TW201006928A - A universal baculovirus surface display system and application inproduction of subunit vaccine - Google Patents

Abstract

A universal baculovirus surface display system (UBSDS). The signal sequence (SS), transmembrane domain (TM), cytoplasmic domain (CTD) derived from the gp64 protein of baculovirus and histidine-tagged respectively was inserted into the pBacSC vector of UBSDS. Wherein, Four restriction sites between the histidine-tagged and gp64 transmembrane domain were established for expression of different foreign proteins. The UBSDS will therefore be a good platform for developing various subunit vaccines against viruses in the future.

Description

201006928 IX. Description of the invention: « [Technical field to which the invention belongs] % The present invention relates to the surface of a foreign gene (4). A general-purpose baculovirus surface presentation system. [Prior Art] In addition to being a bio-pesticide with great potential in organic agriculture, baculovirus is an important source of exogenous egg whites in industrial and medical fields today. The baculovirus is only good for money, and there are about _ species. The invertebrates on 2 are baculovirus hosts, most of which are insects, including Lepidoptera, Hymenoptera, Diptera and Coleoptera, and Lepidoptera Kun A is the main host of baculovirus. The scorpion virus can be subdivided into two subfamilies. The true subfamily (Eubacui 〇virjnae) and the naked genus (Nudibaculovirinae), the true baculovirus subfamily has a fine knife for two The genus granulosis virus (grimw/o illusion y; GV) has a strong polyhedrin promoter (Q nuclear polyhedrosis virus., ΝΡλ/) ·, the nucleoside polyhedrosis virus contains a strong polyhedrin promoter ( Polyhedrin promoter) and pl0 promoter (pl0 proinoter), this

Both promoters are genes of a nuclear polyhedrosis virus non-essential structure, and the damage of the two promoters does not affect the replication and proliferation of nuclear polyhedrosis virus in insect cells, so the polyhedrosis protein of the nuclear polyhedrosis virus The promoter and plO promoter are often used to initiate the expression of foreign genes, so that the foreign gene inserted into the nuclear polyhedrosis virus gene can achieve excellent performance under the transcriptional regulation of the polyhedrin promoter and the pl〇 promoter. the amount. Currently, it is the most commonly used baculovirus for the expression of foreign genes; in addition, it is commonly used in baculovirus-infected host cells for autumn. 201006928 calif or nica multiple nucleocapsid polyhedrosis viruses; AcMNPV) Spdai ovariantissue cells of Spodoptera frugiperda (Sf), commonly referred to as Sf cells; Sf cells can be divided into two cell lines, Sf-9 and Sf-21. The moth-core polyhedrosis virus is highly sensitive, and both Sf-9 and Sf-21 cell lines can be cultured in large quantities, and the recombinant protein produced can be post-translationally modified by appropriate translation. Therefore, the combination of the scorpion scorpion nucleopolyhedrovirus and the Sf cell line is the most widely used insect baculovirus gene expression system. The baculovirus surface rendering system operates through the gp64 gene. Gp64 is the major glycoprotein on the surface of the baculovirus envelope. After the foreign protein gene is genetically recombined with the gp64 glycoprotein gene, the foreign protein can be presented on the surface of the baculovirus envelope. The gp64 protein is mainly composed of an n-terminal signal sequence and a main region, and the main region is further divided into a transmembrane domain (TM) and a cytoplasmic domain (CTD). The gp64 protein is transported by a signal sequence after performance. Go to the cell membrane. The main region is presented on the surface of infected cells in a trimer structure. Until the newly synthesized nucleocapsid is to bud, it interacts with the cytoplasmic region of gp64, causing gp64 to bind to the nucleocapsid and appear on the viral envelope. The conventional baculovirus expression system utilizes the co-transfection reaction of baculovirus to ride the baculovirus of the baculovirus. 6 - 201006928 Construction of the 'co-transfection reaction system utilizes the genetically modified transfer vector and its own defective gene Perform homologous interchange to generate a recombinant baculovirus with an ancient f: defective baculovirus gene in this process, which will also produce a homologous gene; however, it must be further replaced by a virus: Method (4) 2 and virion purification and immunoassay

The construction takes less than a month, which leads to the shortcomings of the traditional restructuring of the baculovirus construction. In order to rapidly produce re-transformed viruses, a known surface-presenting system for dry-type viruses has emerged, such as the stimuli of the Chinese patent application for the induction of avian flu-like symptoms of avian influenza. The Bac-to-Bac® system (manufactured by Invitrogen) is used as a recombinant baculovirus vector, and the Bae_tQ_Bae8 system contains a competent cell carrying a coronavirus shuttle vector (bacmid) and a helper plasmid. Dm〇Bac, the DH1〇Bac is a large intestine strain's helper vector with a gene that can express transp〇sase, and transforms the expression vector with exogenous gene, Tn7R and Tn7L gene jumping points. The method of (transformati〇n) is sent to the competent cell DHlOBac'. The baculovirus shuttle vector also has a Tn7R and Tn7L gene jumping point, and the baculovirus shuttle vector and the gene of the expression vector are translocated by the transposase translated on the helper vector. The gene exchange between the jumping points is such that the foreign gene to be expressed jumps into the baculovirus shuttle vector, and the process of this gene jump exchange is called transposition. There is a lacZ gene between the Tn7R and Tn7L gene hopping points in the baculovirus shuttle vector. When the gene jumps 201006928, the lacZ gene is interrupted, which makes the transformation successful and completes the DX gene hopping DHlOBac colony containing χ-gal and The culture of IPTG* was white on the basis; the DHlOBac colony which was white was selected to directly purify the shuttle vector, and the purification of the plaque was not required, and the time for preparing the recombinant baculovirus was greatly reduced. The known baculovirus surface presentation system utilizes the constructed recombinant vectors Bac-HA and Bac-HA64 to express the avian influenza virus erythrocyte agglutinin ' to prepare an H5N1 avian influenza vaccine; wherein the Bac_HA vector is in a pBacCE plastid Insert gp64 signal sequence (gp64 SS), 6 histidine residues (His6 tag), avian influenza virus erythrocyte lectin gene transmembrane region (HA transmembrane region;

Transmembrane domain; HA ΤΜ) and avian influenza virus erythrocyte cytoplasmic domain (HA cytoplasmic domain; HA CTD) or; f cytoplasmic cytoplasmic domain (gp^ Cyt〇plasmic domain; gp64 CTD). ◎Because the transmembrane region of the known baculovirus surface presentation system uses the erythrocyte lectin gene transmembrane region of avian influenza virus, and the baculovirus transmembrane region is not used, the avian influenza virus erythrocyte agglutination protein expression amount Not-good' causes the known baculovirus surface to appear, and has the disadvantage of exogenous protein #efficiency*; in addition, the H-reducing erythropoietin gene outer region does not have any restriction enzyme cleavage position, and cannot be performed. Elimination or ligation of the avian influenza virus red blood cell lectin gene, while limiting the known baculovirus surface presentation system can only be used to express avian influenza virus erythrocyte agglutinin and can only be used once, making the known rod The viral surface 201006928 and cannot be repeated allows the rendering system to have the disadvantage of showing only avian influenza virus proteins for the expression of different foreign proteins. SUMMARY OF THE INVENTION The present invention is to improve the known baculovirus surface presentation system performance = poor rate, can only express single-type foreign proteins, and can not be reused to express foreign protein defects, and work hard to study Developed a universal baculovirus surface rendering system (universal baeulQvims surfaee)

