to transform or transfect a beggar, the master of the field. Accordingly, the present invention provides recombinant host cells which are produced by transforming a host cell into a recombinant expression vector of t. It is contemplated that the practice of the invention is not limited to the use of particular host 5 cells. In principle, the present invention can be applied to a wide variety of prokaryotic and eukaryotic donor cells 'including: bacterial cells, yeast cells, fungal cells, plant cells, insect cells, mammalian cells, etc., and can be Used to produce useful ribozymes and RNA transcripts, as well as different kinds of proteins, including proteins present in the cytoplasm or periplasmic space 10, proteins present on the cell membrane or extracellular proteins, and Supply enzymes for the industrial, agricultural, food, environmental, aquaculture and animal husbandry industries, especially pharmaceutical proteins and peptides such as interferons, human and animal hormones, immune antigens and antibodies. The host cell which can be used in the present invention may be a prokaryotic or eukaryotic 15 cell' and may be an untransformed/transfected cell, or another having been subjected to at least one specific nucleic acid sequence not disclosed herein. Recombinant nucleic acid sequence transfected/transfected cells. Prokaryotic cells suitable for use in the present invention include, but are not limited to, cells derived from bacteria such as Escherichia coli (five, Bacillus subtilis 20 (Wan Shen (6) like (10) (6)), Lactobacillus species 5* ρ·), Keystrophy species (Streptomy 577·) and Salmonella typhi (Symonella typhi); Cyanobacteria, Aci/nomyceia and so on. Eukaryotic cells suitable for use in the present invention include, for example, fungal cells, protozoa 37 1296285 protozoa cells, plant cells, insect cells, animal cells, and human cells. Examples of suitable fungal cells are yeast cells, such as baker's yeast απν illusion or methanolophilic yeast ( cells of Ρί(:/Η·α. Suitable plant cells are those derived from gynosperms or angiosperms). (angiosperms), preferably monocots and dicots, especially crops, and derived from the roots, stems, leaves or mestems of these plants, and It is cultured in the form of protoplasts (pr〇t〇plasts) or healing tissues (callus). Examples of suitable insect cells are Sf21 cells and Sf9 cells derived from the fruit line S2 cells and 10 derived from the autumn army insects. The animal cells may be cultured cells or cells in vivo, preferably derived from vertebrates, more preferably from mammals, and are organs or tissues derived from such animals, such as kidneys, liver, lungs, ovaries, breasts. , skin, bone network, blood. Representative examples of animal cells include: 15 CHO, COS, BHK, HEK_293, HeLa, NIH3T3, VERO, MDCK, MOLT-4, Jurkat, K562, HepG2, and the like. In a preferred embodiment of the invention, the recombinant host cell is

Sf9 insect cells. Suitable media for host cells for performing DNA recombination techniques are well known in the art of biotechnology. For example, the host cells can be cultured in a fermentation bioreactor, a shake flask, a test tube, a microtiter plate, or a cultured orphan, and the host cells can be cultured under conditions suitable for the growth of the cells (including The culture temperature, the pH of the culture medium, and the dissolved oxygen concentration of the culture are carried out. 38 1296285 Finally, the present invention provides a primer set for culturing a WSSV very early promoter-regulatory region, comprising a forward primer and a reverse primer, the forward primer being substantially selected from the group consisting of The sequence identification number: 15 and the nucleotide sequence of the nucleotide sequence shown in the sequence identification number: 17 are composed of 5 nucleotides, and the reverse primer is basically a nucleotide sequence of sequence identification number: 16. Composition. The side primer set can be used to select a WSSV very early promoter-regulatory region that is evolutionarily homologous to the particular sequence disclosed herein. It is contemplated that all of the materials and methodologies disclosed herein may be used to practice the invention. The invention will be further illustrated by the following examples. A person having ordinary knowledge in the art will be familiar with many modifications that allow these embodiments, as well as the embodiments noted in the present disclosure, which also apply the basic, novel or advantageous features of the present invention. Technology and teaching. Therefore, the scope of the invention is not limited by the specific embodiments set forth herein or elsewhere. Example I·Materials and Methods Gene microarray of virus-wafer preparation 20 Grass shrimp WssV 1994 Taiwan isolate (WSSV Τ_υ (its genome is registered in the NCBI database under accession number AF4405 70) was used in the following In all the experiments described, this WSSVT~1 isolate has been deposited with the Chinese Type Culture Collection on January 11, 1996 according to the Budapest Treaty (CCTCC, Wuhan University, Lushan, Wuhan, Hubei, 430〇) 72, 39 1296285 People's Republic of China), and was given the registration number CCTCC-V96001 (see 1^5,824,535 and 1; 5 6,190,862 granted to Guo Guangxiong et al.) WSSV viral gene microarray (wafer) is based on WSSV T- 1 The genome of the isolate was designed. Briefly, each wafer contained 532 predicted 5 WSSV ORFs and a partial sequence of the grass shrimp/5-actin gene. The specificity derived from these 532 ORFs was used. After the polymerase chain reaction (PCR) of the sex primer, the PCR product having an amplification product size of 200 to 600 bp for each WSSVORFs was spotted in a pre-coated manner in a three-fold manner. Glass slides (U-Vision Biotech Inc., Taiwan), 10 and related shrimps (the grass shrimp actin gene is also used for 3 replicates. The shrimp/5-actin gene is used as a positive control group and is Used to normalize data between slides. Details of preparing viral microarrays are described in various literatures, see 'eg H.-C. Wang, et al· DNA microarrays of the white 15 spot syndrome virus Genome: genes expressed in the gills of infected shrimp," Marine Biotechnology, Fu Xizhong. Preparation of viral gene microarrays - the preparation of the virus inoculum preparation and viral challegene trial is described in From still to α/. (*/999), 20 Dis· Aquat. Org·, 38 (2), 107-114. Cultured WSSV-free shrimp used for CHX (Sigma) treatment and virus challenge tests (Grass shrimp) was generously provided by the Tung-Kang Marine Laboratory (Taiwan Fisheries Research Institute) of the Fisheries Research Institute of the Executive Yuan Agricultural Committee. During the experimental period, at the temperature of 40 1296285 25C With a weight of 3545 g of shrimp farming in 8 containing sterile 33 ppt (one thousandth) of 1000 liters of water FRP (fiber-reinforced plastics on) slots. The WSSV inoculum is the tissue prepared from the collection 5 of experimentally infected shrimp (grass shrimp). To identify the WSSVIE gene, WSSVT-1 infected shrimp were treated with different doses of CHX (12.5 mg/kg, 62.5 mg/kg and 250 mg/kg body weight) by intramuscular injection 2 hours prior to the virus challenge test. In 2% ethanol). Control shrimp were injected with 2% ethanol. In the virus challenge test, shrimp pretreated with CHX and 20% ethanol were randomly infected with a 〇·9% NaCl solution (m0Ck-infected) or infected by intramuscular injection at a dose of 5 〇pL/10 g body weight WSSV inoculum (prepared in 0.9% NaCl solution). Because sputum tissue is a major target of WSSV infection (C.-F· Lo, ef αί· (J996), Dis. AquaL Org·, 27, 215-225·, md H, C. Liu, et al· (1997) ), Dis, Aquat· Org·, 15 53-72), after 8 hours of infection (hpi), pseudo-infected and WSSV-infected sputum sputum tissues were collected and immediately frozen in liquid nitrogen until RNA extraction When. Total RNA was harvested from the collected shrimp tissue using a Rneasykit (Qiagen) according to the manufacturer's protocol. To make the 20 cDNA target, the RNA sample (2 〇 kg) was fluorescently labeled with Cy3dUTP using a CyScribe First cDNA Labeling kit (Amersham Bioscience) according to the manufacturer's instructions. Thereafter, a Microcon YM-30 column (Amicon) was used to remove unincorporated free nucleotides. The Cy3-labeled cDNA thus obtained was concentrated, and in the subsequent experiments, 41 1296285 was used as the microarray target. Hybridization, scanning and statistical analysis of the viral gene m-column I target The Cy3-labeled cDNA target prepared above was introduced to hybridize with all 1:8 points in the WSSV microarray. Then, a five conjugated laser scanner (GenetaCTM LS IV Microarray Sc surface er, Genomic Solutions) was used to scan the microarray. The intensity of the scanned fluorescence was quantified by GenePix 3® (Axon Instruments). For those transcripts that are bound to the microarray probes, they are detected as 10 to the intensity of the sputum, which is converted to a measure of approximate absolute performance by subtracting the background signal levels. The signal level of the positive control group (grass stone-actin gene) was used to normalize viral gene expression results between different arrays. The gene expression of WSSV is based on the normalized ratios of the normalized gene table I5 on the CHX-pretreated WSSV-infected wafer relative to the corresponding gene in the pseudo-infected wafer. It was decided to draw. RT-PCR analysis of CHX-insensitive genes confirmed microarray results for microarray results at the highest level of CHX treatment (250 mg/kg body weight), those in WSSV-infected samples The intermediate level 20 is reverse transcriptase-polymerase chain reaction (RT-PCR) for ORFs that are at least 1.5 times higher than those in the pseudo-infected sample. In the RT-PCR analysis, the template was prepared from the original batch of total RNA (ie, from 250 mg/kg CHX-pretreated WSSV-infected shrimp) and an additional batch of RNA (extracted from 42 1296285 WSSV-attacked shrimp pretreated with 20% ethanol). The RNA samples (20 pg) were treated at 37 ° C with ribonuclease (Rnase)-free DNase I (Roche) for 1 hour to remove the total RNA sample prepared. Any viral genomic DNA contaminant. A portion of total RNA (~10 pg) 5 was used to synthesize the first strand of cDNA by using Superscript II reverse transcriptase (Invitrogen) and a (dT) primer in a 20 μί reaction mixture. . After the RT reaction, a portion (2 pL) of the reaction product (containing approximately 1 pg of cDN A) was dispensed to correspond to those WSSV genes that were expressed in the infected shrimp treated with CHX-pretreatment (ie, The specific primer set of the IE gene candidates shown in the microarray analysis result 10 was subjected to pCR. As a positive control group of the CHX treatment group, PCR was also carried out using a gene-specific primer set dnapolF/dnapolR (Table 1) based on a previously reported early WSSV late gene, ie, W And was designed to

