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WO2006005222A1 - Proteine p40 obtenue par genie genetique et utilisation - Google Patents

Proteine p40 obtenue par genie genetique et utilisation Download PDF

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Publication number
WO2006005222A1
WO2006005222A1 PCT/CN2004/000776 CN2004000776W WO2006005222A1 WO 2006005222 A1 WO2006005222 A1 WO 2006005222A1 CN 2004000776 W CN2004000776 W CN 2004000776W WO 2006005222 A1 WO2006005222 A1 WO 2006005222A1
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WIPO (PCT)
Prior art keywords
protein
nucleotide sequence
genetically engineered
amino acid
white spot
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2004/000776
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English (en)
Chinese (zh)
Inventor
Xiaolin Meng
Jinping Xu
Hongxia Li
Jian Wang
Wei Lu
Xu Cao
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GREEN LIFE LABORATORY Ltd
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GREEN LIFE LABORATORY Ltd
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Priority to CN200480043768.1A priority Critical patent/CN101023095B/zh
Priority to PCT/CN2004/000776 priority patent/WO2006005222A1/fr
Publication of WO2006005222A1 publication Critical patent/WO2006005222A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/18011—Nimaviridae
    • C12N2710/18022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the prevention and treatment of white spot syndrome of clam shell organisms. More particularly, the present invention relates to genetically engineered proteins that can control shrimp white spot syndrome and uses thereof.
  • Chinese shrimp farming has entered a peak in the 1990s, with a breeding area of 160,000 hm 2 , an annual breeding capacity of more than 10 million, an annual output of more than 200,000 tons of shrimp, annual foreign exchange - more than 500 million US dollars, consecutive Maintaining world leadership in the year. Since 1993, due to the outbreak of shrimp virus disease, white spot syndrome of shrimp, the Chinese shrimp farming industry has experienced a significant decline. The shrimp farming industry has entered a trough, and the total production of cultured shrimp has dropped sharply.
  • the global demand for shrimp is at least 1.2 million tons, and the trade volume is more than 7 billion US dollars. It has formed four major markets in Japan, the United States, the European Union and Southeast Asia. In recent years, the demand for shrimp in the international market has been increasing, and the price has risen steadily. However, the shrimp white spot syndrome has led to a decline in the production of shrimp.
  • Shrimp white spot syndrome is a disease caused by a virus (white spot syndrome virus, called WSSV), which can rapidly form an epidemic in shrimp populations and cause devastating blows to shrimp culture.
  • WSSV white spot syndrome virus
  • the literature reveals only the sequencing of VP28, VP26, VP24, and their nucleic acid sequences.
  • the literature also indicates that VP28 and VP19 are decidual proteins.
  • this article does not report the full-length amino acid sequence of VP19, nor does it report the gene sequence encoding VP19.
  • This document also reports the preparation of WSSV vaccines. It is generally indicated that the vaccine includes a combination of two or more kinds of proteins of VP28, VP26, VP24, and VP19.
  • the vaccine includes VP26 and VP28, with or without the addition of VP24. However, there is no indication of the need to join VP19.
  • WO0222664 Antigenic Proteins of Shrimp White Spot Syndrome Virus and Use thereof (International Application Date, September 14, 2001), the applicant of which is AKZO NOBEL, the Netherlands.
  • This article discloses the antigenic proteins VP19 and VP13 derived from WSSV, and the use of these proteins as vaccine components, antibodies to these proteins, using these antibodies as components of vaccines, and also revealing the use of these proteins for the prevention and treatment of white spot syndrome.
  • Vaccine containing kits for antibodies or nucleic acids. In the vaccine disclosed in this document, VP13 and/or VP19 are contained. It is also mentioned that VP19 alone has delayed the death of shrimp after suffering a WSSV attack. However, this document does not address the combination of VP28 and VP19.
  • One object of the present invention is to provide a genetically engineered protein P40 having an amino acid sequence at least 70% homologous to the amino acid sequence of SEQ ID NO. 1, and having a molecular weight of about 40 kilodaltons.
  • the genetically engineered protein P40 has an antigen 'I' which is resistant to the white spot syndrome virus of shrimp.
  • One of the objects of the present invention is to provide a genetically engineered protein P40, which encodes an amino acid sequence consisting of two parts, an amino acid sequence having at least 96% homology with the white spot syndrome virus protein VP19, with sputum and white spot syndrome.
  • the viral protein VP28 has an amino acid sequence of at least 99% homology.
  • One of the objects of the present invention is to provide a genetically engineered protein P40 which encodes an amino acid sequence consisting of two parts, an amino acid sequence having at least 96% homology with the white spot syndrome virus protein VP19, and a white spot syndrome virus.
  • the protein VP28 has an amino acid sequence of at least 99% homology with two linked amino acid residues between the two fragments.
  • One of the objects of the present invention is to provide a nucleotide sequence encoding the genetically engineered protein P40 having a nucleotide sequence which is at least 50% homologous to the nucleotide sequence of SEQ ID NO.
  • One of the objects of the present invention is to provide a nucleotide sequence encoding the genetically engineered protein Shield P40 having a nucleotide sequence at least 70% homologous to the nucleotide sequence of SEQ ID NO.
  • One of the objects of the present invention is to provide a nucleotide sequence encoding the genetically engineered protein Shield P40 having a nucleotide sequence which is at least 80% homologous to the nucleotide sequence of SEQ ID NO.
  • One of the objects of the present invention is to provide a nucleotide sequence encoding the genetically engineered protein P40, which consists of two parts, i.e., at least 90% homologous to the coding nucleotide of the white ⁇ syndrome virus protein VP19.
  • One of the objects of the present invention is to provide a method for producing a DNA fragment comprising the nucleotide sequence encoding the genetically engineered peptone 40 of the present invention.
  • One of the objects of the present invention is to provide a host cell comprising a nucleotide sequence encoding the genetically engineered peptone 40 of the present invention, or a DNA fragment encoding the genetically engineered peptone 40 of the present invention.
  • One of the objects of the present invention is to provide a method for constructing an E. coli host cell which comprises the nucleotide sequence of the genetically engineered peptone 40 of the present invention.
  • One of the objects of the present invention is to provide an immune vaccine against white spot syndrome, comprising the protein ⁇ 40, or a nucleotide sequence encoding the protein ⁇ 40 or a functional DNA fragment thereof, or comprising the a nucleotide sequence or a function thereof, a host cell of a 1" DNA fragment, or a mixture of two or more of them.
  • One of the objects of the present invention is to provide a method for preparing an immune vaccine for white spot syndrome Characterized by the method comprising: protein ⁇ 40, or encoding said a nucleotide sequence of a protein P40 or a functional DNA fragment thereof, or a host fine sequence comprising the nucleotide sequence or a functional DNA fragment thereof, or a mixture of two or more thereof, and pharmacology The acceptable carriers are mixed together.