Such as play system; U is DS), which can increase the amount of foreign protein expression on the surface of the virus' and the general-purpose baculovirus surface presentation system can be reused to express a variety of foreign proteins for multiple applications. Preparation of unit vaccines. The main object of the present invention is to provide a general-purpose baculovirus surface presentation system which selects four high-efficiency restriction enzyme sites of melon, I, I, I and five (7) and I into a recombinant load. The six histidine residues and the gP64 transmembrane region are ligated or eliminated by different foreign genes, so that the present invention has the effect of being reusable for expressing different foreign proteins. The invention provides a general-purpose baculovirus surface presentation system, which uses the gp64 transmembrane region of the baculovirus itself to express the foreign protein, so that the invention has the specificity of exogenous protein and the foreign protein in the baculovirus. The effect of the amount of surface performance. Another object of the present invention is to utilize the Bac-to-Bac system for the simple protein purification process, so that the present invention has the effect of greatly shortening the time required for protein purification. 201006928

The present invention provides a general-purpose baculovirus surface presentation system, which comprises a baculovirus gp64 protein message sequence, a transmembrane region gene, a cytoplasmic region gene and six histidine sequences, and Four high-efficiency restriction sites of I, I, cadaver and five coiM were selected between the six histidine sequences and the gp64 transmembrane region to make the general-purpose baculovirus surface presentation system Different exogenous proteins can be expressed in large quantities on the baculovirus envelope for the preparation of different subunit vaccines. The above and other objects, features, and advantages of the present invention will become more apparent <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; First, the general-purpose baculovirus surface presentation platform pBacSC: the construction

The invention discloses a general-purpose baculovirus surface presentation platform pBacSC

The first step: the pFastBac Dual vector (manufactured by Invitrogexi Co., Ltd.) carrying the Tn7R skip gene, the Tn7L skip gene, the polyhedrin promoter and the pl〇 promoter. A cytomegalovirus immediate early promoter-enhanced green fluorescent protein (CMV-EGFP) is inserted downstream of the polyhedrin promoter in the pFastBac Dual vector to construct a pBacCE vector; and the pBacCE vector plO The gp64 histidine (His-gp64) mature region gene was inserted downstream of the promoter to construct a pBacCEH vector.

The second step of constructing the universal baculovirus surface presenting platform pBacSC of the present invention: taking the pBacCEH vector as a template, using primer A 201006928 (sequence number 1: 5'-ATATCCCGGGATGCTACTAGTAAATC AGTCACAC-3), and with 6 A histidine (GTG) primer B (SEQ ID NO: 2: 5'-TAGC CTCGAGGTG GTG GJG GTG GTG GTGCGC -3') undergoes a polymerase chain reaction to amplify the gp64 signal sequence (gp64 signal sequence; gp64 SS) And histidine tag (His tag), the expected size of the amplified product should be 147 bp. In addition, pBacCEH is used as a template, with primer F (sequence number 3: 5'-ATATGGTACCTTAATATTGTCTATTACGGTTTC -3,) and with I (CTCGAG) ^ Xba 1 ( TCTAGA) - Pst I ( CTGCAG) and EcoR 1 (GAATTC) The primer for the restriction enzyme cleavage sequence CTD (SEQ ID NO: 4: 5'- GGTCTCGAG TCTAGA CTGCAG GAMTCTTCATGTTTGGTCATGTAGTTA -3^) polymerase chain reaction to amplify the gp64 transmembrane domain (gp64 TM) and gp64 cytoplasmic region (gp64) Cytoplasmic domain; gp64 CTD), the size of the amplified product should be 123 bp. 'The polymerase chain reaction is used to select the four high-efficiency restriction sites of me and z (7) and I into the recombinant vector. The recombinant vector can be repeatedly used to express the efficacy of various foreign proteins; and the gp64 transmembrane region of the baculovirus itself is used to express the foreign protein, so that the recombinant vector can increase the specificity and expression of the foreign protein (Yang et et Al, 2〇〇7) 'The products of the secondary polymerase chain reaction were separately subjected to agarose gel electrophoresis analysis to confirm that the gene fragment cloned into the recombinant vector was correct. As shown in Figure 1, the gp64 message sequence _ histidine marker (size 147 bp) gene fragment and the -11 cytoplasmic region (123 bp in size) were blocked by restriction enzyme cleavage and ligation. Gene fragment into a gift-bearing coffee-tea with a cytomegalovirus green glory white gene, ^• into a generic baculovirus surface rendering platform, and the platform two named pBacSC' and use the platform pBacSC The system for expressing foreign proteins is called the imiversal baculovirus surface display system (UBSDS). The general-purpose baculovirus surface presentation platform pBacSC uses the design of the 穿64 transmembrane region of the baculovirus itself to improve the stability and performance of the foreign protein on the viral envelope. In addition, on the surface of the general-purpose baculovirus, the platform pBacSC was selected between the six histidine sequences and the gp64 translocation region, and the four high-efficiency restriction enzymes were selected for JOw II, household to I, and five (3) and I. The design of the universal baculovirus surface presentation platform pBacSC allows different foreign genes to be constructed into pBacS (: platform, and the exogenous gene can also be removed by restriction enzyme cleavage, and then another The construction of the foreign gene; and further, the pBacSC platform can repeat the construction of the foreign gene which is not identical. After the construction of the pBacSC platform is completed, the correctness of the pBacSC platform is further identified. 2. Universal baculovirus Identification of the surface presentation platform pBacSC • Identification of the universal baculovirus surface presentation platform pBacSC takes the generic baculovirus presentation platform pBacSC as a template, with A/B introduction group and F/CTD introduction group A polymerase chain reaction was performed on the pBacSC platform to determine whether the gp64 message sequence-histidine tag gene fragment and the gp64 transmembrane region-gp64 cytoplasmic region gene fragment have been successfully constructed into a pBacCE vector; As shown in Figure 2, where -12 — 201006928

Lane Μ is a standard band; Lane 1 is a product amplified by the A/B primer group for the lion platform, and its size is 147 bp; Lane 2 is the F/CTD primer group amplified for the pBacSC platform. The product, the large J is 123 bp, and the result can be confirmed that the 卽64 message sequence histidine tag gene fragment and the gP64 transmembrane region _gp64 cytoplasmic region gene fragment have been successfully constructed on the pBacCE vector, and the determination has been successful. Construct a pan-like baculovirus surface presentation platform pBacsc. After determining that the pBacSC platform was successfully constructed, the pBacSC platform was further utilized to express different foreign proteins, and the inventors used the pBacSC platform to express the poultry virus virus and the swine fever virus protein. Third, the use of pBacSC platform to express different foreign proteins using pBacSC: the cjC and σΒ protein of poultry virus. The orC and σΒ proteins of poultry virus have the function of inducing group specificity and neutralizing antibodies. The first step of the present invention for expressing poultry virus AC and σΒ protein using pBacSC is: designing a σ CF primer according to the sequence of the poultry virus gene (SEQ ID NO: 5: 5'-ATAGGTCGACTTAATGGCGGGTCTCAATCCATCGCAGC • 3,) / aC-R primer ( SEQ ID NO:6: 5,-GCGCGAATTCTTCGG TGTCGATGCCGGTACGCACGG-3') Introduction group and σ BF (SEQ ID NO: 7: 5'-GCGCTCGAGTTAATG GAGGTACGTGTGC CAAAC -3,) ΑτΒ-R (SEQ ID NO: 8: 5, - CCGGGAATTCTTC CCAACCACACTCCACTTCAGTG -3,) The primer group, and the aC-F/aC-R primer group and the σΒ-FMB-R primer group were used to amplify the poultry virus of the poultry Leo-13-201006928:-based polymerase chain reaction. σ (: and j 翻 翻 翻 pBaeSC performance poultry rio virus σ from (four) two steps: miscellaneous system of poultry virus virus ° ^ σ Β gene to limit endonuclease enzymes = at the same time pBacSC planted 10,000 ^ The heart and the cleavage enzyme are the main 'knife'. As shown in Fig. 3, the pBacSC vector is ligated with the poultry rio disease and the (10) gene to construct a recombinant baculovirus surface and a pool. Construct weight