Chen et al. (2002), Virology, 301, 136-147). 15

43 1296285 Table 1. Primer ORF/gene primer sequence (5'-3') used to screen the wssv IE gene by microarray and CHX treatment WSSV T-1 genome sequence coordinate amplification product size (bp) ORF126&quot; W 126F: gactctacaaatctctttgcca (SEQ ID NO: 1) 126SP1: ctacctttgcaccaattgctag (SEQ ID NO: 2) 65729—65750 66209—66230 502 bp ORF242/^2 242F1: ataccaacaaccccagaa (SEQ ID NO: 3) 242R1: atggggcgggatacaaaa (Sequence Identification Number :4) 131117—131134 131332—131349 233 bp 0RF418/^3 418F1: gctggaggaggcttgttgat (SEQ ID NO: 5) 418R1: gggccagaaatgccttacag (SEQ ID NO: 6) 242832—242851 243081—243100 269 bp DNA pol dnapolF: tgggaagaaagatgcgagag (Order歹] J identification number: 7) dnapolR: ccctccgaacaacatctcag (serial identification number: 8) 26292-^26311 26858 — 26877 586 bp VP28 vp28F: ctgctgtgattgctgtattt (sequence identification number: 9) vp28R: cagtgccagagtaggtgac (sequence identification number: 10) 278914 —278933 279450 — 279468 555 bp Intergenic IC-F2: cag Actattaatgtacaagtgcg (SEQ ID NO: 11) IC-R3: gaatgattgttgctggttagaacc (Serial Identification Number: 12) 126597-^126619 125494-125517 1126 bp Shrimp/3 - Actin Actin FI: gaygayatggagaagatctgg (Serial Identification Number: 13) Actin Rl: ccrgggtacatggtggtrcc (SEQ ID NO: 14) — 686 bp