  • One of the objects of the present invention is to provide a shrimp feed characterized in that the shrimp feed comprises the protein P40, or a nucleotide sequence encoding the protein P40 or a functional DNA fragment thereof, or A host cell of the nucleotide sequence or a functional DNA fragment thereof, or a mixture of two or more of them.
  • One of the objects of the present invention is to provide an antiserum characterized in that the antiserum is prepared by using the genetic engineering protein P40 as an antigen.
  • One of the objects of the present invention is to provide a white spot syndrome detection kit, characterized in that the detection kit comprises the antiserum.
  • FIG. 4 SDS-PAGE electrophoresis of genetically engineered strains expressing fusion membrane proteins.
  • Lane 1 E.coli BL21(DE3) (pET22) strain before induction;
  • Lane 2 E. coli BL21 (DE3) (pET22) strain 5 hours after induction; Lane 3: E. coli BL21 (DE3) (pET22) was not induced;
  • Figure 5 shows the results of SDS-PAGE electrophoresis after purification of the scorpion scorpion protein
  • lanes 1 -8 protein step-wise collection after purification of the Ni column
  • FIG. 6 Schematic diagram of the test strip structure
  • Figure 7 Schematic diagram of the results of rapid detection of shrimp white spot syndrome virus by test strips; (1) a positive result;
  • a genetically engineered protein P40 provided by the present invention has an amino acid sequence at least 70% homologous to the amino acid sequence of SEQ ID NO. Sexually, its molecular weight is about 40 kilodaltons, and the genetically engineered protein P40 has immunogenicity against the white spot syndrome virus.
  • the shaded portion indicates the sequence of the vector, the other is the gene sequence, and the box indicates the two amino acids extrapolated by the cleavage site of VP19 and VP28.
  • the amino acid sequence of the genetically engineered protein P40 of the present invention can be modified and changed within a certain range, and the obtained modified protein or protein fragment has the same biological function as the genetically engineered protein shield P40, especially the protein antigen. No change in sex can stimulate the body to produce a similar immune response. Methods for modifying and modifying proteins are conventional methods and are familiar to those skilled in the art.
  • proteins with more than 70% amino acid sequence homology are often interpreted in biology as proteins with the same biological function. Modifications and mutations to the amino acid sequence of the protein within this range are not considered to alter the biological properties of the protein, including:
  • 6 consecutive histidines can be added at the N-terminus or C-terminus to facilitate purification of the protein by Ni affinity chromatography; or the enterokinase site can be added at the N-terminus, and the protein shield can be cleaved by enterokinase.
  • the above method of modifying and changing the protein is a conventional method. It is familiar to those skilled in the art.
  • the protein or immunological fragment obtained by the above method having 70% homology with the genetic engineering protein shield of the present invention has the same biology as the genetic engineering protein P40 of the present invention.
  • the function that is, can stimulate the shrimp to produce a specific anti-WSSV immune response, can stimulate the animal to produce WSSV decidual fusion protein specific antibodies, can be used to achieve one or more of the purposes of the present invention.
  • the genetic engineering protein P40 of the present invention has a molecular weight of about 40 kilodaltons. Part or portion of changes occurring amino acid sequence, molecular mass vary.
  • the White Spot Syndrome Virus (WSSV) of the present invention is isolated from the prawn of the North Sea of China, and the genetic sequence homology between the isolates of different regions of WSSV varies greatly depending on the gene, wherein the decidua The sequence homology of the protein is between 72% and 99%.
  • an amino acid sequence having at least 96% homology to the WSSV decidual protein VP 19 and an amino acid sequence having at least 99% homology to the decidual protein VP28 can be fused together by direct or indirect means.
  • the fusion proteins all have epitopes of all WSSV decidual proteins VP19 and VP28, all having the same biological functions as the genetically engineered protein P40 of the present invention, and can be used to achieve one or more of the purposes of the present invention.
  • the Applicant performed amino acid sequencing of the proteins VP 19 and VP28 of Shrimp White Spot Syndrome Virus.
  • Table 1 The basic situation of gene sequencing of VP19 and VP28 is given in Table 1 shown below.
  • a genetically engineered protein P40 which encodes an amino acid sequence consisting of two parts, an amino acid sequence having at least 96% homology with the white spot syndrome virus protein VP19, and
  • the white spot syndrome virus protein VP28 has an amino acid sequence of at least 99% homology with two linked amino acid residues between the two fragments.
  • vpl9 and vp28 can be ligated in vitro according to the cleavage site of the vector and the sequence of the gene, in one embodiment of the present invention.
  • the ligation method uses EcoR I to link the two genes.
  • a fusion protein P40 is prepared.
  • the amino acid sequence of the fusion protein is SEQ ID NO.
  • a genetically engineered protein P40 which is a genetically engineered fusion protein, the N-terminal and C-terminal amino acids of which are vector-encoded, the N-terminus is MKYLLPTAAAGLLLLAAQPAMAMDIGINSD P, C-terminal KLAAALEHHHHHH.
  • the present invention selects the pET22b(+) expression vector to prepare the genetic engineering protein P40, and the host cell is Escherichia coli BL21 (DE3).
  • the target gene is cloned into a vector, which is controlled by strong transcription and translation signals of phage T7, and the expression requires T7 RNA polymerase from the host cell.
  • a lac operator sequence is immediately downstream of the T7 promoter.
  • the host cell BL21 (DE3) is a lysogen of phage DE3, and its transcription of T7 RNA polymerase is also controlled by the lac promoter. Therefore, the lac repressor can inhibit the expression of the target gene by controlling the vector promoter, and can also inhibit the transcription of the host T7 RNA polymerase and inhibit the leakage expression of the target gene caused by any RNA polymerization.
  • this protein of interest can reduce the toxicity of the protein of interest to the cells.
  • the target gene Under non-inducing conditions, the target gene can be completely silenced without transcription.
  • the target protein can account for up to the total number of cells. More than 50% of the protein.
  • the excessive synthesis of genetically engineered products in bacterial cells will inevitably affect the growth and metabolism of host cells, while the damage of host cell metabolism inhibits the production of foreign gene products. Synthesis. In the pET22-E.
  • coli BL21 (DE3) expression system when the host cell grows in a large amount, the expression of the foreign gene is inhibited, and when the biomass of the host cell is saturated, the induced expression of the genetically engineered protein is performed, or It is desirable to easily attenuate protein expression by reducing the concentration of the inducer.
  • the expression level is high, and the expression level of the genetic engineering protein can reach 30-50% of the total amount of the bacterial protein.
  • the N-terminal sequence (30 amino acids) contributes to the correct folding of the protein, increases the solubility of the protein, avoids the formation of inclusion bodies, and protects the protein of interest from the protease in the host cell, prolonging the genetically engineered protein.
