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Fusion protein of porcine pseudorabies virus and preparation method, application and vaccine of fusion protein

A porcine pseudorabies virus, fusion protein technology, applied in antiviral immunoglobulins, veterinary vaccines, biochemical equipment and methods, etc., can solve the problems of limited immune efficacy, long production cycle, high cost, and achieve good immunogenicity , low preparation cost, good immune effect

Active Publication Date: 2019-01-04
天康生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although inactivated vaccines are safe, their immune efficacy is limited and they cannot achieve the goal of purifying the disease
There are hidden dangers in the safety of attenuated vaccines, long production cycle and high cost
In addition, porcine pseudorabies virus gene-deleted live vaccines and whole-virus inactivated vaccines grow on adherent cells and require animal-derived proteins, which brings risks to vaccine manufacturing

Method used

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  • Fusion protein of porcine pseudorabies virus and preparation method, application and vaccine of fusion protein
  • Fusion protein of porcine pseudorabies virus and preparation method, application and vaccine of fusion protein

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preparation example Construction

[0049] A method for preparing the above fusion protein, comprising expressing the gene encoding the fusion protein in a mammalian expression system. The preparation method can realize the high-quality expression of the above-mentioned fusion protein in large quantities, and is simple to operate and suitable for large-scale production. The mammalian expression system can be, for example but not limited to, HEK 293-F cells, HEK 293-E cells, HEK 293-T cells, CHO cells or COS cells.

[0050] In a preferred embodiment of the present invention, the gene encoding the fusion protein is expressed in HEK 293-F cell expression system. HEK293 is a stable cell line obtained after transfection of human embryonic kidney cells by adenovirus Ad5. HEK293-F is a derivative cell line of HEK293, which has easy transfection, high expression, natural glycosylation modification, allows correct protein folding and related translation Post-modification and other advantages.

[0051] In some embodimen...

Embodiment 1

[0065] Embodiment 1 encodes the fusion protein sequence of porcine pseudorabies virus

[0066] The genes of the classic strain (SC strain) and the current epidemic strain (JS strain) were selected from Genebank as the research objects. Through comparative analysis, the dominant epitope was selected as the component of the vaccine antigen. According to the codons of HEK 293-F cells partial tropism, and this sequence is carried out codon optimization and modification, obtains the nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2, the agarose gel electrophoresis figure of PCR enrichment gB sequence is as follows figure 1 As shown, the agarose gel electrophoresis image of the PCR-enriched gD sequence is as follows figure 2 shown. The middle of the gB and gD sequences is connected with the sequence shown in SEQ ID NO.3 to achieve the purpose of further improving the broad spectrum of the fusion protein antigen and increasing the expression of the antigen.

Embodiment 2

[0067] The construction of the recombinant vector of embodiment 2 expression fusion protein

[0068] The sequence encoding the fusion protein synthesized above was cloned into the eukaryotic transfer vector pcDNA3.1 by inserting Mlu I and Hind III sites. Use T4 DNA ligase to ligate overnight at 16°C to obtain ligation products, transform Escherichia coli competent DH5α, spread on LB plates containing ampicillin, culture overnight at 37°C, and pick positive colonies on LB plates containing ampicillin Cultivate in culture medium and extract plasmid. The correct recombinant plasmid was obtained through PCR, double enzyme digestion and sequencing verification.

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Abstract

The invention relates to the technical field of biology, in particular to a fusion protein of porcine pseudorabies virus and a preparation method, application and vaccine of the fusion protein. The fusion protein of porcine pseudorabies virus comprises a gB section and a gD section, wherein the gB section is expressed by the nucleotide sequence shown in SEQ ID NO.1; and the gD section is expressedby the nucleotide sequence shown in SEQ ID NO.2. The sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are the sequences obtained through contrast and analysis by selecting genes of classical strains and current epidemic strains as research objects, and codon optimization and modification are preformed on the sequences, so that the broad spectrum of fusion protein antigen is further improved and theantigen expression amount is increased. The invention further provides a preparation method and application of the fusion protein, and the vaccine for preparation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein of porcine pseudorabies virus and its preparation method, application and vaccine. Background technique [0002] Porcine pseudorabies (porcine pseudorabies, PR) is caused by pseudorabies virus (Pseudorabiesb virus, PRV), one of the important infectious diseases that seriously endanger the healthy development of my country's pig industry. The virus belongs to the linear double-stranded DNA virus of the herpesviridae herpesvirinae subfamily, and the G+C content of the viral DNA is relatively high. There are currently 11 known viral glycoproteins, namely essential glycoproteins and non-essential glycoproteins. [0003] Pseudorabies has a wide range of susceptibility. Newborn piglets are mostly fatal infections, similar to other susceptible species, and mostly die of central nervous system diseases. Adult pigs infected with the disease often show respiratory diseases, ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K16/08C12N15/62C12N15/85G01N33/569A61K39/295A61K39/245A61P31/22
CPCA61K39/12A61K2039/552A61K2039/70A61P31/22C07K14/005C07K16/085C07K2319/00C12N15/85C12N2710/16722C12N2710/16734G01N33/56994
Inventor 贺笋李俊辉师小潇张伟潘晓梅石丁锋程兰玲王遵宝豆智华
Owner 天康生物制药有限公司
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