As a quality control for examining WSSV genomic DNA contaminants, PCR was also performed on CHX-pretreated WSSV-infected RNA samples without reverse transcription. The PCR product 5 amplified from WSSV genomic DNA was used as a positive control for PCR and was also used as a related size marker. Promoter Activity Assay For each WSSV IE gene candidate identified by RT-PCR, a transient reporter assay was performed. Off 44 1296285 In both analyses, Sf9 insect cells (Invitrogen) were transfected with several promoters with an EGFP reporter gene. As a starting point for this analysis, the plastid pIZAIE/V5-His is a commercially available plastid pIZ/V5-His (Invitrogen), which is located at the multiple colonization site (MCS) by deletion 5 The previous 0pIE2 (OpMNPV 2) promoter was remodeled. Subsequently, an EGFP gene (BD Biosciences) was inserted into the MCS of pIZAIE/V5-His to generate a first derivative plastid pIZAIE/V5-EGFP-His. Thereafter, 5' of each WSSV IE gene candidate was amplified from WSSV genomic DNA by using PCRs containing primers (see Table 2) with appropriate restriction endonuclease 10 recognition sites at their 5' ends. The portion of the untranslated region (5, UTRs) (~1 kbp). Each of the resulting PCR products was purified using a GFX PCR product purification kit (Roche) to limit digestion by endonuclease and then inserted into the EGFP-based 15 factor in pIZAIE/V5-EGFP-His MCS. The resulting plastid was named pIZAIE/WSSVx/V5-EGFP-His, where "X" represents an ORF selected by RT-PCR analysis. A plastid, pIZ/V5-EGFP-His constructed by culturing the EGFP gene into the plastid PIZ/V5-His was used as a positive control plastid, and a 2 〇 corresponding AIE plastid That is, pIZME/V5-EGFP-His was used as a negative control group. Another negative control plastid was constructed by introducing the EGFP gene and the WSSV promoter region into the plastid pIZAIE/V5-EGFP-His to generate pIZME/WSSVdnapol/V5-EGFP-His. For a promoter sequence candidate that provides a positive signal, its reverse 45 1296285 sequence was used to construct an additional control plastid named pIZME/WSSVxrev/V5-EGFP-His. Finally, in addition to these 1 kbp forward and reverse plastids, each volume can have a high level of EGFP, and its promoter is constructed by a 5 segment of a 2 kbp directional fragment. The driven EGFP-reported gene plastid was further analyzed. The correctness of all these strains was confirmed by DNA sequencing. These primers for the construction of these plastids are listed in Table 2.

Table 2. Injectors used to construct transient expression vectors for promoter activity analysis

The plastid primer sequence (5'-3')/restriction enzyme* is small (bp, ρΙΖΔΙΕ/WSS V126-1 k/V5-EGFP-His F: cg£^Mi£gagatcctagaaagaggagtg (sequence identification number·· 15)/ EcoRl R: ccg£i££^£cttgagtggagagagagagc (Catalog ID: 997 pIZAIE/WSSV126-2k/V5^EGFP-His F: cggMli£gatgatggtgatgtttctagg (Serial Identification Number·· 17)/EcoRl R: ccg£l£^ £cugagtggagagagagagc (SEQ ID NO: 16)/XhoI 2063 pIZAIE/WSSV126rev/V5-EGFP-His F: cgg^£cttgagtggagagagagagc (Serial Identification Number: 18)/£coRl R: ccgctceaegacatcctaeaaaeaeeagtg (^ ^»| Identification Number: 19) /X/ioI 997 pIZAIE/WSSV242/V5&gt;EGFP-His F: eeeetaccetcttcaacatcttcttgttcg ID: 20) ΑίΓρηΙ R: ataagaatgcsecceccatsaaeatctctgegaaatg (Sequence ID: 21)/Λ^ίΙ 987 pIZAIE/WSSV418/V5-HGFP-His F: cseaattcetcecacatetgtctaaacttc Identification number: 22) / £cMI R: ccectceaecaacaaecctcctccaecc ID: 23) /X/^I 841 pIZAIE/WSSVdnapol/V5-EGFP-His F: tagaectcacttctccteacactcttsactsat H Identification number·· 24)/Sacl R: gtggaagagggtga Tggagctggagatgatcatc (SEQ ID NO: 25) 571: The restriction enzyme cleavage site of these primers is underlined. 10 For DNA transfection, Sf9 insect cells were seeded in a 24-well culture dish (3xl05 cells/well) and grown overnight at 27 °C in Sf-900 II SFM without 46 1296285 serum medium (Invitrogen). . The plastid DNA was diluted to 1 pg/pL in TE buffer (pH 8.0), and the liposome vector of sf9 cells was transfected (1 gg plastid DNA/well) according to the manufacturer's protocol.

Cellfectin Reagent (Invitrogen) is coming. After 72 hours of transfection, the EGFP fluorescence signal was observed under an Olympus 1X71 inverted fluorescence microscope and an Olympus DP50 digital microscope camera was used for photo recording. At that time, harvest and dissolve the cells. Φ The cell lysate thus obtained from each well was adjusted to an equal amount of 10 total protein' and EGFP was analyzed by Western blotting. For Western blot analysis, the total protein of each cell lysate was separated on a 15% SDS-PAGE, transferred to a PVDF membrane (Osmonics) and used anti-EGFP monoclonal antibody (B-2, Santa Cruz) Biotechnology) or anti-human/3-actin polyclonal antibody (η &gt; 496, 15 Santa Cruz Biotechnology) for probe detection. Blots were visualized using an enhanced chemiluminescent-light (ECL) _ detection kit (NEN Life Sciences), which was combined with horseradish peroxidase (horseradish) Peroxidase goat anti-mouse IgG or goat anti-rabbit IgG was used as a secondary antibody to produce a very early gene transcription of the 5' and 3' endpoints of very early gene transcripts. The 5' and 3' regions of the present are obtained by rapid amplification of the cDNA 5, /3, (MA Fro / anger (10) d al ·, (1988), Proc · Natl · Acad · Sci · USA · 85, 8998-9002), this 47 1296285 uses a commercial 5,/3, RACE kit (Roche) with avian myeloblatosis virus (AMV) reverse transcriptase (Roche, included in the kit). The RNA samples used in this study for 5,/3, RACE analysis were isolated from shrimps infected 24 hours after WSSV infection and then treated with RNase-free DNase I (Roche). Suitable gene-specific primers for rapid amplification of the 5'/3' end of the cDNA are listed in Table 3. The final amplified product was cloned into the pGEM-T Easy vector (Promega) and sequenced. The sequence of the insert is compared to the sequence of the WSSV genomic DNA. 10 Table 3. Specific primers for ORF126 5' RACE and 3' RACE _ primer sequence (5'-3')_ usage