  • the half-life of the gene improves the stability of the genetically engineered protein.
  • the C-terminus contains 6 consecutive histidines, which facilitate the isolation and purification of proteins. Histidine is a rare amino acid in the general protein, and the introduction of six consecutive histidines has a high specificity.
  • the His6 tail has strong binding ability to metal ions, and has little effect on the structure and function of the target protein. Generally, the biological activity can be maintained, and the biological activity of the target protein can be directly studied without removing the His6 tail.
  • E. coli expression system is a safe expression system. It has successfully expressed a variety of medical proteins with E. coli expression system, which is safe and non-toxic, non-toxic and harmless to humans, animals and the environment.
  • Conducive to industrial production In all genetic engineering expression systems, the production cost of E. coli is the best, and there are already formed fermentation equipment and production methods. The expression system adopted by the invention is advantageous for realizing industrial production. Ampicillin is the cheapest antibiotic that can further reduce production costs.
  • One of the objects of the present invention is to provide a nucleotide sequence encoding the genetically engineered protein ⁇ 40 having a nucleotide sequence which is at least 50% homologous to the nucleotide sequence of SEQ ID NO.
  • any change in the amino acid sequence of a genetically engineered protein is attributable to changes in its nucleotide sequence.
  • the organism consists of a variety of proteins, and the amino acids that make up the protein are only about 22.
  • the nucleotide sequence encoding the protein is arranged in a different order by the single nucleotides (A, T, G, C) of 4. Combined composition. Therefore, in theory, any change in nucleotide sequence has the potential to alter the amino acid it encodes, thereby altering the structure and function of its encoded protein.
  • the amino acid codons of the organism are degenerate, that is, different codons can encode the same amino acid.
  • GCA, GCC, GCG, and GCT all encode Ala (alanine).
  • the biological significance of this kind of merger is that in the process of DNA replication, the genetic shield can be changed by internal or external conditions or factors. If these changes immediately cause amino acid changes, it will directly affect the protein, especially the function. The structure and function of proteins. Most changes in nucleotide sequence are not conducive to mutations in the organism, and the annexation of codons greatly reduces the mutation of nucleotides to protein function. Impact. In addition, the annexation of codons is also responsible for the fact that the amino acid mutation rate is much lower than the nucleotide variation rate.
  • nucleotide sequence of the genetically engineered protein P40 of the present invention can be mutated, but the encoded protein or immunological fragment has the same biological function as the genetically engineered protein P40, that is, the protein antigenicity does not change, Can stimulate the body to produce a similar immune response.
  • mutations include:
  • A) Synonymous mutation A mutation of an individual nucleotide, but the amino acid encoded by it does not change, so all the biological characteristics of the encoded protein are the same.
  • hemagglutinin factor Xa can be added to specifically recognize and cleave the 4 peptide sequence Ile-Glu-Gly-Arg.
  • the above methods for modifying and changing nucleotides are conventional methods, such as PCR, which are familiar to those skilled in the art and can be obtained without creative labor.
  • the nucleotide sequence obtained by this method which has 50% homology with the nucleotide sequence of the present invention has the same biological function as the nucleotide sequence of the present invention, that is, both can encode WSSV ruthenium membrane
  • the fusion protein which stimulates the shrimp to produce a specific anti-WSSV immune response, can stimulate the animal to produce a specific antibody to the WSSV decidual fusion protein, and can be used to achieve one or more of the purposes of the present invention.
  • a nucleotide sequence encoding the genetically engineered protein P40 is provided, the nucleotide sequence of which is at least 70% homologous to the nucleotide sequence of SEQ ID NO.
  • nucleotide sequence encoding the genetically engineered protein P40 the nucleotide sequence of which preferably has at least 80% homology to the nucleotide sequence of SEQ ID NO. Sex.
  • the nucleotide sequence encoding the genetically engineered protein P40 of the present invention can be changed within a certain range, and the obtained nucleotide sequence has the same biological function as the nucleotide sequence encoding the genetically engineered protein P40, in particular The antigenicity of the encoded protein has not changed, and it can stimulate the body to produce a similar immune response.
  • nucleotide sequences with more than 70% homology can be judged to have obvious similarities, homology.
  • the above methods for modifying and changing nucleotides are conventional methods, such as PCR, which are familiar to those skilled in the art and can be obtained without creative labor.
  • the obtained nucleotide sequence having 70% homology, preferably 80% homology with the nucleotide sequence of the present invention has the same biological function as the nucleotide sequence of the present invention, ie Both can encode WSSV membrane fusion protein, can stimulate the shrimp to produce a specific anti-WSSV immune response, can stimulate the animal to produce WSSV membrane fusion protein specific antibodies, can be used to achieve one or more purposes of the present invention.
  • a nucleotide sequence "J" encoding a genetically engineered protein P40 is provided, the sequence consisting of two parts, that is, at least 90 with the coding nucleotide of the white spot syndrome virus protein VP19.
  • Decidual membrane proteins are important determinants of viral infection and pathogenicity, and are also important regions of antigenic determinants.
  • WSSV has two decidual protein genes, vpl9 and vp28.
  • vp28 may be more critical in the invasion of viruses.
  • the WSSV of the present invention is isolated from the prawn of the North Sea of China, and the genetic sequence homology between the isolates of different regions of WSSV is greatly different due to different genes, wherein the nucleotide sequence homology of the decidual protein is 48%-99%.
  • nucleotide sequence having at least 90% homology to the WSSV decidual protein gene vpl9, and a nucleotide sequence having at least 95% homology to ⁇ vp28 can be fused directly or indirectly Together, the resulting fusion gene has all the epitopes of the WSSV decidual membrane proteins vpl9 and vp28, and all of the proteins encoding the same biological function as the genetically engineered protein P40 of the present invention can be used for realization.
  • a method for producing a nucleotide sequence encoding a genetically engineered protein P40 wherein the nucleotide sequence of the fusion gene is SEQ ID NO. 2, wherein the positions of the vpl9 and vp28 genes are different ( J is 94-456, 462-1116.
  • Other gene fusion methods such as vpl9 post-vp28 fusion.
  • vpl9 and vp28 can each form their own advanced structure, still It is possible to encode all the epitopes of cysticin.
  • a nucleotide sequence encoding the genetically engineered protein Shield P40 is provided, the encoded amino acid sequence consisting of two parts, namely, the white spot syndrome virus decidual protein vpl9 and vp28 genes, two The gene was fused by the EcoR I (GAATTC) site, and the fused gene was inserted into the expression vector through two sites, BamH I ( GGATCC ) and Hind III ( AAGCTT ).
  • V pl9 and vp28 can be ligated in vitro according to the cleavage site of the vector and the sequence of the gene, in one embodiment of the present invention.