The second step of the present invention using pBacSc to express the poultry virus virus and the white heart of the poultry is the B^t (the KBae system constructs the ^s σ (the vector and the pBaeS (the specific gene fragment on the ^B vector) The transplanted carrier is exchanged on the shuttle vector of the competent cells by transposition, and the transposed cells are cultured for two to three days, and the transfected white colonies are selected, and the polymerase chain reaction is used to amplify the aC and σΒ genes. The fragment is ritually recognized and a large number of culture shafts are mixed, and then the recombinant shuttle vector DNA of the strain is added; the cell transfection reagent (eellfeetin reagent) is mixed with the recombinant sulphonate at room temperature for 3G minutes, and the mixture is taken. The solution was added to a cell culture plate (weU) containing the Sf_9 cell strain, and after 5 hours of incubation at 27 C, the mixture was withdrawn, and the mixture was added to the mixture containing ίο% fetal calf and three-in-one antibiotic &amp; culture solution, and then placed. The 27 C incubator was cultured for 3 days, centrifuged, and the supernatant was taken. The supernatant contained BacSC-cjC and BacSC-σΒ recombinant baculovirus particles. The BacSC-aC recombinant baculovirus can express the region with gp64 signal. , aC structural protein, gp64 wear Recombinant 201006928 protein in the membrane region and in the 64 cytoplasmic region; and the BacSC-σΒ recombinant baculovirus can express a recombinant protein with a gp64 signal region, a σΒ structural protein, a gp64 transmembrane region, and a gp64 cytoplasmic region and the BacSC -aC and BacSC-σΒ recombinant baculovirus all carry a histidine marker and then infect the Sf-9 cell line with the BacSC-aC and BacSC-σΒ recombinant baculovirus, and the cells with pH 5 5 after three days of infection The culture medium (TNM-FH) stimulated the Sf-9 cell line infected with BacSC-aC and BacSC-σΒ recombinant baculovirus, observed whether cell fusion occurred under a microscope, and observed whether green was observed under a fluorescence microscope. Fluorescence. ' As shown in Figures 4A1, 4A2 and 4A3, Figure 4A1 shows Sf_9 cell line not infected with BacSC-aC and BacSC-σΒ recombinant baculovirus as a control group, Figure 4A2 And Figure 4A3 shows that the Sf_9 cell line infected with BacSC-σΒ and BacSC-aC recombinant baculovirus respectively can observe the phenomenon of cell fusion in the 4A2 and 4A3 images; and 4B1, 4B2 and 4B3 The control group was observed by fluorescence microscope, and was infected with BacSC-σΒ. As a result of the Sf-9 cell line and the Sf-9 cell line infected with BacSC-aC, the control group did not produce any fluorescence phenomenon, and the Sf-9 cell line infected with BacSC-σΒ was infected with BacSC-aC. The Sf-9 cell line showed green fluorescence; the cell fusion and green fluorescence were used to confirm that the BacSC-σΒ and BacSC-oC recombinant baculovirus had successfully infected the Sf-9 cell line. After determining that the BacSC-σΒ and BacSC-oC recombinant baculovirus have successfully infected the Sf-9 cell line, the fourth step of the present invention using the pBacSC to express the poultry virus σ(: and σΒ protein: further confirming the recombination rod —15 — 201006928 The prion BacSC-σC and BacSC-σΒ can express the poultry virus and σΒ protein. The recombinant baculovirus BacS (:_ σ 〇BacSC-σΒ and without aC and σΒ protein will be constructed. The control level BacSC of the gene infects Sf-9 insect cells, and after three days of infection, it is further analyzed by Western blot analysis. For the results, please refer to Figure 5A and Figure 'The Lane 1 is the standard strip (Marker). Lane 2 is Sf_9 insect cells infected by BacSC-σΒ and BacSC-aC respectively. 3 Ο Ο are Sf-9 insect cells infected with BacSC, and Lane 4 is not infected with any Sf-9 insect cells. No sf 9 insect cells and Sf-9 insect cells showing BacSC sensation were found to be recognized by 浐crC and anti-σΒ primary antibody (jC D11 monoclonal antibody or anti-chicken = antibody); Sf-9 insect cells infected with BacSC-σΒ and BacSC-aC at 38 and 3 The size of 6 KD appears to be recognized by the primary antibody; the results of the Western blot analysis can confirm that the Cao 纟 virus BacSC-crC, BacSC-σΒ can express the poultry rio virus and the ο* B protein. The pBacSC is used to express the poultry virus σ (the fifth step of the protein: further confirming the general-purpose baculovirus surface presentation system (UBSDS) to present the recombinant proteins cjC and crB on the viral envelope. Take the M0I 10 virus dose BacSC- σ C, Bacsc_ cr β and

Bac-SC, infected with Sf-9 insect cells, cultured against infected Sf-9 insect cells under appropriate conditions for two days, and then reacted with anti-sigma (: and anti-cjb primary antibody) and then immunofluorescence (flU〇rescein isQthiQeyanate;FITC) labeling and method observation of a common focal microscope to confirm the performance 201006928 • #recombination 0^ &amp;σΒ protein expressed in the position of insect cell JL. For example, the 6th '帛6 ®仙焦焦Visceral plagiarism • Observations 'The results showed that Sf9 insect cells infected with the recombinant virus BacSC (control group) showed no protein recognized by the primary antibody; whereas after infection with the recombinant viruses BacSC_JC and BacSC-σΒ 'The appearance of heavy and protein on the surface of the cell membrane was observed. This result proves that the expressed recombinant proteins iJC and σΒ can be expressed on the surface of sf_9 insect cells. Further, the recombinant baculovirus BaeSC_cjC, BaeSC-σΒ and Bac-SC purifies the virus by ultra-high speed centrifugation with a concentration of sucrose, and observes whether the recombinant protein is expressed on the viral envelope by immunogold electron microscopy. The recombinant protein can be effectively presented on the baculovirus envelope; as shown in Figure 7, the results of antibody identification by immunogold electron microscopy showed that BacSC (control group) did not have any gold particles on the virus surface. And ΒΝ8(:_σ(: and BacSC 〇_B does have gold particles on the surface of the baculovirus. Based on the above results, it can be confirmed that the ubiquitous baculovirus surface rendering system (UBSDS) can indeed reorganize The protein aC and σΒ are present on the viral envelope. The poultry virus (7C and σΒ protein are used in the experimental animal immunization test of the subunit vaccine. The poultry virus JC of the invention and the jB protein are applied to the subunit vaccine. An experimental animal immunoassay is carried out by using an experimental animal having no specific pathogen of six to eight weeks of age to perform an immunoassay of the poultry virus σ (: &amp; σ Β protein, wherein the experimental animal is a mouse, the strain of the experimental mouse For BALB/c. Take BacSC-aC and BacSC-σΒ as experimental groups; and take -17 to 201006928

BacSC was a negative control group; the experimental group and the negative control tissue recombinant virus were respectively infected with Sf-9 cells, and the sf-9 cells infected with the recombinant virus were carried with the recombinant virus protein on the cell membrane; Whether it can effectively induce an immune response' Therefore, the cells infected with the recombinant baculovirus are subjected to an immunoassay, and the serum antibody titer is tested by an enzyme-binding immunosorbent assay (ELISA). The results are shown in Figure 8, and the results of the ELISA reader reading ODmo showed infection with BacSC.