126SP1: ctacctugcaccaattgctag (sequence identification number: 2) 5, RACE

126SP2: gtacagtactgtccatgtcgat (sequence identification number: 26) 5, RACE

126SP3: cctcttcatcacctcaatacc (SEQ ID NO: 27) 5, RACE

128SP1: gagactgatcgacatggacagtac (SEQ ID NO: 28) 35 RACE Temporary analysis of WSSV very early gene transcripts by RT-PCR WSSV-attacked shrimp (grass shrimp) at 〇hpi (that is, at the time before infection) And samples are taken at 2, 4, 6, 8, 12, 18, 24, 36, 48, 60 and 72 hpi. Total RNA was removed from the ascites of shrimp harvested at various time points, purified by TRIzol Reagent (Invitrogen), and then treated with RNase-free DNase I (Roche) to remove any residue. DNA. The synthesis of the first strand of cDNA was performed using the (dT) primer, and 2 μί (~1 gg) of the cDNA was subjected to PCR in a 50-μί reaction mixture containing a suitable primer set (see Table ^ for comparison). , WSSV heart and 2〇叩2 (§ The gene fragment was also amplified from the same template by the primer group dnapolF/dnapolR and vp28F/vp28R, respectively. One shrimp million muscle egg 48 1296285 white primer group, muscle The kinesin F1/actin sR1 was used as an internal control for RNA quality and amplification efficiency. To confirm the absence of WSSV DNA contaminants in these RNA samples, a WSSV genomic DNA-specific primer set, IC-F2/IC-R3 (which are regions between five genes derived from the WSSV genome) were also used as a quality control group. These primers were used to amplify the target ORF/gene sequence. It is shown in Table 1. Analysis of very early gene promoters and coding regions According to the genome of WSSV T-1 (NCBI accession number · AF440570), the nucleotide sequences of these ORFs and the upstream region of these ORFs are Use 10 computer programs NNPP (Reese, MG·, EecKman, FH· 1995. New neural network algorithms for improved eukaryotic promotor site recognition. In: The seventh international genome sequencing and analysis conference. Hilton Head Island, SC; MG Reese, et al. (1996), Large scale sequencing 15 specific neural networks for promotor and Splice site recognition. In: Hunter, L., Klein. TE (Eds.), Biocomputing: Proceedings of the 1996 Pacific symposium. World Scientific Publishing, Singapore) and the GenBank/EMBL, SWISSPROT, PIR and EMBOSS databases were analyzed. 20 Π Results The microarray and CHX treatment were used to screen the WSSVIE gene. When the WSSV DNA (ORF/gene) microarray was used to examine viral gene expression, the protein synthesis inhibitor CHX present in WSSV-infected shrimp was It is expected to result in a specific accumulation of 49 1296285 RNA transcripts of very early genes. This is because, although transcription of all other viral genes requires viral proteins as transcription factors, the synthesis of these proteins has been inhibited by the presence of CHX.

Figure 1 shows that in 3 viral challenge tests with different doses of CHX (12.5 rng/kg, 62.5 mg/kg, and 250 mg/kg), infection was compared to mock infection. A scatter plot of the normalized Cy3 fluorescence intensity (ie, performance level) of the 532 WSSV ORFs located on the microarray. Each of the plotted points is based on a three-repeat microarray result corresponding to a single WSSV ORF and represents the ratio of the Cy3 fluorescence level to the expression level of β_actin. With 45. The proximity of the contour (Proximity to the 45. line of equivalence) indicates a similar level of performance in infected and uninfected conditions. The differential expression cut-off line (1.5:1) is shown in group C of Figure 1. As can be seen from Figure 1, the differential expression level of the WSSV gene is gradually increasing close to the 45 in the presence of increasing doses of CHX. Contour. The results observed not only confirmed that CHX treatment successfully inhibited viral protein synthesis, but also suggested that for most of these 532 WSSV ORFs, the WSSV gene transcription does depend on the presence of one or more viral proteins. Orf, which is considerably unaffected by the highest CHX dose (i.e., having a differential performance level greater than 1 · 5:1), is considered a candidate for the IE gene. Although data points close to the initial points of the scatter plots could not be clearly identified&apos; computer analysis identified 60 IE gene candidates from Group C of Figure 1. 50 1296285 Order of CHX-insensitive genes _ recognition analysis The 60 IE gene candidates identified above were further subjected to RT-PCR analysis. Figure 2 shows the agarose gel electrophoresis RT-pCR junction of 〇RFs (ORF126, ORF242, ORF418) of three WSSV IE gene candidates, in which the WSSV DNA polymerase gene (also called 70/) was used as a control. The group, the primer set used to amplify the target ORF/gene sequence, is listed in Table 1, and the results are based on total RNA extracted from whales of WSSV-infected shrimp at 8 hpi. Path 1 shows RT-PCR results for the 250 mg/kg CHX-pretreatment group; Run 2 shows 10 RT-PCR results for the vehicle-pretreatment group (only 20% ethanol); Trail 3 shows 250 for no reverse transcription reaction PCR results of the mg/kg CHX-pretreatment group; and PCR 4 shows the PCR product amplified from the WSSV genomic DNA (PCR positive control); and the Μ is 100 bp DNAp ladder (Lambda Biotech Inc., Taiwan) The results obtained by 〇 identified three CHX-insensitive 〇RFs, namely 15 ORF126, ORF242 and ORF418. Repeated alignment of total RNA samples from different individual 3% infected shrimps confirmed that the CHX-insensitivity observed in Figure 2 was consistent. The possible sequence coordinates of these three 〇rfs in the genome of three known ~^¥ isolates are summarized in Table 4. It can be seen that none of the three ORFs deleted in the three known WSSV isolates. 51 1296285 Table 4. Sequence coordinates of three identified CHX-insensitive ORFs within the genome of three known WSSV isolates. Taiwan isolate (WSSV T-1)* Thai isolate ** Chinese isolate* ** NCBI registration number NCBI registration number NCBI registration number AF440570 AF369029 AF332093 ORF126 65711~66385 81077~81751 32125~32799 ORF242 131023~131349 146706~146380 97436~97762 ORF418 242850~243032 256954~257136 207904~208086 *:Separated from Taiwan Infected grass shrimp, its complete genome sequence is directly sent to GenBank for storage; and the term "T-1 strain, is in L.-L. d (2 offering 2), 5 30 people j? 36 -W7 was first described. **: Isolated from infected grass shrimp imported from Thailand in 1996, see CW vim Hulten et al., Virology· July 20, 2001, 286 (1): 7-22. **: Infected spotted scorpionfish from Tongan, Xiamen, east China, in October 1996, see, j 10 Virol” Dec. 2001, 75 (23): 11811-11820 The promoter activity analysis of the 〇WSSV IE gene candidate is shown in Figure 3, at the transfection At 72 hours, the green fluorescent signal produced by the expressed EGFP (enhanced green fluorescent protein) was observed to be present only in pIZME/WSSV126-lk/V5-EGFP-His or 15 pIZMEAVSSV126-2k/V5. -EGFP-His (this rain-recombinant plastid is constructed into sf9 cells transfected with the 1 kbp and 2 kbp promoter sequences of WSSV ORF126 according to the present invention, respectively), and in the positive control plastid ( Sf9 cells transfected with pIZ/V5-EGFP-His). The plastids of the 20 promoter sequences, which are respectively constructed to contain two other WSSV IE gene candidates, namely piZME/WSSV242/V5-EGFP-His and 卩12 八压/\¥83乂418/\^5$0 ??- Yeah, providing negative results in the 8£9 insect cells they transfected (data not shown). ORF126 was thus named WSSV W (very early gene #1). It was also surprisingly observed that both the 1 kbp and 2 kbp promoter sequences of the WSSV gene produced a higher 52 1296285 EGFP fluorescence signal than the positive control plastid pIZ/V5-EGFP-His. The plastid of the control group contained the promoter of the insect virus QpMNPV [Orgyia Pseudotsugata multicapsid nucleopolyhedrosis virus] gene, that is, the positive 2 promoter (see Figure 3). Similar results were found in Western blot analysis (Fig. 5 4). In addition, consistent results were observed in the repeated implementation of these two different promoter activity assays (data not shown). It is also observed from Figure 3 that the green-free fluorescent signal is sf9 insect transfected by the plastid ρΙΖΔΙΕ/WSSV126rev/V5-EGFP-His (which is constructed as a reverse sequence of the 1 kbp promoter sequence containing the wssv ORF126). 10 cells are produced. This fact suggests that the promoter of the WSSV /el gene is preferably operably linked to a target gene in a forward orientation. The 5' and y-end maps of the iel transcript were cloned into the pGEM-T Easy vector. Analysis of the RACE product revealed that 6 of the 7 first randomly selected strains were selected. Within 15, the 5' end is located at 52 nt (G) upstream of the putative ATG start codon, while in the other selected strain, the 5' end is at 51 nt (τ) (Fig. 5). This implies that -52 nt G represents the primary transcriptional starting point. Upstream (-26 nt) of the transcription start site (-82 nt to -78 nt relative to the ATG translation start), a putative TATA box (TATAA) was found. The NNPP of the upstream sequence of the putative t-recorded start address [the neural network for promoter prediction] has a high probability of being predicted. The basic promoter region is between -92 nt and -43 nt in front of the putative translation start codon (Figure 5). In addition, sequence analysis of the selected 3, RACE product revealed that poly(4) was added by 53 1296285 at a site located 17 nt downstream of the A AT AAA polyadenylation signal (Figure 5). Figure 6 shows that a very similar 5' UTR pattern was also found in the WSSV dnapol-based M (L, L. Chen et al. (2002), Virology 301, 5 136-147) with anti-WSSV ηΛ and rr2 groups.舀(M.-F· Tsai et al· (2000),