  • BamH I GGATCC
  • Hind III AAGCTT
  • a nucleotide sequence encoding a genetically engineered protein P40 comprising the vector upstream and downstream of the sequence: Upstream ATGAAATACCTGCTGCCGACCGCTGCTGCTG GTCT GCTGCTCCTCGCT GCCCAGCCGG CGATGGC CATG GAT A TCGGAATTAATTC GGATCCT, downstream AAGGTTTGCGGCCGC A CTCGAGCACCAC CACCACCACCACTGA, wherein GGATCC and A AGCTT are restriction sites for gene ligation.
  • the present invention selects the pET22b(+) expression vector to prepare the genetic engineering protein P40, and the host cell is Escherichia coli BL21 (DE3).
  • the advantage of the system is: tight control of the expression of the target protein to reduce the toxicity of the target protein to the cells.
  • the expression level is high, and the expression level of the genetic engineering protein can reach 30-50% of the total amount of the bacterial protein.
  • Fusion expression The N-terminal sequence contributes to the correct folding of the protein and increases the solubility and stability of the protein.
  • the C-terminus contains 6 consecutive histidines, which facilitate the isolation and purification of proteins. High security and industrial production.
  • a DNA fragment and a method of making the same are provided. This fragment contains the nucleotide sequence encoding the genetically engineered protein P40 of the present invention.
  • the DNA fragment according to the invention can be obtained by artificial synthesis, the DNA fragment of the present invention can be directly synthesized by a DNA synthesizer, or the DNA fragment of the present invention can be synthesized in stages, and these synthetic products have the present invention.
  • One or more of the objects described herein can be achieved by the same biological function of the DNA fragments.
  • PCR amplification using the DNA fragment of the present invention as a template, or using a plasmid, a vector, and a host squamous packet containing the DNA fragment of the present invention as a template to obtain a DNA fragment by PCR amplification, and these PCR products have a
  • One or more of the objects of the present invention can be achieved by the same biological function of the DNA fragments of the present invention.
  • the above method is a method commonly used in molecular biology, and is familiar to those skilled in the art, and can be obtained without creative labor, and the obtained DNA fragment is regarded as the same organism as the nucleotide sequence involved in the present invention. Learning function, one or more objects of the invention can be further achieved by genetic engineering methods.
  • a host cell comprising a nucleotide sequence encoding the genetically engineered protein P40 of the present invention, or a DNA encoding the genetically engineered protein P40 of the present invention is provided. Fragment.
  • the DNA fragments involved in the present invention can be transferred into host cells by chemical or biological methods, such as transformation, transduction, site, gene gun injection, etc.
  • Cells include, but are not limited to, E. coli, insect cells, mammalian cells, yeast, and their genetic localization can be on the chromosome, or they can exist alone or in addition to other genetic material outside the chromosome.
  • an E. coli host cell comprising the nucleotide sequence of the genetically engineered protein P40 of the present invention, designated as (E. coli) BL21(DE3)-pET The -22b(+)-p40 strain, which is numbered CCTCC M204050 in the China Center for Type Culture Collection.
  • a method of constructing an E. coli host cell comprising the nucleotide sequence of the genetically engineered protein P40 of the present invention is provided.
  • WSSV virus genome is a double-stranded DNA molecule, and the genome sequences of different strains of viruses are quite different. Among them, VP24 and VP26 The protein is a capsid-associated protein, and the VP19 and VP28 proteins are decidual-associated proteins.
  • the preparation method of WSSV virus genome has been reported in many literatures, and is familiar to those skilled in the art, and does not require creative labor. In one embodiment of the invention, a method of making a WSSV viral genome is provided.
  • the preparation of the vp28 and vpl9 genes can be obtained by several methods, such as by PCR using the genome of the virus as a template, or by restriction endonuclease digestion of the gene group. Gene fragments can also be obtained directly from genomic libraries or cDNA libraries.
  • a method for producing the vp28 and vpl9 genes by PCR is provided, and the amplification primers are designed according to the published WSSV sequence in GeneBank.
  • the target genes vp28 and vpl9 were amplified by conventional PCR using the WSSV genome as a template.
  • the PCR product was recovered and cloned into a T vector and transformed into E. coli JM109 for gene proliferation and preservation.
  • C) splicing P40 open reading frame Recombinant containing vp28 and vpl9 gene is digested with appropriate restriction enzymes, and the corresponding fragment is recovered. The fusion gene is constructed according to the correct reading frame (ORF), and the constructed P40 is sequenced. The risk certificate is correct.
  • the ligated P40 open reading frame is inserted into the expression vector by restriction enzyme ligation, and the constructed recombinant expression vector is transformed into E. coli.
  • the transformation methods include but are not limited to electroporation. , calcium chloride Conversion method.
  • a method for transforming calcium chloride is provided, wherein the expression vector used is pET22b(+) and the host cell is Escherichia coli BL21(DE3)»
  • the expression vector used is pET22b(+) and the host cell is Escherichia coli BL21(DE3)»
  • the above molecular biology methods of operation are those skilled in the art. As is familiar, reference can be made to Molecular Cloning: A Laboratry Manual, Cold Spring Harbor Lab (CSHL) Press, 2001 by Joe Sambrook, David Russell et al.
  • an immune vaccine against white spot syndrome virus which comprises the genetically engineered protein P40 of the present invention, or a nucleotide sequence encoding the genetically engineered protein P40, Or a DNA fragment encoding the genetically engineered protein P40, or a host cell of the invention, or a mixture of two or more thereof.
  • vertebral pushers have a sound immune system that produces many anti-disease substances against viruses and other pathogens, and that ridgeless pushers lack the immunity to obtain a virus. Specific resistance.
  • Japan's Venegas et al.'s study of Japanese shrimp found that shrimp has a disease-like mechanism similar to the immune system, the quasi-immune system.
  • Survivors of Japanese prawn leukoplakia outbreaks had a survival rate of 94% when vaccinated with WSSV.
  • Survivors after manual injection of WSSV virus the survival rate of live virus injected with WSSV after 32 days, the survival rate was as high as 66-74%.
  • the increase in antiviral capacity is not due to the fact that the survivor is a natural mutant, but because there is a similar immune system against the disease, the quasi-immune system.
  • the quasi-immune system of the shrimp When the virus is infected, the quasi-immune system of the shrimp is activated.
  • the quasi-immunological response of prawn is related to the stimulation of 4 ⁇ organisms. It has been proved that when inactivated Vibrio spp (Vibrio species) is injected into the prawn and Japanese worms, the prawn itself will be effective. The immune effect, against the reinvasion of the inactivated Vibrio spp.
  • the genetically engineered protein P40 of the present invention can also be used as a subunit vaccine for the prevention of white spot syndrome. Prawns do not have the immune system of higher animals, nor the immune response of higher animals such as immune memory.