Sf-9 cells and Sf-9 cells were injected with experimental mice, and the ELISA values were less than 〇·4; and Sf-9 cells infected with BacSC- σ C and Sf infected with BacSC-σΒ The OD65 血清 value of the serum produced by the -9 cell injection test mice was as high as 1.〇 or more. From this result, it was revealed that the Sf-9 cells carrying the recombinant viral protein and σΒ could indeed cause an immune response in mice. In addition, the ARV vaccine (commercial poultry Rio S1133 live vaccine) was used as the control group, and the recombinant viruses BacSCjC, BacSC-jB and BacSC-(jC+aB (ie, simultaneous injection of recombinant viruses Baesc-jc and BacSC-σΒ) were used. For the experimental group, and with BacSC and phosphoric acid

The experimental group of the Phosphate ^ test group and the control virus protein protein were injected intraperitoneally. Two weeks after the experimental mice were tested for abdomen, another additional injection was performed, and six weeks after the additional injection, the experimental mice were collected for enzyme-binding immunosorbent assay and virus towels and tests. Results As shown in Fig. 9, the ELISA coffee was used to interpret 〇D. The results showed that the elisa value of the experimental mice Jinqing, which was injected into the BacSC and PBS negative control groups, was less than 0.4. The experimental mice injected with BacSC-aC cut B had blood/month ELISA values of 2.2. The results of the virus neutralization test are as shown in Section 1.®: ???, where the highest in the cut group B is 'the antibody strength is up to 10%. The results show that the recombinant virus BacSC_σC and

BacSC-σΒ has a significant effect on the immune response, so that -sc· σ C+ σ B (ie simultaneous injection of recombinant virus such as _ ^ c and _ can be used with adjuvants or excipients for application as a poultry virus virus subunit The marker bacillus itself can be used as an adjuvant for the single sapling. J uses pBacSC to express the porcine prion protein. The first step of the invention is that the pBaeSC expresses a porcine such as a venom protein. E〇_f (sequence number 11:5,_

TTGAACTCGAGGCCGAAAATATAACTCAA-3- ) /EO-R ( 12 : 5f-GCATAGAATTC AGCGCCAAACCAGGT TTT-3 ) and E2-F (SEQ ID NO: 5'-CAGTACTCGAG GCACAAGGCCGGCTAGCC-3') /E2_r (SEQ ID NO: 14: y_ ◎ CCAAGAATTCAAATTCTGCGAAGTAATC-3, In the primer group, the polymerase chain reaction was carried out using the classical swine fever virus gene as a template to amplify the E0 gene (675 bp in size) and the E2 gene (1074 bp in size). The amplified swine fever virus gene and the E2 gene were cleaved by restriction endonuclease five (3) and I and H. I, and the pBacSC vector was cleaved with five coi? I and /V I endonucleases. The pBacSC vector was ligated with the CSFV E0 gene and the E2 gene to construct a recombinant baculovirus vector pBacSC-Εθ and pBacSC-E2. The second step of the present invention for expressing a swine fever virus protein using pBacSC is 201006928. The specific gene fragments on the constructed pBacSC_E〇 and PfiacSC-E2 vectors are interchangeably inserted by transposition using the Bac-to-Bac® system. On the shuttle vector of competent cells, the transfected competent cells are cultured for two to three days, and the transfected white colonies are selected, and the E0 and E2 gene fragments are amplified by polymerase chain reaction to confirm and cultured in large quantities. The successfully transfected strain 're-extracting the recombinant shuttle vector of the strain', the cellfectin reagent and the recombinant shuttle vector DNA were mixed for 3 minutes into the temperature_', and the mixture was added to the sf_9-containing cell line. In a cell culture plate, the mixture was cultured at 27 ° C for 5 hours, and the mixture was extracted, and a TNM-FH culture solution containing 10% fetal calf serum and a 3-in-1 antibiotic was added to the mixture, and then placed at 27 °. The culture incubator was incubated for 3 days, and the supernatant was centrifuged, and the supernatant contained the BacSC-EO and BacSC-E2 recombinant baculovirus of the second generation. The third step of the present invention for expressing hog cholera virus protein using pBacSC is to reinfect fresh Sf-9 cells with BacSC-EO and BacSC-E2 recombinant baculovirus as seed venom, and collect infected sf_9 cells three days later. SDS-PAGE electrophoresis and Western blot analysis (western bl〇t) were performed. The primary antibody is porcine sputum positive serum 'secondary antibody is horseradish peroxidase (HRp) labeled goat anti-mouse IgG, the results please refer to the ha chart and the iiB chart, the Lane 1 are standard bands (Marker), Lane 2 Sf-9 insect cells infected with BacSC-EO and BacSC-E2, respectively, Lane 3 are Sf-9 insect cells infected with BacSC, and Lane 4 is Sf-9 insect cells not infected, and the results show None of the Sf-9 insect cells and Sf-9 insect cells infected with BacSC were recognized by the primary antibody against 201006928 crC and anti-σΒ; and Bac:sc-E0 and

BacSC-E2-infected Sf-9 insect cells showed a reaction recognized by primary antibody at the large and small positions of 43 and 50 KD, respectively. The results of the Western blot analysis confirmed the recombinant baculovirus BacSC-EO, BacSC. -E2 can express poultry virus E0 and E2 proteins. In order to prove that the recombinant protein Swine fever 毋 protein line is expressed on the cell membrane of insect cells, the fourth step of using the pBacSC to express the Swine Fever virus protein of the present invention is carried out: the porcine sputum positive blood is used as the primary antibody to immunize Fluorescent (fluorescein isothiocyanate; FITC)-labeled goat anti-pigs were used as secondary antibodies; the recombinant proteins carrying CSFV E0 and E2 were further calibrated for conjugated focusing microscopy. The results are shown in Fig. 12, and it can be seen that the E〇&amp;E2 protein is expressed on the cell membrane of the insect cell. The fifth step of using the pBacSC to express the classical swine fever virus protein of the present invention is to combine the purified refolded secret-anti-anti-legged monoclonal antibody, and then with the knee gold-labeled goat anti-mouse IgG Antibody binding Ο Last I over 2% of the phosphorheic acid droplets were counterstained by electron microscopy. The results were as shown in the figure to observe the binding of gold particles to the envelope f of the recombinant baculovirus, while the negative control group had no gold particles. On the envelope membrane bound to the virus, the recombinant E0 and E2 proteins were expressed on the recombinant baculovirus envelope membrane. The experimental animal immunization test for pig subcutaneous E2 protein is applied to the sub-unit vaccine. The invention adopts the test animals of six to eight weeks old without specific pathogens to enter the virus E2 egg, which is small, and the experiment is small. The rat strain is BALB/C. The first step of the experimental animal immunization test for the pig prion E2-21-201006928 protein of the invention is applied to the sub-unit vaccine: the BacSC-E2 is used as the experimental group, and the CSFV (commercial pig-toothed live vaccine) is used as the control group. BacSC and PBS were used as control groups. Four groups of the experimental mice were intraperitoneally injected with the experimental group, the control group and the control group. Two weeks after the intraperitoneal injection of the mice, another additional injection was performed, and the blood of the experimental mice was collected for enzyme-binding immunosorbent assay and virus neutralization test six weeks after the additional injection. The result is as shown in Figure 14.