Wro沁277, 92-99). Since the transcription initiation sites of these four genes are consistent with the arthropod initiation elements, that is, (eight/&lt;:/1^8 (0/1[)1[(1&gt;.(:/1^^ And P. Cherbas (1993), Insect Biochem·Mol·Biol” 23, Sh90), it can be reasonably inferred that all of these viral genes have transcriptional essential elements that allow them to be transcribed by at least the arthropod host RNA polymerase II. Temporary analysis of ΨSSV iel gene transcription by RT:_?CR A RT-PCR transient analysis showed that the transcript was first detected at 2 hpi and persisted until 72 hpi (Figure 7). The intensity of the PCR 15 product band increases with time, reaching a maximum at 18 hpi and then at a performance level of one. As a comparison, the other two WSSV genes are also And vp2S [- a WSSV major envelope protein gene, see X-/f. L(9), with α/. (2005), Λ Ww/·, Am· 2005, 79 (7), with 0- The transcript of 9] was not detected until 20 4 hpi. Π · The very early (ΙΕ) gene of the virus was discussed at the beginning of a virus. Immediately after infection or reactivation, the genes of this group were identified by the experimental boundary 54 1296285 by their ability to produce transcripts even in the presence of inhibitors of protein synthesis (FX· Zhu et al· (1999) ), J. Virol, 73, 5556-5567). In the present invention, before the challenge of shrimp (grass shrimp) with wssv, a protein synthesis inhibitor, CHX, was used to pretreat the cockroach (grass shrimp) Shrimp were injected with 3 different doses of CHX (12.5, 62.5 and 250 5 mg per kg of body weight), and as expected, when the dose of CHX was increased, the number of expressed WSSV genes was reduced ( Figure 1). However, in the microarray analysis, even at the highest CHX dose (250 mg/kg body weight, the untrained shrimp treated at this dose lasted approximately 12 hours in the previous trial, Under the data, there are still 60 10 WSSV ORFs that produce a fairly high number of transcripts (group c in Figure 1). RT-PCR reduces the number of IE gene candidates to only 3 and consistently displays Out of CHX-insensitivity (Figure 2). Observed IE gene candidates One possible reason for the initial number (or false positive) may be that, in vivo, CHX does not synchronously and completely inhibit the expression of all I5 IE genes in each cell, especially for the m gene with a strong promoter. Lu. As a result, the appearance of any one of the viral gene transcription factors may trigger a cascade of downstream genes, and it is highly probable that the transcription of early and late genes will start very quickly. On the other hand, referring to group C of Fig. 1, it can be noted that not only the 丨5:1 2 〇 differential expression cutwff criterion is somewhat arbitrary, and the absolute expression levels are also As the dose of CHX increases, it is greatly reduced, which makes it more difficult to accurately quantify the fluorescence intensity data. Therefore, it can be reasonably assumed that there are still other IE genes which are not included in the 6〇 55 1296285 candidates identified in the group C of Fig. 1 . In the literature, Cao reported that several very early genes of baculovirus were expressed in a range of insect cell lines early in the process of viral infection, and were usually transcribed by the host cell transcriptional mechanism across species (2λΖλ 5 Hegedus Et al. (1998), Gene, 207, 241-249·, YG· Zhao and Ρ· 五状/如(10)&quot;999), /fzacf Me?/·Price from, S,. Therefore, the IE gene may be important in host-scale decisions. As far as the Applicant is aware, WSSV infection in insect cell lines has yet to be confirmed by the document. However, the wssv promoter identified herein was able to drive transient EGFP expression in non-host Sf9 cells (Figure 3). The WSSV 7 promoter therefore must share a sequela sequence for invertebrate transcription factor recognition. Thus, based on the fact that the WSSV-1 promoter can be activated even by a transcription factor of a non-decapod host, it can be reasonably assumed that WSSV can infect a wide range of hosts, carapace Class and, possibly, non-crustaceans (7: W. (i997), WorM /.