  • the invasion of the virus requires specific binding of the virus to the cell surface receptor, and the genetic engineering fusion membrane protein of the present invention can be competitively combined with the viral cell receptor, thereby affecting the adsorption and invasion of the virus. Therefore, the genetically engineered protein P40 of the present invention can be used as a "quasi-vaccine" for the control of shrimp WSSV virus.
  • the genetically engineered protein P40 for vaccine preparation is a fusion membrane protein VP19 and VP28.
  • the fusion protein has all amino acid coding sequences of two proteins, and has antigenic determinant sites of two proteins, which enhances the engineering protein P40. Immune effect.
  • the expression system used in the present invention is an E.
  • the main active ingredient of the shrimp WSSV vaccine of the present invention is the genetic engineering protein P40, which is easily biodegraded and utilized in the environment, so the shrimp WSSV vaccine of the present invention has the advantages of not polluting the environment and being safe for humans and animals. And will not affect the aquatic ecological environment, and is conducive to the sustainable development of the aquaculture industry.
  • the anti-white spot syndrome virus immunization vaccine provided by the invention has the main active component of the genetic engineering protein P40, and the protein can be used alone or in the host cell of the genetic engineering protein, or includes the fusion gene.
  • an immune vaccine against white spot syndrome virus is provided, the main active ingredient of which is genetically engineered protein P40, which may also include pharmacologically acceptable during production and use.
  • the preparation of the shrimp WSSV vaccine can be carried out according to the conventional method for preparing aquatic products.
  • the main active ingredient of the vaccine is the genetic engineering protein P40, and the protein has a short effective period in the environment, so that various additives can be added as needed.
  • Or adjuvants including protein antioxidants, stabilizers, preservatives, solvent oils, activators, synergists, UV absorbers, etc., for the purpose of protecting engineered proteins and increasing the immune effect, such adjuvants commonly used in the art.
  • insoluble aluminum salts such as chrome ⁇ , etc.
  • oil-water emulsion such as Freund's adjuvant
  • peanut oil emulsion adjuvant soil temperature 80, liposome, polysaccharide (such as lipopolysaccharide, zymosan), propolis and so on.
  • the materials are mixed and used, including but not limited to feeding, injecting, soaking or spraying.
  • the scope of application includes all common shrimp species, especially the artificial breeding of the traits, including but not limited to: Penaeus vannamei under the Chinese worm (commonly known as Penaeus orientalis).
  • Penaeus penicillatus, Penaeus merguiensis, Penulas merguiensis, Penaeus semisulcatus, and Penaeus semisulcatus commonly known as grasshopper, Penaeus foot woi ow
  • Penaeus japonicus commonly known as Penaeus japonicus
  • a shrimp feed is provided, the main of which is The active ingredient is a genetically engineered protein P40, or a host cell producing the genetically engineered protein P40, or a plasmid including a genetically engineered protein P40 fusion gene, or a DNA fragment including a genetically engineered protein P40 fusion gene, or two or more of them
  • the method for preparing the shrimp feed of the present invention may be carried out by a method well known to those skilled in the art. E.g., including but not limited to the addition of the raw material feed for shrimp in a conventional gene engineering P40 proteins of the present invention is prepared as shrimp feed.
  • the genetically engineered protein of the invention and the commercially available conventional P40 mixed with shrimp feed is prepared as shrimp feed.
  • an antiserum which is prepared using the genetically engineered protein P40 as an antigen.
  • the purified genetically engineered protein P40 can be used as an antigen and an adjuvant to directly immunize an animal to prepare an antiserum.
  • the preparation of an antiserum can be used for preparing an antiserum, including but not limited to rabbits, rats, mice, and horses. The method is familiar to those in the field.
  • the antiserum prepared by the method of the present invention has the following advantages: It is more specific than the polyclonal antibody and does not produce a false positive result because it is prepared by using the purified genetically engineered protein P40 as an antigen.
  • a method of preparing rabbit antiserum wherein the antiserum prepared by the method has a neutralizing effect on WSSV of 100%.
  • a kit for detecting white spot syndrome of shrimp is provided, characterized in that the test kit comprises the anti-blood according to the present invention.
  • the invention applies the immunocolloidal gold chromatography technology to the detection of WSSV, and the prepared colloidal gold detection test paper can quickly and accurately diagnose the sample in the field.
  • the rapid diagnostic test strip is a new in vitro diagnostic technology developed on the basis of monoclonal antibody technology, colloidal gold immunochromatography and new material technology since the 1990s. It has developed rapidly in recent years and has been widely used in biomedical fields, especially in medical tests.
  • the technique mainly fixes a specific antigen or antibody on a nitrocellulose (NC) membrane in a strip shape, and the colloidal gold labeling reagent is adsorbed on the binding pad, and when the sample to be tested is added to the sample pad at one end of the test strip, Moving forward by capillary action, dissolving the colloidal gold labeling reagent on the binding pad and reacting with each other, and then moving to the region of the immobilized antigen or antibody, the complex of the analyte and the gold standard reagent is specifically combined with it. Interception, gathered on the test strip, and visually colored results were obtained with visually detectable colloidal gold markers.
  • NC nitrocellulose
  • a test kit which can be used for the detection of shrimp WSSV, the main active ingredient of which is the antiserum of the genetically engineered protein P40.
  • the antiserum of the present invention can be used for preparing all WSSV detection kits based on the principle of antigen-antibody reaction, including an ELISA test kit and a colloidal gold detection kit.
  • the preparation methods of these kits are familiar to those skilled in the art, and the advantages of the preparation of the serum of the present invention are as follows: (1) Compared with the polyclonal antibody, it is highly specific and does not produce false positive results because it It was prepared by using the purified genetically engineered protein P40 as an antigen.
  • a colloidal gold detection kit is provided, which kit can be used for the detection of shrimp WSSV, the main active ingredient antiserum of which is coated on a solid matrix material.
  • the antibody concentration and the size of the colloidal gold particles are adjusted according to actual needs, and the size, shape, material, and assembly method of the test strip of the present invention can be adjusted. Familiar with those skilled in the art.
  • the colloidal gold test strips of the present invention contain two key components, a gold standard WSSV antibody and an anti-WSSV antibody.
  • a method for rapidly detecting a white spot syndrome virus (WSSV) colloidal gold test strip which can be used for rapidly detecting WSSV in a sample, and the detection method has a reaction Fast, easy to operate, easy to observe results, labeled diagnostic reagent strips can be stored for a long time, ready to use, and reagents and sample usage is very small, no equipment is needed, simplifying the cumbersome routine operation process, and reducing Due to the error caused by the operation, it is especially suitable for a large number of base units.
  • WSSV white spot syndrome virus
  • Example 1 Preparation of WSSV virus genome: The lung, stomach and heart of shrimp were taken on ice, and 10 volumes of TN buffer (50 mmol/L Tris-HCl, 0.4 mmol/L NaCl, pH 7.4) was added.