It is indicated that OR5 is interpreted by an ELISA reader. The results showed that the ELISA values of the serum of the experimental mice injected with BacSC and PBS control group were less than 〇5, and the EUSA value of the serum of the experimental mice injected with CSFV control group was less than ι 〇; and the serum of experimental mice injected with BacSC-E2 experimental group. The ELISa value is as high as 1.5 or more. between

The results of the virus neutralization test are shown in the following table:

The virus neutralizing antibody titer can be seen from the above table, the BacSC-E2 sum test; the virus neutralizes the antibody power. From the above results, it is known that the effect of the immune reaction with the recombinant virus such as Guanzheng 2 is remarkable, so that BacSC_E2 can be used together with an adjuvant or an excipient to apply the vaccine as a swine fever virus subunit, and the baculovirus itself can be used as the vaccine. Adjuvant for unit vaccine. As described above, the general-purpose baculovirus surface presentation system of the present invention, which is #(四)H ί~ ! and _ high efficiency limit - 22 - 201006928, is ligated into a recombinant vector, so that the present invention can be reused. • The ability to express a variety of foreign proteins. Although the present invention has been disclosed in the above-described preferred embodiments, it is not intended to limit the invention, and various modifications and changes can be made without departing from the spirit and scope of the invention. The scope of the invention is defined by the scope of the appended claims. Ο 〇 23 — 201006928 [Simple description of the diagram] Figure 1: Construction of the performance platform pBacSC. Fig. 2: Electrophoresis pattern of gp64SS-His6 and gp64TM-CTD gene fragments expressing the platform pBacSc was amplified by polymerase chain reaction. Figure 3: Construction of the recombinant vectors pBacSC-σΒ and pBacSC-ac. Figures 4A1 to 4B3: Fluorescence microscopic observation of BacSC-σΒ and BacSC-aC recombinant baculovirus-infected Sf-9 cell lines. Figure 5A and Figure 5B: Detection of recombinant baculovirus BacSC_aC, BacSC-crB showed poultry virus (jc and crB protein breakdown map. Figure 6: Recombinant proteins aC and crB are present in the viral envelope. Microscopic observation Fig. 7: Recombinant protein aC and crB are presented in the immunogold microscopic observation of the viral envelope. Figure 8: Recombinant and σΒ viral protein injection test animal enzyme combined immunosorbent assay results. Fig. 9: Results of enzyme-binding immunosorbent assays of experimental animals injected with recombinant virus-infected insect cells. Figure 10: Infection of insect cells infected with recombinant virus-infected animal-toxicity test results. - Figure 11 and 11Β图: Detection of recombinant baculovirus BacSC_E〇 and BacSC-E2 showed the breakdown of prion protein. Figure 12: Recombinant proteins E0 and E2 presented in the glory microscopic observation of the viral envelope. Figure 13: Recombinant protein E0 And μ presented in the immunoglobulin of the viral envelope 24-201006928 Electron microscopic observation. Figure 14: Enzyme-binding immunosorbent assay results of recombinant E2 viral protein injection test animals . The main element REFERENCE NUMERALS (None)

——25 — 201006928

Sequence Listing. &lt;11〇&gt;National Pingtung University of Science and Technology. &lt;120>General-purpose baculovirus surface rendering system and its application to subunit vaccines&lt;130> 95144532 &lt;160〉 7 &lt;170&gt ; Patentln version 3.5

&lt;210> 1 &lt;211> 69 〇 &lt;212> DNA <213> baculovirus &lt;400&gt; 1 ttcatgtttg gtcatgtagt taactttgta attatattaa ttgtgatttt atttttgtac tgtatgatt 69

&lt;210> 2 &lt;211> 981 &lt;212&gt; DNA &lt;213> Avian retrovirus &lt;220&gt;&lt;221&gt; CDS &lt;222>(1)...(981) &lt;;400〉 1 atg gcg ggt etc aat cca teg cag ega aga gag gtc gtc age ttg ata

Met Ala Gly Leu Asn Pro Ser Gin Arg Arg Glu Val Val Ser Leu lie 1 5 10 15 ctg tea ttg act teg aac gtg aat ata agt cat ggc gat ttg aeg ccg

Leu Ser Leu Thr Ser Asn Val Asn lie Ser His Gly Asp Leu Thr Pro 20 25 30 ate tat gaa egg ctg acc aat eta gaa gcg tet aeg gag tta tta cat lie Tyr Glu Arg Leu Thr Asn Leu Glu Ale Ser Thr Glu Leu Leu His 35 40 45 48 96 —26 — 144 192201006928 cgc tcc att tcc gat ata tcc act act gtc tea aat att tet gca aat • Arg Ser lie Ser Asp lie Ser Thr Thr Val Ser Asn lie Ser Ala Asn 50 55 60 « tta Caa gac atg acc cat aca ttg gat gat gta act get aat tta gac Leu Gin Asp Met Thr His Thr Leu Asp Asp Val Thr Ala Asn Leu Asp 65 70 75 80 ggt ttg agg acc act gtt act gca ett cag gat tcc gtc tcc att Ctg Gly Leu Arg Thr Thr Val Thr Ala Leu Gin Asp Ser Val Ser lie Leu 85 90 95 tet aca aat gtg act gac tta aeg aac aga tcc tet geg cac geg geg ◎ Ser Thr Asn Val Thr Asp Leu Thr Asn Arg Ser Ser Ala His Ala Ala 100 105 110 ata eta tet tea ett cag act aeg gtt gac gga aac tcc act gee ate lie Leu Ser Ser Leu Gin Thr Thr Val Asp Gly Asn Ser Thr Ala lie 115 120 125 tcc aat ttg aag agt gat ate teg teg Aac ggt tta ge t att aca gat Ser Asn Leu Lys Ser Asp lie Ser Ser Asn Gly Leu Ala lie Thr Asp 130 135 140 ctg cag gat cgt gtt aaa tea ttg gag tet acc geg agt cat ggt eta Leu Gin Asp Arg Val Lys Ser Leu Glu Ser Thr Ala Ser His Gly Leu 145 150 155 160 tet ttt teg cct ccg ett agt gtc get gac ggc gtg gtt tea tta gac Ser Phe Ser Pro Pro Leu Ser Val Ala Asp Gly Val Val Ser Leu Asp 165 170 175 atg gac ccc tac ttc tgt Tet caa ega gtt tet tta aca tea tac teg Met Asp Pro Tyr Phe Cys Ser Gin Arg Val Ser Leu Thr Ser Tyr Ser 180 185 190 geg gag get caa eta atg caa ttt egg tgg atg gca egg ggt act aac Ala Glu Ala Gin Leu Met Gin Phe Arg Trp Met Ala Arg Gly Thr Asn 195 200 205 gga tea tet gat acc att gac atg acc gtt aac get cac tgt cat gga Gly Ser Ser Asp Thr lie Asp Met Thr Val Asn Ala flis Cys His Gly 240 288 336 384 432 480

528 576 624 —27 — 672 720201006928 210 215 220 • aga cgc act gat tat atg atg teg tee aeg gga aat etc aeg gtc act Arg Arg Thr Asp Tyr Met Met Ser Ser Thr Gly Asn Leu Thr Val Thr • 225 230 235 240 agt Aac gtc gtg tta tta acc ttc gat tta agt gac ata aeg cat ate Ser Asn Val Val Leu Leu Thr Phe Asp Leu Ser Asp lie Thr His lie 245 250 255 cca tea gac eta gca cgt ett gtt ccc agt geg gga ttc caa get geg Pro Ser Asp Leu Ala Arg Leu Val Pro Ser Ala Gly Phe Gin Ala Ala 260 265 270 〇teg ttc cct gtg gac gta tea ttc acc cgc gat tet geg act cat geg Ser Phe Pro Val Asp Val Ser Phe Thr Arg Asp Ser Ala Thr His Ala 275 280 285 tac caa geg tat ggg gtg tac teg age tea cgt gtc ttc aca att act Tyr Gin Ala Tyr Gly Val Tyr Ser Ser Ser Arg Val Phe Thr lie Thr 290 295 300 ttc cca acc gga ggt gat ggt aca geg aac Att cgt tee ttg acc gtg Phe Pro Thr Gly Gly Asp Gly Thr Ala Asn lie Arg Ser Leu Thr Val 305 310 315 320 cgt acc ggc ate gac acc taa ©Arg Thr Gly lie Asp Thr 325 768 816 864 912 960 981 &lt;210 〉 3 &lt;211> 326 &lt;212> PRT &lt;213>Avian retrovirus &lt;400〉 3