MicrobioL Biotech., 13, 433-442; DV Lightner (1996), A handbook of pathology and diagnostic procedures for disease of penaeid shrimp. World Aquaculture Society, Baton Rough, 20 LA··, C.-F· Lo, et al · (1996), Dis· Aquat· Org·, 27, 215-225). For the other two identified CHX-insensitive genes, ORF242 and ORF418 (their promoters are unable to drive EGFP in transfected Sf9 cells), we conclude that some other specific transcription factors may It is required for the promoters of these two IE gene candidates to function. 56 1296285 These factors are presumed to be decapods and are not present in Sf9 cells. These two candidates may be shrimp-cell specific and should therefore be analyzed in shrimp cells before being excluded from the IE gene. The TATA element is a major regulator of many baculovirus early promoters and consists of a Α/τ-rich element at 25~31 nuclear oxalic acid upstream of the transcriptional promoter (GW· Blissard). And GF Rohnnarm (1991), J, Virol, 65, 5820-5827\ GW· Blissard et cil. (1992) Virology, 190, 783-793, JA Dickson and PD·Friesen (1991), J. Virol· , 65, 4006-4016·, LA· Guarino and MW· 10 Smith (1992), J. Virol·, 66, 3733-3739·, SS Pullen and PD· Friesen (1995), J. Virol” 69, 156- 165) The TATA combination of I and the transcriptional initiator is an essential element of the RNA polymerase II promoter. The WSSV promoter region also conforms to this pattern (Fig. 5), suggesting that, like most insect rods The early viral gene, WSSV transcription 15 is mediated by host RNA polymerase. Figure 6 shows that WSSV^^/, rrj? and rr2 also conform to this pattern. Their transcription initiation sites are consistent with CAGT/CAGT related elements. (U·C/ze/i α/· (2002),

301,136-147\ L· Cherbas and Ρ· Cherbas (1993), Insect Biochem· Mol· Biol” 23,81-90·, SS· Pullen and PD Friesen 2〇&quot;995), X VzroZ·, 69, 3575-35S3), and they are located 25 to 28 nucleotides downstream of the TATA box, suggesting that these three genes should also be transcribed by host RNA polymerase II. However, these are all chx treatment sites. The suppressed genes were not expressed in the transfection assay (for eg 叩, see Figure 3, and for rd and rr2, the data are not shown). Therefore, it is speculated that: 57 1296285 r&quot; and rr2 require successful transcription of viral proteins. The presence of a transcription factor (whether in addition to the host transcription factor used by RNA polymerase II or a substitute for the primary transcription factor used by polymerase II). The results of the promoter activity assay (Figure 3) suggest that The sequence upstream of the WSSV-J coding region 5 domain is very efficient in the expression of activating genes in sf9 cells. As can be seen from Figure 8, 5, UTR (untranslated region) contains several sequences that are compatible with GATA. Consensus sequence of element (A/T) GATA (G/A). This can be important because the GATA element is identified as a binding site for the transcription factor, for example, within the baculovirus OpMNPV IE gene, from the 10 dynamometers (Ρ·Η·Kogan and GW Blissard (1994) , J. Virol 68, 813-822. Figure 8 also shows several other possible regulatory elements, including: 2 direct repeats 歹丨J (CACACACA and CTCTCTCTCT), 2 short sequence repeats (TTTCTGG and CCAGAAA) The baculovirus early gene promoter requires 15 elements (upstream regulatory elements) (CGTGC) 〇 RL Dan (2 muscles?), /· G^z· Wro/·, (Ti 7), “S27dS42), rod-shaped Viral late promoter promoter (TTAAG) (LAMW (1990), Virology, 179, 1-8·, TD Morris and LK· Miller

Gmg 7 deduction, 747453), and a palindromic sequence that produces a function like a transcriptional increase of 2 protons (Cr McMurray et al. (1991), Proc. Natl. Acad. Sci. USA, 88, 666-670; C. Rasmussen et aL (1996), Virology, 224, 235-245) 〇 In particular, since the palindromic sequence is located at the translation start address of 58 1296285, swim 1,000 nt more Farther, it may be helpful to explain the difference between the expression levels of pIZME/WSSV126-2k/V5-EGFP-His and pIZME/WSSV126-lk/V5-EGFP-His in transfected Sf9 cells ( image 3). 5 On the other hand, the 5, RACE analysis performed on RNA extracted from WSSV-infected shrimp at 24 hpi did not produce evidence to show that the TTAAG baculovirus late promoter promoter was produced. It is like a function for the initiator of WSSV id transcription. Finally, the 10 BLAST analysis of the GenBank/EMBL, SWISSPROT and HR databases predicted that the 7 coding region contained the Cys2/His2-type Zinc finger m〇Uf. This element has a role in DNA binding and suggests that 7 is capable of producing a transcription factor. Progressive research should explore the use of W as a transcription factor for those wssv genes. All the patents and literature materials used in the bow of the book in the book, and in the case of the Γ Γ Γ 以 以 以 以 以 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 . Coffee (4), Ben (10) just (including the definition of (4) defined in 20, it is intended to be described as an example, which can be understood, it can be made into a + 沾 啄 啄 涵盖 涵盖 涵盖 涵盖 涵盖 涵盖 涵盖 涵盖 涵盖 涵盖 ” ” ” ” ” ” The original (four) of the present invention is intended to contain M, / New Zealand, which is often followed by the embodiment and can be used by the art to which the present invention pertains and changes in the essential features described above. The scope of the patent application scope attached to the document inspection. 59

Claims (1)