  • TN buffer 50 mmol/L Tris-HCl, 0.4 mmol/L NaCl, pH 7.4
  • homogenize in an ice bath centrifuge at 8000 r /min for 5 min, take the supernatant, add proteinase K (100) Ltg / ml), DS (50 mmol / L KCl; 10 mmol / L Tris-Cl PH8.3 Gelatin 0.1mg/ml; NP-40, 0.45%; T ween 20, 0.45%), boiled in boiling water for about 15min, immediately ice bath for 5min, centrifuged at 12,000r/min for 10min, and take the supernatant as template DNA. Store at the remaining 4 °C.
  • PCR oligonucleotide amplification primers were designed based on the published WSSV sequences in GenBank.
  • Vpl9 upstream primer gga tec atggccaccacgactaac ac (lower line is BmaH I cleavage site), downstream primer aa ttc ctg cct cct ctt ggt gt (downline is EcoR I drunken site),
  • vp28 upstream primer aga gaa Ttc atg gat ctt tct ttc ac (the lower line is the EcoR I alcohol cleavage site), and the downstream primer cac aag ctt ta etc ggt etc agtgc (the lower line is the Hindlll alcohol cleavage site 'point).
  • the target gene vp28 and vpl9 were amplified by conventional PCR method, and the PCR product was detected by gel electrophoresis to determine whether its size was as expected. Recovering the PCR product and cloning it into the T vector, and transforming the large intestine rod Bacterial ( . JM109, picking up colonies and quickly extracting the scutellum for restriction enzyme digestion, the positive clones were named pUCm-T19 and pUCm-T28 for gene proliferation and preservation.
  • Example 3 vp28+v l9 fusion gene Preparation of the recombinant pUCm-T19 and pUCm-T28 containing the V p28 and vpl9 genes were digested with EcoK I (37 ° C, 4 hr), 1.0% agarose electrophoresis, and the pCCm-T19 was digested by the gel. The fragment (about 450 bp) and the large fragment after pUCm-T28 digestion (about 3300 bp), the two fragments of the cut were separately recovered by a gel purification kit (Omega product), and the large fragment of pUCm-T28 was dephosphorylated.
  • the ligation product 30 ⁇ 1 was transformed into E. coli competent JM109, single colony was identified, and the recombinant plasmid pUCm-T (19+28) DNA was taken to determine the nucleotide sequence.
  • the sequencing primer is the M13 promoter primer, and the sequence assay results verify the vp28 and vpl9 enzymes. The ligation result is correct.
  • Example 4 Construction of the genetically engineered strain expressing the fusion protein (VP28+VP19) ndUi and ⁇ -fiber HI were double-digested with pUCm-T (19+28) and Expression vector pET-226(+), fully digested, 1.0% agarose gel electrophoresis, tapping recovery The target band, back to the j
  • the construction process of the recombinant plasmid is shown in Figure 2.
  • Ligation system T4 DNA ligase buffer 5 ⁇ 1, pUCm-T (19+28) small fragment DNA 25 1, pET-226(+)DNA 5 ⁇ 1, ⁇ 4 DNA ligase 4/ ⁇ 1, sterile double distilled water 11 ⁇ 1 ), connected at 16 ° C for 15 hr.
  • the ligation product was transformed into E. coli BL21 (DE3) competent cells, and the recombinants were identified by Hind III and a HI double restriction enzymes (reported as BL21 (DE3) -pET-22b(+)-vp(19+ 28)) Recombinant, the result of enzyme digestion is shown in Figure 3.
  • Example 5 Expression of genetically engineered fusion protein
  • the glycerol-preserved strain BL21(DE3)-pET-22b(+)-p40 was streaked on an LB plate supplemented with ampicillin, and the culture was over the next day; Colonies were inoculated into fresh 50 ml LB rich liquid medium supplemented with ampicillin, shaken at 230 rpm, 37 ° C for 8-10 hr, and stored at 4 ° C overnight.
  • Example 6 Purification of genetically engineered fusion protein
  • the fermented cells frozen at - 20 °C were repeatedly frozen and thawed several times, then disrupted by ultrasonication, centrifuged at 18,000 rpm for 20 min, and then the supernatant was subjected to Ni column and purified by Ni column.
  • the protein is further dialyzed, concentrated, and finally concentrated to about 380 ⁇ l/ ⁇ 1 (protein/saline), which is used as an antigen to immunize the rabbit.
  • the results of SDS-PAGE electrophoresis for a small amount of sample were as shown in Figure 3.
  • Example 7 Preparation of Shrimp WSSV-Specific Antibody
  • the prepared antigen was about 200 g, and after mixing with an equal volume of complete Freund's adjuvant, the rabbit was subjected to lymphatic injection. Two weeks later, the immune response was boosted with 200ittg of purified antigen. After a week, I took the blood and took the blood. The blood was placed at 37 ° C for 30 min and then placed in a 4 ° C water tank overnight. The next day, after 8 minutes of high-speed centrifugation at 8,5000 rpm, serum was collected. Add 0.02% sodium azide to the serum to prevent corrosion, mix and store at -70 °C, before use. C inactivated for 30 min.
  • Example 8 Preparation of colloidal gold test strips (1) Preparation of colloidal gold particles of 20 ⁇ 30 nm: The configuration was carried out by using the four-nano reduction method of citric acid. For the method, see [Shen care, etc., modern immunological experimental technology, Wuhan: Hubei Science and Technology Press, 1998]. Heat 1000ml of deionized water to boil, add 4ml gold chloride 2.5ml and 1% citric acid three while stirring. Sodium 15ml, heated and stirred until the solution turns into wine red, continue to boil to a suitable concentration, and after cooling, adjust the pH to 7.2-7.5 with 0.2 mol/L potassium carbonate. Filter with a 0.22 ⁇ m filter sterile syringe and store the filtrate at 4 °C.
  • NC antibody solid phase nitrocellulose membrane
  • the antibody of WSSV membrane protein was diluted to 3 mg/ml with a solid phase solution.
  • the goat anti-rabbit antibody was diluted to 1 mg/ml with a solid phase solution.
  • 4 & 2 antibodies were taken 1 ⁇ l and spotted on a nitrocellulose membrane.
  • the solid phase antibody nitrocellulose membrane is placed in a blocking solution, Incubate for 1 hour in a 37 °C water bath.
  • the solid phase antibody nitrocellulose membrane was taken out and washed in the washing solution twice, air-dried at room temperature, and stored at 4 to 8 ° C for use.
  • the assembly of the test strip is as shown in Fig. 4.
  • the water-absorbing glass fiber, the gold-labeled antibody conjugate-glassy cellulose film, and the water-absorbing filter paper are adhered to the inside of the white plastic plate in order, and overlapped with each other by 2 mm.