Met Ala Gly Leu Asn Pro Ser Gin Arg Arg Glu Val Val Ser Leu lie 15 10 15

Leu Ser Leu Thr Ser Asn Val Asn lie Ser His Gly Asp Leu Thr Pro —28 — 201006928 20 25 30 lie Tyr Glu Arg Leu Thr Asn Leu Glu Ala Ser Thr Glu Leu Leu His 35 40 . 45

Arg Ser lie Ser Asp lie Ser Thr Thr Val Ser Asn lie Ser Ala Asn 50 55 60

Leu Gin Asp Met Thr His Thr Leu Asp Asp Val Thr Ala Asn Leu Asp 65 70 75 80

Gly Leu Arg Thr Thr Val TTir Ala Leu Gin Asp Ser Val Ser lie Leu 85 90 95

Ser Thr Asn Val Thr Asp Leu Thr Asn Arg Ser Ser Ala His Ala Ala 100 105 110 lie Leu Ser Ser Leu Gin Thr Thr Val Asp Gly Asn Ser Thr Ala lie 115 120 125

Ser Asn Leu Lys Ser Asp lie Ser Ser Asn Gly Leu Ala lie Thr Asp O 130 135 140

Leu Gin Asp Arg Val Lys Ser Leu Glu Ser Thr Ala Ser His Gly Leu 145 150 155 160

Ser Phe Ser Pro Pro Leu Ser Val Ala Asp Gly Val Val Ser Leu Asp 165 170 175

Met Asp Pro Tyr Phe Cys Ser Gin Arg Val Ser Leu Thr Ser Tyr Ser 180 185 190 —29 201006928

Ala Glu Ala Gin Leu Met Gin Phe Arg Trp Met Ala Arg Gly Thr Asn 195 200 205

Gly Ser Ser Asp Thr lie Asp Met Thr Val Asn Ala His Cys His Gly 210 215 220

Arg Arg Thr Asp Tyr Met Met Ser Ser Thr Gly Asn Leu Thr Val Thr 225 230 235 240

Ser Asn Val Val Leu Leu Thr Phe Asp Leu Ser Asp lie Thr His lie 245 250 255 o

Pro Ser Asp Leu Ala Arg Leu Val Pro Ser Ala Gly Phe Gin Ala Ala 260 265 270

Ser Phe Pro Val Asp Val Ser Phe Thr Arg Asp Ser Ala Thr His Ala 275 280 285

Tyr Gin Ala Tyr Gly Val Tyr Ser Ser Ser Arg Val Phe Thr lie Thr 290 295 300 ◎ Phe Pro Thr Gly Gly Asp Gly Thr Ala Asn lie Arg Ser Leu Thr Val 305 310 315 320

Arg Thr Gly lie Asp Thr 325

&lt;210> 4 &lt;211> 1104 &lt;212> DNA &lt;213> Avian retrovirus &lt;220&gt;

&lt;221> CDS — 30 — 48 201006928 &lt;222〉 (1)...(1104) • &lt;400&gt; 4 atg gag gta cgt gtg cca aac ttt cac teg ttc gtt gaa gga ata aca * Met Glu Val Arg Val Pro Asn Phe His Ser Phe Val Glu Gly lie Thr 15 10 15 tet age tat ttg aag act cct get tgc tgg aat gca cag aca gee tgg

Ser Ser Tyr Leu Lys Thr Pro Ala Cys Trp Asn Ala Gin Thr Ala Trp 20 25 30 gac act gtg act ttt cac gtc cct gat gta att aga gtt ggc aat geg

Asp Thr Val Thr Phe His Val Pro Asp Val lie Arg Val Gly Asn Ala 35 40 45 o tat tgt tgc tet caa tgt tgt ggt gta ett tat tac ggg act ctg ccc

Tyr Cys Cys Ser Gin Cys Cys Gly Val Leu Tyr Tyr Gly Thr Leu Pro 50 55 60 geg gat gga aat tac ttc cct cat cac aaa tgc cat cag caa cag tac

Ala Asp Gly Asn Tyr Phe Pro His His Lys Cys His Gin Gin Gin Tyr 65 70 75 80 agg acc gat acc cca ctg etc egg tat gtg ega att ggc aga aeg act

Arg Thr Asp Thr Pro Leu Leu Arg Tyr Val Arg lie Gly Arg Thr Thr 85 90 95 ^ gag cat ctg ttg gac caa tat get gtt geg ctg gag tet att get gat C# Glu His Leu Leu Asp Gin Tyr Ala Val Ala Leu Glu Ser Lie Ala Asp 100 105 110 cac tat gat gaa ate agt caa ege atg gtc gat gag cca gag aac gat

His Tyr Asp Glu lie Ser Gin Arg Met Val Asp Glu Pro Glu Asn Asp ' 115 120 125 gaa gtc geg ccc ett gac att gta aeg cgt act gaa tet ate ega agt

Glu Val Ala Pro Leu Asp lie Val Thr Arg Thr Glu Ser lie Arg Ser 130 135 140 gat aag aeg gtt gac ccg gac ttt tgg act tac egg ett gag ega cgt

Asp Lys Thr Val Asp Pro Asp Phe Trp Thr Tyr Arg Leu Glu Arg Arg 145 150 155 160 96 144 192 240 288 336 384 432 — 31 — 480 528201006928 tct gat gat tct cgt cga gac ate gee gca tea tgc tgg aga atg att Ser Asp Asp Ser Arg Arg Asp lie Ala Ala Ser Cys Trp Arg Met lie * 165 170 175 ' gat gca tea tea cgt agt etc act ett cca aat tgt ett gtg tee ccg Asp Ala Ser Ser Arg Ser Leu Thr Leu Pro Asn Cys Leu Val Ser Pro 180 185 190 tct ttg cat tct cgt tee gtc ttt ggt caa atg caa aeg acc acc acc Ser Leu His Ser Arg Ser Val Phe Gly Gin Met Gin Thr Thr Thr Thr 195 200 205 ata tac gat gtt geg geg teg gga aag gee Gtt aaa ttt tct ccg atg lie Tyr Asp Val Ala Ala Ser Gly Lys Ala Val Lys Phe Ser Pro Met 〇210 215 220 gtg get aca eta teg caa cgt gat get ggc cct gta atg ett geg aat Val Ala Thr Leu Ser Gin Arg Asp Ala Gly Pro Val Met Leu Ala Asn 225 230 235 240 get gac cca geg gaa ggt gta tat tea ttt tgg aeg teg cac ttc gee Ala Asp Pro Ala Glu Gly Val Tyr Ser Phe Trp Thr Ser liis Phe Ala 245 250 255 ttc tea c Cg etc att ggt gga gtt ggg att aeg gga cag tac get cgt Phe Ser Pro Leu lie Gly Gly Val Gly lie Thr Gly Gin Tyr Ala Arg 260 265 270 gag tea tac cat cac gtg ggt cat cca gtg ett ggg agt ggt aag aag Glu Ser Tyr His His Val Gly His Pro Val lie Gly Ser Gly Lys Lys 275 280 285 __ geg tea cac tac aga aat ctg ttt atg gaa tea tgg cgt ggg tgg tea Ala Ser His Tyr Arg Asn Leu Phe Met Glu Ser Trp Arg Gly Trp Ser _ 290 295 300 aag tea get ttc gca tgc get aca gga atg gag cca get gaa tgt gaa Lys Ser Ala Phe Ala Cys Ala Thr Gly Met Glu Pro Ala Glu Cys Glu 305 310 315 320 tct cgt ctg agg gga cat get ege act Atg ett gga ege tct ctg ccg Ser Arg Leu Arg Gly His Ala Arg Thr Met Leu Gly Arg Ser Leu Pro 325 330 335 576 624 672 720 768 816 864 912 960 a 32 - 1008 1056 201006928 aac gtc tgt gac gac gag gtt get Cag cag tet ggc gee gtg eta aeg