1296285 Renovation of each year of the year P, No. 094100625, the patent application amendment page 10, the scope of application: - isolated white spot virus (WSSV) very early promoter - regulatory area 匕 basically one A nucleotide sequence selected from the group consisting of: (I) a nucleotide sequence of sequence identification number: 29; (II) a 5,_ truncated fragment of a nucleotide sequence of (1) , which has at least 92 nucleolytic residues from the 3, end of the sequence identification number: 29; (111) a nucleic acid sequence which is derived from the use of a white spot virus (WSSV) base, and The DNA is amplified as a template and a polymerase chain reaction (pCR) of a primer set having a forward primer and a reverse primer, the forward primer being substantially selected from a sequence identification number: 15 And the nucleotide sequence of the nucleotide sequence shown in the sequence identification number: 17 is constituted, and the reverse primer is basically composed of a nucleotide sequence of the sequence identification number·16; (1V) A nucleic acid analog of the nucleotide sequence of (1), which is associated with the core of (1) The acid sequence has at least about 60% sequence identity and is conserved in a region corresponding to 92 nuclear % acid residues from the 3' end of the sequence ID: 29, and can drive a a nucleic acid analog operably linked to the target gene of the nucleic acid analog; and (v) - a 5,-truncated fragment of (ii) having at least about 60 with the truncated fragment % sequence identity and in one of its corresponding to the sequence identification number: 29, 3, the end 63, the 094,100, 625 patent application, the revised page, the date of revision: 92 February 17, 19 The region of the base is singular and can drive the expression of a target gene operably linked to the nucleic acid analog. 2. The isolated WSSV very early promoter-regulatory region as claimed in claim 1 , which is basically composed of a 5, _ truncated fragment of the nucleotide sequence (1), wherein the 5, _ truncated fragment has at least the 3' end of the sequence identification 5 tiger 29 16 nucleotide residues. The isolated WSSV very early promoter _ regulatory region of the first aspect of the patent, which consists essentially of a 5, _ truncated fragment of the nucleotide sequence (1), wherein the 5, _ truncated fragment Having at least 250 nucleotide residues from the 3' end of sequence identification number: 29. 4. The isolated WSSV very early promoter-regulatory region of claim 1 of the patent scope, which is basically A 5,- truncated fragment of the nucleotide sequence (1), wherein the 5,-truncated fragment has at least 500 nucleotide residues from the 3' end of the sequence ID:29. 5. The isolated wssv very early promoter _ regulatory region of claim 1 of the patent scope, which consists essentially of a 5, _ truncated fragment of the nucleotide sequence (1) 'where the 5'- The truncated fragment has at least one nucleotide residue from the 3' end of the sequence identification number. 6. The isolated wssv very early promoter _ regulatory region of claim 1 in which the WSSV genomic DNA to be used as a template is extracted from a single accession number CCTCC-V96001 The Taiwan isolate WSSVT-1, which is deposited in the China Center for Type Culture Collection. 1296285 Revision No. 094100625 Patent Application Revision Date: 97 dry February 19 7. The isolated WSSV very early promoter regulatory region of Patent Application No. 1, which was obtained by chemical synthesis. 8. The isolated WSSv very early promoter regulatory region of Patent Application No. 1, which is obtained by recombinant DNA technology. 5 9. The isolated WSSV very early promoter regulatory region of claim 1 wherein the nucleic acid analog of (iv) or (v) comprises - corresponding to the 3' end of the sequence identification number: 29 The region of the 92 nucleotide residue • domain. 10. A recombinant expression vector comprising: a first target gene encoding a selected gene 1 〇 product, and an isolated WSSV very early promoter-regulatory region as in claim i Linked to the first target gene. 11. A recombinant expression vector according to claim 10, wherein the isolated WSSV very early promoter-regulatory region is located upstream of the gene in the forward orientation of I5. Φ 12. The recombinant expression vector of claim 10, further comprising at least one of: an additional promoter located at a position spaced apart from the wssv very early promoter-regulatory region a sequence, a second target gene encoding a second gene product, a transcription start 20 site, a transcription termination site, a ribosome binding site, a secretion letter 5 tiger coding sequence, an RNA junction site a Shine-Dalgarno sequence, a marker gene, a reporter gene, an antibiotic resistance gene, a translation termination site, an insertion site, an enhancer sequence, a polyadenylation site, and a regulatory sequence. </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> </ RTI> </ RTI> <RTIgt; Gene products in the following groups: enzymes, therapeutic polypeptides, epitopes, and antibodies. 