  • WSSV antibody solid phase oleic acid cellulose membrane and anti-WSSV antibody solid phase nitrocellulose membrane strip fixed with tape, cut into 5 m wide strips with a paper cutter, and stored at 4 ° C for use.
  • test samples Take the prawns to be inspected, peel off the shrimp shells, take about 1 gram of shrimp body and mash them, add a small amount of pure water to mix, and use healthy against the bad.
  • Example 10 Neutralization of WSSV by fusion membrane anti-serum protein preparation and use method The prepared antiserum and oil emulsion were mixed at a ratio of 1:1, and then mixed with the feed to feed the prawn, and the dosage was 100 ml/ Kg shrimp, simultaneous WSSV virus attack, set virus attack control and emulsifier control, each group of 50 insects, while ⁇ ⁇ a repeat, 3 days after the day to calculate the number of deaths, until the 20th, to calculate the control effect. Feeding temperature 20-26 ° C. Control effect: As shown in Table 2 below, the test results showed that antiserum drugs have a good control effect on WSSV. Table 2
  • Example 11 Engineering Fermentation Strains of Genetically Engineered Strains Genetically Engineered Strains E. coliBL21 (DE3)-pET-22b(+)-p40 Fermentation Initial Culture Medium: Fermentation medium 14L was prepared according to the following formula, adjusted to pH 7.0, 121 The fermenter was sterilized in place at °C for 20 minutes.
  • Feeding medium 4L was prepared according to the following formula, 121 ° C, 20 minutes, before being used in a sterilized feeding bottle and adding ampicillin to a final concentration of 200 mg / L.
  • Process record Fermentation medium was sterilized at 121 °C for 20 minutes in the fermenter, cooled and stabilized at 36.5 °C, inoculated with 3% LB seeds, and added with ampicillin.
  • Penicillin to a final concentration of 200 mg / L and pre-sterilized MgS0 4 solution.
  • the initial mixing speed is 300RPM, observe the change of dissolved oxygen, ensure that the DO value is above 30%, and increase the stirring speed when the DO drops below 30%.
  • the DO increased rapidly under the condition of constant oxygen supply, indicating that the nutrients in the initial medium had been exhausted, the stirring speed was increased to a predetermined maximum speed of 500 RPM, and the feed medium was started to flow.
  • the supplementary mode of the feed medium is periodic automatic feeding. Observe the change of DO value after feeding, adjust the feeding speed, and ensure the DO value is maintained at 30%-40%.
  • Ni 2+ column chromatography sample processing 30 g of wet cells obtained by centrifugation of fermenting cell fermentation broth, resuspended in 60 ml of 1 X Binding Buffer (0.5 M NaCl + 5 mM imidazole + 20 mM Tris. HC1 pH 7.9), ultrasonically disrupted 3 x 30 minutes (effective break time is 30 minutes), the broken protein suspension was centrifuged at 15000 RPM for 30 minutes, the supernatant was taken, and filtered with 0.45 ⁇ filter, using 1 x Binding Buffer Capacitance to 100 ml Ni 2+ column chromatography samples.
  • 1 X Binding Buffer 0.5 M NaCl + 5 mM imidazole + 20 mM Tris. HC1 pH 7.9
  • Ni 2+ column Pretreatment of Ni 2+ column
  • the bed was cleaned with 10 beds of 1 X Binding Buffer, 20 beds of sterile water; 10 beds of 1 X Charge Buffer ( 5 mM NiS0 4 ), combined with Ni 2+ ; 10 beds 1 ⁇ Binding Buffer After balancing the bed, spare (1 bed is the volume of the medium in the column:).
  • Column chromatography The concentrated sample was a peristaltic pump in a cyclic manner by Ni 2+ column, so there are 6 X His protein samples sufficiently mobile phase 2+ and Ni 2+ column combination of Ni, Ni 2+ from the column after an overnight The outlet collects samples that are not bound to Ni 2+ (through the leak).
  • the column was cleaned using a 50 bed 1 X Binding Buffer; 75 beds of 60 mM imidazole; 75 beds of 100 mM imidazole; a 30 bed 150 mM imidazole gradient to wash a small sample remaining in the column and a non-target protein bound to the Ni 2+ column.
  • the distribution of the target protein in the eluate was examined by SDS-PAGE.
  • Ni 2+ column was eluted with 1 X Elute Buffer to wash all the proteins in the column; then the Ni 2+ on the column was washed away with 1 X Strip Buffer (100 mM EDTA + 0.5 M NaCl + 20 mM Tris ⁇ HC1 pH 7.9 ) ion.
  • 1 X Strip Buffer 100 mM EDTA + 0.5 M NaCl + 20 mM Tris ⁇ HC1 pH 7.9
  • Example 13 Determination of ⁇ 40 content in ⁇ >40 purified samples The l 1 ml Ni 2+ column was eluted, the collected sample was loaded into a treated dialysis bag, and PBS was added to a final volume of 60 ml. The PEG was concentrated to dryness and PEG was concentrated after addition of PBS. The PEG was concentrated and concentrated until the total volume of force p into the PBS reached 300 ml.
  • the concentrated dialysis bag was rinsed with ddH 2 0, placed in a 1000 ml PBS beaker, and allowed to stand overnight. 50 ml of PBS was added to the well-transparent dialysis bag, and concentrated to dryness with PEG. The protein in the dialysis bag was rinsed out by adding 1.6 ml of PBS, and the above operations were carried out at 4 °C. The sample before dialysis treatment was quantified by Bradford method.
  • WSSV suspension for injection of test materials.
  • WSSV-infected W. serrata frozen at -20 °C was placed on ice to remove carapace, appendage, hepatopancreas, and head chest (sputum, liver, heart, etc.) Organs), cut into small pieces with surgical scissors, placed in a glass homogenizer, a total of 10, and added PBS 1:5 (W / V). Homogenize in an ice bath with a glass homogenizer. The homogenate was centrifuged at 12000 rpm for 20 min at 4 °C.
  • the supernatant was sterilized by filtration through a 0.45 ⁇ m filter, and 1.5 ml of EP tube was dispensed and stored at -20 °C until use. A total of 40 ml of WSSV virus suspension was obtained.
  • 100 ⁇ ⁇ / head WSSV suspension back to the original crayfish, infected at 22-24 ° C, 72 hours mortality rate reached 35.4%, 96 hours mortality rate of 89.6%, 108 hours of mortality 100%.
  • Procambarus clarkii with an average body length of 8cm, purchased from the bazaar, 10 bowls per pot in the plastic room (50cm X 35cm X 15cm), and no healthy shrimps that continue to die for two weeks.
  • the normal prawn bait was fed, and two groups were treated, and each group was treated with 10 shrimps.