Asn Val Cys Asp Asp Glu Val Ala Gin Gin Ser Gly Ala Val Leu Thr 340 345 350 1104 tee ctg cag aag act acc aag ttc act gaa gtg gag tgt ggt tgg taa

Ser Leu Gin Lys Thr Oir Lys Phe Thr Glu Val Glu Cys Gly Trp 355 360 365

&lt;210> 5 &lt;211&gt; 367 &lt;212&gt; PRT &lt; 213 &gt; Avian retrovirus o &lt;400&gt;

Met Glu Val Arg Val Pro Asn Phe His Ser Phe Val Glu Gly lie Thr 15 10 15

Ser Ser Tyr Leu Lys Thr Pro Ala Cys Trp Asn Ala Gin Thr Ala Trp 20 25 30

Asp Thr Val Thr Phe His Val Pro Asp Val lie Arg Val Gly Asn Ala 35 40 45 ◎

Tyr Cys Cys Ser Gin Cys Cys Gly Val Leu Tyr Tyr Gly Thr Leu Pro 50 55 60

Ala Asp Gly Asn Tyr Phe Pro His His Lys Cys His Gin Gin Gin Tyr 65 70 75 80

Arg Thr Asp Thr Pro Leu Leu Arg Tyr Val Arg lie Gly Arg Thr Thr 85 90 95

Glu His Leu Leu Asp Gin Tyr Ala Val Ala Leu Glu Ser lie Ala Asp 100 105 110 —33 — 201006928

His Tyr Asp Glu He Ser Gin Arg Met Val Asp Glu Pro Glu Asn Asp 115 120 125

Glu Val Ala Pro Leu Asp lie Val Thr Arg Thr Glu Ser lie Arg Ser 130 135 140

Asp Lys Thr Val Asp Pro Asp Phe Trp Thr Tyr Arg Leu Glu Arg Arg 145 150 155 160 o Ser Asp Asp Ser Arg Arg Asp lie Ala Ala Ser Cys Trp Arg Met lie 165 170 175

Asp Ala Ser Ser Arg Ser Leu Thr Leu Pro Asn Cys Leu Val Ser Pro 180 185 190

Ser Leu His Ser Arg Ser Val Phe Gly Gin Met Gin Thr Thr Thr Thr 195 200 205 lie Tyr Asp Val Ala Ala Ser Gly Lys Ala Val Lys Phe Ser Pro Met 210 215 220 ◎

Val Ala Thr Leu Ser Gin Arg Asp Ala Gly Pro Val Met Leu Ala Asn 225 230 235 240

Ala Asp Pro Ala Glu Gly Val Tyr Ser Phe Trp Thr Ser His Phe Ala 245 250 255

Phe Ser Pro Leu lie Gly Gly Val Gly lie Thr Gly Gin Tyr Ala Arg 260 265 270

Glu Ser Tyr His His Val Gly His Pro Val lie Gly Ser Gly Lys Lys —34 — 201006928 275 280 285

Ala Ser His Tyr Arg Asn Leu Phe Met Glu Ser Trp Arg Gly Trp Ser 290 295 300

Lys Ser Ala Phe Ala Cys Ala Thr Gly Met Glu Pro Ala Glu Cys Glu 305 310 315 320

Ser Arg Leu Arg Gly His Ala Arg Thr Met Leu Gly Arg Ser Leu Pro 325 330 335 〇 Asn Val Cys Asp Asp Glu Val Ala Gin Gin Ser Gly Ala Val Leu Thr 340 345 350

Ser Leu Gin Lys Thr Thr Lys Phe Thr Glu Val Glu Cys Gly Trp 355 360 365 &lt;210&gt; 6 &lt;211&gt; 1035 &lt;212&gt; DNA &lt;213>Pestivirus ❹ &lt;220&gt;&lt;221&gt; CDS &lt;222> (1)...(1035) * &lt;400&gt; 6 gca caa ggc egg eta gcc tgc aag gaa gat tac agg tac gca ata teg Ala Gin Gly Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala lie Ser 15 10 15 tea acc gat gag ata ggg eta ett ggg gcc gga ggt etc acc acc acc Ser Thr Asp Glu lie Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr 20 25 30 48 96 tgg aag gaa &quot;tac Aac cac gat ttg caa ctg aat gac ggg acc gtt aag 144 —35 a 192201006928

Trp Lys Glu Tyr Asn His Asp Leu Gin Leu Asn Asp Gly Thr Val Lys 35 40 45 gcc agt tgc gtg gca ggt tcc ttt aaa gtc aca gca ctt aat gtg gtc ' Ala Ser Cys Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val 50 50 60 agt agg agg tat ttg gca tea ttg cat aag aag get tta ccc act tcc Ser Arg Arg Tyr Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser 65 70 75 80 gtg aca ttc gag etc ctg ttc gac ggg Acc aac cca tea act gag gaa Val Thr Phe Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu 85 90 95 o atg gga gat gac ttc agg tcc ggg ctg tgc ccg ttt gat aeg agt cct Met Gly Asp Asp Phe Arg Ser Gly Leu Cys Pro Phe Asp Thr Ser Pro 100 105 110 gtt gtt aag gga aag tac aat aeg acc ttg ttg aac ggt agt get ttc Val Val Lys Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe 115 120 125 tat ctt gtc Tgc cca ata ggg tgg aeg ggt gtc ata gag tgc aca gca Tyr Leu Val Cys Pro lie Gly Trp Thr Gly Val He Glu Cys Thr Ala 130 135 140 gtg age cca aca act ctg aga aca gaa gtg gta aag acc ttc agg aga Val Ser Pro Thr Thr Leu Ar g Thr Glu Val Val Lys Thr Phe Arg Arg 145 150 155 160 gac aag ccc ttt ccg cac aga atg gat tgt gtg acc acc aca gtg gaa Asp Lys Pro Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu * 165 170 175 Aat gaa gat tta ttc tat tgt aag ttg ggg ggc aac tgg aca tgt gtg Asn Glu Asp Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val 180 185 190 aaa ggc gag cca gtg gtc tac aca ggg step on g eta gta aaa caa Tgt aga Lys Gly Glu Pro Val Val Tyr Thr Gly Gly Leu Val Lys Gin Cys Arg 195 200 205 240 288 336 384 432 480 528 576 —36 — 624 672201006928 &quot;tgg &quot;tgt ggc ttc gac ttc gat ggg cct gac gga etc Ccg cat tac ccc Trp Cys Gly Phe Asp Phe Asp Gly Pro Asp Gly Leu Pro His Tyr Pro ' 210 215 220 ata ggt aag tgc att ttg gca aat gag aca ggt tac aga ata gta gat lie Gly Lys Cys lie Leu Ala Asn Glu Thr Gly Tyr Arg lie Val Asp 225 230 235 240 tea aeg gac tgt aac aga gat ggc gtt gta ate age aca gag ggg agt Ser Thr Asp Cys Asn Arg Asp Gly Val Val lie Ser Thr Glu Gly Ser 245 250 255 cat gag tgc ttg ate Ggt Aac aeg act gtc aag gtg cat gca tea gat His Glu Cys Leu lie Gly Asn Thr Thr Val Lys Val His Ala Ser Asp 〇260 265 270 gaa aga ttg ggc cct atg cca tgc aga cct aaa gag att gtc tet agt Glu Arg Leu Gly Pro Met Pro Cys Arg Pro Lys Glu lie Val Ser Ser 275 280 285 720 768 816 864 get gga cct gta aag aaa acc tee tgt aca ttc aac tac aca aaa act Ala Gly Pro Val Lys Lys Thr Ser Cys Thr Phe Asn Tyr Thr Lys Thr 290 295 300 912 ttg aag aac agg tac tat gag ccc agg gac age tac ttc cag caa tat Leu Lys Asn Arg Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gin Gin Tyr 305 310 315 320 960