14. A recombinant expression vector as claimed in claim 1 which is 5 pIZME/WSSV126-2k/V5-EGFP-His. 15. A recombinant expression vector according to the first aspect of the patent application, which is pIZME/WSSV126-lk/V5-EGFP-His. A recombinant host cell which is produced by transforming a host cell with a recombinant expression vector as in claim 10 of the patent application. 1〇η·Recombinant host cell according to claim 16 of the patent application, which is selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells, insect cells, crustaceans, and non- Crustacean cells as well as human cells. 18. A recombinant host cell as claimed in claim π, which is a Sf9 Kun 15 insect cell. Lu 19. A primer set for culturing a regulatory region of a very early promoter of wssv, which comprises a forward primer and a reverse primer, the forward primer being substantially selected from a sequence identification number: 15 It is composed of a nuclear (four) sequence among the nuclear (four) sequences shown by sequence identification number: 17, and the reverse primer is basically composed of a nucleotide sequence of sequence identification number: 16. 66 1296285 tggtgtaatcgctgttgtgggcggagcatatttgtgta | tataa | gagcccgtgttagctcc -61 stupid following i l · 2,3,5,9,10 tcgattcagtcacaagagcgcacacacacgcttataactagctctctctctccactcaag -1 atggcctttaattttgaagactctacaaatctctttgccaatatggacttgacggctggc 60 MetAlaPheAsnPheGluAspSerThrAsnLeuPheAlaAsnMetAspLeuThrAlaGly 20 acaacaacagaccctacccgccccaatatcatattctttgaaagtctactccccaactct 120 ThrThrThrAspProThrArgProAsnllellePhePheGluSerLeuLeuProAsnSer 40 ggtattgaggtgatgaagaggcgtctcgtacggcaaggaaagtgtgggaattttqaagca 180 ^ -12 6SP3 GlylleGluValMetLysArgArgLeuValArgGlnGlyLysCysGlyAsnPheGluAla 60 agtggaggtgctatgtcgtatttctggctcgaagataatgcagaagatatggagaatctc 240 SerGlyGlyAlaMetSerTyrPhetrpLeuGluAspAsnAlaGluAspMetGluAsnLeu 80 aacagtggttcccatgtcaagacaaactgcttggcattattccttcaagagtttatcagc 300 AsnSerGlySerHisValLysThrAsnCysLeuAlaLeuPheLeuGlnGluPhelleSer 100 _128SP1 ►_ aactggattgaagagactgatcgacatggacagtactgtacttttccccaatacatggac 360 ^ - 126SP2 AsnTrpIleGluGluThrAspArgHisGlyGlntyrCysthrPheProGlnTyrMetAsp 120 ggtggggatggttcacgtgggggatattttacttcgctagccatgaaatggatggctagg 420 GlyGlyAspGlySerArgGlyGlyTyrPheThrSerLeuAlaMetLysTrpMetAlaArg 140 gatgtgactttctttgtgtttgttgataggaataatactgtagaaaatgcggcatccata 480 AspValThrPhePheValPheValAspArgAsnAsnThrValGluAsnAlaAlaSerlle 160 tqqatqtaccaaaaactactagcaattggtgcaaaggtagtaaaggtgattgttgacaat 540 ^ - 126SP1 TrpMetTyrGlnlysLeuLeuAlalleGlyAlaLysValValLysVallleValAspAsn 180 gcatcaaacccaatgttttctgtatgtaatgcgtgtaggtgcaagtacccaggcccagtg 600 AlaSerAsnProMetPheSerValCysAsnAlaCysArgCysLysTyrProGlyProVal 200 tcatacgttattgaaggccatggagtgggtcattctgatttgacatgtgatgagatttct 660 SerTyrVallleGluGlyHisGlyValGlyHisSerAspLeuThrCysAspGluIleSer 220 ggattctttgtataataaaaccccataagaaacaataatcttttttattcaacacccatg 720 poly A signal + GlyPhePheVal * No clones are: 2,3,5, address 6224 added poly A 1296285 1296285 fduu4J6u4JJJnJ5iti55a5asunJuas (d4J54Jw4-) c &gt; ualy4 -) - p4J5u4J5fdu4Jas4Jcdcntrl4J4Jfd :: &gt; ulT) 4J4Jcdas (tiaJ4-) clJcd4-) mcnuuu4Jc &gt; 4J55fdcdfdasnJ5 (xscdu55asl4JIul4JI4-) l4-) l54JIa3lcnl4JIcnl6wfdl64JIaslcnl SCS9CSII &gt; Ur ^ £ • 1- \ si / 4 I&gt;e+ 5ρ·ραϋαϋ·ρίΰΓΰΓΰυα5·υυ·ρϋΓϋ5ΗΓϋΡΊ4-)·υ·ι-): ΓϋΓϋΓϋΓϋ-ρ:ί·ρυυ5ι5·ριΓΰιι5ΙΓΰΗιυι·ρ1υι{ϋιυ—ϋ1·ριυι·ριυι·ριιυιι·ριυι-ριιυιΙΡΊ(ϋ· ρυ (ϋ &lt; ϋ-ρΓΰ-ρ · Μυσ1ϋρυ1ΓϋιυιΓϋιυ1Γΰιυισ &gt; υ5 (ΰ5Γΰ (ϋυΓϋυ · ρ5Γϋυ · υ · ρ (ϋ5ϋ · υυυ4-) ^ vHvl β9π ^ Shu φ ^^ - ίτ hinder ^ -ilgliB spider medium inch 91ucnoiaJwcnJJlJIuuucrlnJBaslpJJIcdlJJIfduCJl ^ cnJJwwaswcdutTlpcnDIuDIcncnJJcnaJJJcnJJucnuJJccaJJJcnJJmcrlwtTSfdcdEaswuuJJwftiJJJJwasuwufdJJJJDlypiDupJJ- ucdaJcnBaslmJJIJJIcnJJrdu-lJtTJ Z|^1^罅柄寸VDIH-f«cn4J4J4Jcn4Jucntn4Jfdasfdfdcn4JfdD1pucn4J4Junippcr)cn4Jcnfdumupuuaspu4Jp4J55cncna5cn-p4Ju+Jas4J-u4J4JCJl4JasuaJcn:D4JaJ4-):DaJ4J 4J4Jufd6cnas54J4JfdmaJnJIHcdlcnlmulul4Ju654Jmcd inch VDCN-4J4J4JwnJ56fdcn4J4Jcnmas4Juuo: i4-) fd: Dunscncd-po: Du4J5u5uu64J64J4-) tJ) cducnaJa36f «aJcls4J4Jcn4J6cnfdcn4J4Jn3;: DaJuu: Dcd4Jasas4Jcd4J: i4JfduaJuasucnufdcn4Jcncn4J4Ju4JuaJu4Juu4J inch 9ΓΟ-cdwuJJcnnssascnscnuufdfdfdmJJuJJcnEcnwtTIcnpaJwcnftiuutJlcnJJfdEWuJJaJu-poascnJJuyJJuJJuJJoaiyaJupcdusJJ-UJasJJasftiuJJJJuuucnwcnuBcncnwJJcncncnuJJcnJJfdcncdcnDIucncd inch 9 inch - BCTIusnsuJJHJJDlJJJJBccuiDcnJJmuwcnJJwasascnpcncnJJfdfdJJuEcnwcnJJfdJJwJJJJpfdowucnaJucdIJIJJasufduucrlaJeuuJJuuaJJJuJJuDItJlaJfd.IJJJuupuuJJuyfdcnEUajJJuuwflJaJcn VIVO inch 9 SIcn4Jfdut7) cnua5Dl4JaJ4J4-) fd54-) nJcncnfdcd5fdl4JIaJIcnl4JI (tcnfdu; D4JaJ (Ti-efti6ascnascn4J: Du: D (TJ5cdnJas4Jun34J (d4JctiBu055JJ4JaJasu: icdcrl4J4-) rd4J5cncnaJaJfdcncn4Jufd4JaJ-IJ4-) aJcnuu4J4Ju4J4J5 VHVO inch 99-4-) uulp4JIn5l54JInJ4Jasc «54Jucn4Jcncn4J4J4J4Jfd; D4-) 5u5cn4JaJaiuu4J4-) fd56utl54Jr«4-)6::&gt;uuaJcn664J4J4Jrdcnfd55cnfd5fdu6tti5fd4JaJcr)uttJ4Jfd4J4J655pcnfd:i6f«ufd554J4JJJ4J4JaJaJ5 inch 9I&gt;- c7) cnu5u4JcnD164Jfdm4JucnuCIJfd654Ju4Jrtuctiulajl4JIcdl5cdlfdcnma15aJasuu5m4J4JaJnJcxlaJpniuucnupcnfc3rs4JaJmut7lcncn5asu: ipu4J: 15cn4Jcnu4J: D-IJufdascnaJp5Dl4Jcn4J4Jcnp55fd - inch 900-cnftiDlcnaJo ^ JJJJufdasfdasEfdfduuftiJJucduJJuJJwcnaJuuaJwfduuaJuascnumcnfdcnuiJJJowiDWuJJJJmcnfdcnuaJasrduuuunscncnJJJJaJuJJfduuJJccwuaJuaJEcnDIfduftiaJscnJJunJfdcn inch 9 6- m4Jid5uas55aJrd6 (TiasB5B ^ BB ^ Bi ¥¥ B ^ 55 ^ iFiB55fdu4J6ai4J; DUaJaJcn4J4J4Jcti4JaJasu-pmuaJnJ5fd5cn5fd6cn4-) as4JronJ; D4Jas34Jfd: DuaJfd4J4Jcdu4Jfdumcncn4J4-)? 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