  • a set of 10 shrimps was injected three times with PBS 100 ⁇ l/head, and the control group was fed normal bait. Observe the number of dead shrimps processed per group per day.
  • Example 15 Application of genetically engineered fusion envelope protein in WSSV control - Procambarus clarkii was attacked by oral genetic engineering protein P 40 against WSSV injection
  • Procambarus clarkii with an average body length of 8cm, purchased from the bazaar, and 10 bowls per pot in the built-in (50cm X 35cm X 15cm) plastic, cultured for two weeks without healthy shrimp that continue to die , what to feed the prawn.
  • Micromp bait No. 0
  • E. CO/ BL21 (DE3 )-pET-22b (+) Prepared for the preparation of the insects.
  • Co/ BL21(DE3)-pET-22b(+) was fermented by shake flask and the cells were collected. 100 g of wet cells were resuspended in 600 ml of PBS, repeatedly frozen and thawed, sonicated, centrifuged, and the supernatant was taken.
  • the wet weight per gram of the worm is the co-z'BL21(DE3)-pET-22b(+) cell disrupted supernatant. After drying in the shade, store at -20 °C for later use.
  • the P40 package was inactivated by the bait injection group, and the survival rate was 43.33% compared with the BL21 (DE3) coated bait injection group and the normal bait injection group.
  • the results showed that the original genetic engineering protein p40 of Oryza sativa had a good protective effect on WSSV injection infection.
  • Shrimp bait No. 0
  • the practical method is to use the P40 engineering protein-coated slanting material and the BL 21 DE 3 bacterial cell disrupting solution to coat the prawn coated with the prawn bait in the laboratory, and the normal prawn bait is sooner or later. Feeding, feeding for 2 days, on the third day, feeding the sputum and head and chest tissue blocks of W. serrata infected with WSSV. After that, the other two groups were fed P40 engineering protein coated bait, BL21 (DE3) bacterial cell disruption supernatant coated prawn bait, normal prawn bait, total 3 groups of treatment, the first 2 groups of 20 heads / each group, the first 3 groups of 7 heads.
  • the P40 package was fed to the infected group with a lower mortality rate.
  • the survival rate of the infected group was 70%, compared with the normal bait feeding survival group. The rate is 80%.
  • the results showed that P. vannamei was well-protected by oral infection of WSSV.
  • Normal bait feeding P40 package is fed by bait BL21 (DE3) package is dyed bait feeding
  • Rate of death rate rate (%) rate (%)
  • Shrimp bait No. 0
  • the wet weight per gram of prawn oblique material is .co/z'BL21 (DE3)-pET-22b(+) bacterial cell disrupted supernatant. After drying in the shade, store at -20 °C for later use.
  • the actual method is to use the P40 engineering protein-coated bait, BL21 (DE3) cell suspension, and the prawn bait for the healthy prawn cultured in the squid bait.
  • the normal prawn bait is fed sooner or later.
  • the animals were fed continuously for 2 days.
  • the bracts and head and chest tissue blocks of the K. pneumoniae infected with WSSV were fed.
  • the P40 engineering protein coated materials, the BL21 (DE3) bacterial cell disruption supernatant coated prawn oblique material, and the normal prawn were treated separately, and the first two groups were treated with 20 groups/group. The latter group of 7 heads.
  • Depository address (including postal code and country name)
  • This column has a cup filling port

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Abstract

L'invention concerne une protéine P40 obtenue par génie génétique, une séquence nucléotidique et une séquence d'acides aminés codant pour celle-ci. L'invention concerne également un vaccin utilisé contre le virus associé au syndrome du point blanc, contenant ladite protéine P40 obtenue par génie génétique. L'invention concerne, de plus, un kit de diagnostic associé à ladite protéine P40 obtenue par génie génétique, permettant de détecter le virus associé au syndrome du point blanc chez les crevettes.
PCT/CN2004/000776 2004-07-09 2004-07-09 Proteine p40 obtenue par genie genetique et utilisation Ceased WO2006005222A1 (fr)

Priority Applications (2)

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CN200480043768.1A CN101023095B (zh) 2004-07-09 2004-07-09 基因工程蛋白质p40及其应用
PCT/CN2004/000776 WO2006005222A1 (fr) 2004-07-09 2004-07-09 Proteine p40 obtenue par genie genetique et utilisation

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114805597A (zh) * 2022-03-07 2022-07-29 河北农业大学 一种重组TetR蛋白及其制备方法和试剂盒以及牛奶中四环素类抗生素残留的检测方法

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Publication number Priority date Publication date Assignee Title
WO2001009340A1 (fr) * 1999-08-03 2001-02-08 Akzo Nobel N.V. Proteines issues du virus de la maladie des points blancs (ichthyopthirius) et ses utilisations
WO2002022664A2 (fr) * 2000-09-15 2002-03-21 Akzo Nobel N.V. Proteines antigeniques du virus de la maladie du point blanc (wssv) de la crevette et utilisations de ces proteines
WO2003000900A1 (fr) * 2001-06-22 2003-01-03 Akzo Nobel N.V. Vaccin contre le virus de la maladie du point blanc
WO2003017780A1 (fr) * 2001-08-27 2003-03-06 Advanced Bionutrition Corporation Apport de produits de lutte contre les maladies en aquaculture et agriculture au moyen d'aliments nutritionnels contenant des proteines bioactives produites par des virus
KR20040026426A (ko) * 2002-09-24 2004-03-31 주식회사 단바이오텍 흰반점바이러스 감염의 예방 및 치료를 위한 수용성 항체단백질 및 이를 포함한 알, 및 이들의 제조방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001009340A1 (fr) * 1999-08-03 2001-02-08 Akzo Nobel N.V. Proteines issues du virus de la maladie des points blancs (ichthyopthirius) et ses utilisations
WO2002022664A2 (fr) * 2000-09-15 2002-03-21 Akzo Nobel N.V. Proteines antigeniques du virus de la maladie du point blanc (wssv) de la crevette et utilisations de ces proteines
WO2003000900A1 (fr) * 2001-06-22 2003-01-03 Akzo Nobel N.V. Vaccin contre le virus de la maladie du point blanc
WO2003017780A1 (fr) * 2001-08-27 2003-03-06 Advanced Bionutrition Corporation Apport de produits de lutte contre les maladies en aquaculture et agriculture au moyen d'aliments nutritionnels contenant des proteines bioactives produites par des virus
KR20040026426A (ko) * 2002-09-24 2004-03-31 주식회사 단바이오텍 흰반점바이러스 감염의 예방 및 치료를 위한 수용성 항체단백질 및 이를 포함한 알, 및 이들의 제조방법

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114805597A (zh) * 2022-03-07 2022-07-29 河北农业大学 一种重组TetR蛋白及其制备方法和试剂盒以及牛奶中四环素类抗生素残留的检测方法

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