Atg ett aag ggt gag tat cag tac tgg ttt gac ctg gat geg act gac Met Leu Lys Gly Glu Tyr Gin Tyr Trp Phe Asp Leu Asp Ala Thr Asp 325 330 335 1008 ege cac tea gat tac ttc gca gaa ttt Arg His Ser Asp Tyr Phe Ala Glu Phe 340 345 1035 &lt;210> 7 &lt;211&gt; 345 &lt;212&gt; PRT &lt;213&gt; Porcine Virus &lt;400&gt; 7 -37 201006928

Ala Gin Gly Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala lie Ser 15 10 15

Ser Thr Asp Glu lie Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr 20 25 30

Trp Lys Glu Tyr Asn His Asp Leu Gin Leu Asn Asp Gly Thr Val Lys 35 40 45

Ala Ser Cys Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val 50 55 60 〇

Ser Arg Arg Tyr Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser 65 70 75 80

Val Thr Phe Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu 85 90 95

Met Gly Asp Asp Phe Arg Ser Gly Leu Cys Pro Phe Asp Thr Ser Pro 100 105 110

Val Val Lys Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe 115 120 125

Tyr Leu Val Cys Pro lie Gly Trp Thr Gly Val lie Glu Cys Thr Ala 130 135 140

Val Ser Pro Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg 145 150 155 160

Asp Lys Pro Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu 165 170 175 —38 — 201006928

Asn Glu Asp Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val 180 185 190

Lys Gly Glu Pro Val Val Tyr Thr Gly Gly Leu Val Lys Gin Cys Arg 195 200 205

Trp Cys Gly Phe Asp Phe Asp Gly Pro Asp Gly Leu Pro His Tyr Pro 210 215 220 lie Gly Lys Cys lie Leu Ala Asn Glu Thr Gly Tyr Arg lie Val Asp 〇 225 230 235 240

Ser Thr Asp Cys Asn Arg Asp Gly Val Val lie Ser Thr Glu Gly Ser 245 250 255

His Glu Cys Leu lie Gly Asn Thr Thr Val Lys Val His Ala Ser Asp 260 265 270

Glu Arg Leu Gly Pro Met Pro Cys Arg Pro Lys Glu lie Val Ser Ser 275 280 285 ❹

Ala Gly Pro Val Lys Lys Thr Ser Cys Thr Phe Asn Tyr Thr Lys Thr 290 295 300

Leu Lys Asn Arg Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gin Gin Tyr 305 310 315 320

Met Leu Lys Gly Glu Tyr Gin Tyr Trp Phe Asp Leu Asp Ala Thr Asp 325 330 335

Arg His Ser Asp Tyr Phe Ala Glu Phe 340 345 —39

Claims (1)

201006928 X. Patent application scope: 1 - an expression vector characterized by having the ability to be repeatedly used to express different foreign genes, wherein the vector comprises: at least one restriction enzyme cleavage site; a baculovirus transmembrane region sequence , which is composed of the nucleotide sequence shown by SEQ ΙΓ" i; a message sequence of a baculovirus; a cytoplasmic region sequence of a baculovirus; a promoter region; and six histidine message sequences; wherein the at least-restricted cleavage site is constructed in the six histidine message sequence and the baculovirus transmembrane region sequence between. 2 m full-time (4) The heavy beer-like carrier described in item i, its exogenous DM insert, which contains the gamma-exogenous protein 3, according to the weight of the patent application, the restriction enzyme cleavage system It is selected from the group consisting of the orphan virus vector 'its £coRl. a Ϊ, Pst I inverse 4, according to the weight of the patent scope i): = domain contains baculovirus 5, according to the scope of claim 2, the exogenous protein gene system poultry rio </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> The recombinant baculovirus vector according to the above, wherein the foreign protein gene is a recombinant baculovirus vector according to the second aspect of the patent application, wherein the foreign protein gene is a pig. The prion Ε 2 gene. 9. A host cell comprising the expression vector of the ninth aspect of the patent application. The host cell according to claim 9 wherein the cell line is an insect cell. The host cell according to claim 9, wherein the cell line recombinant protein is a cell. The method for preparing avian virus (jC and (7. B recombinant virus protein method) is characterized by the obtained c The rC and σΒ recombinant viral proteins may present an immune response to the experimental animal, and the method comprises the following steps: (a) constructing the poultry virus gene into at least one restriction enzyme cleavage position of the expression vector of the first application patent; (b) transfecting the recombinant expression vector obtained in step (a) into insect fine β cells; • (C) cultivating the infected insect cells obtained in the step (b) to express and secrete the sigma of the poultry virus (: And σΒ recombinant virus protein. 13. A recombinant protein of poultry virus, the amino acid sequence of the aC recombinant protein of the poultry virus, sEq ID no : 3, 201006928 14. A σΒ recombination of a poultry virus Protein, the amino acid sequence of the σΒ recombinant protein of the poultry virus, seq id NO : 5, a poultry virus virus subunit identification vaccine comprising: the poultry rio of SEQ ID NO: 3 The amino acid sequence of the viral recombinant protein; q the amino acid sequence of the poultry virus σΒ recombinant protein represented by SEQ ID NO: 5; and an adjuvant or an excipient. Item 15 of the poultry virus virus subunit identification vaccine, wherein the adjuvant is a baculovirus. U. A method for preparing a swine fever virus protein, which comprises Lu (a) a swine fever virus The Ε0 gene is constructed into at least one restriction enzyme cleavage site of the expression vector of the first item of the patent application; 00, the recombinant expression vector obtained in the step (a) is transfected into the insect cell; '(G) is cultured under appropriate conditions The infected Kun-worm cells obtained in the step (b) are allowed to express and secrete the E0 recombinant virus protein of the classical swine fever virus. 18. A method for preparing an E2 recombinant viral protein of a porcine lean virus, comprising: U) constructing an E2 gene of a porcine lean virus, such as at least one restriction enzyme cleavage position of a performance vector of the patent scope - 42 - 201006928 (b) transfecting the recombinant expression vector obtained in step (a) into an insect cell; (c) cultivating the infected insect cell obtained in the step (b) under appropriate conditions to express and secrete the piglet Virus E2 recombinant viral protein. 19. An E2 recombinant protein of the lean virus, wherein the base sequence of the E2 recombinant protein of the porcine lean virus is represented by SEQ ID NO: 7. ^20. A swine fever virus subunit identification vaccine comprising: the amino acid sequence of the classical swine fever virus E2 recombinant protein of SEQ ID NO: 7; and an adjuvant or excipient. 21. The swine fever virus subunit identification vaccine according to item 20 of the patent application scope, wherein the adjuvant is a baculovirus. 〇 —43 —