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Novel porcine reproductive and respiratory syndrome virus variant GP5 recombinant protein and preparation method and application thereof

A respiratory syndrome and recombinant protein technology, applied in the field of the new porcine reproductive and respiratory syndrome virus variant strain GP5 recombinant protein and its preparation, can solve the problems of cumbersome operation, difficulty in expressing GP5 protein, and repeated trials, etc. Achieve the effect of improving immunogenicity, improving the body's humoral immunity and cellular immunity

Active Publication Date: 2015-10-14
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immunization effect of the subunit vaccine targeting GP5 protein is not satisfactory, the possible reasons are: (1) GP5 protein is a glycosylated envelope protein, which contains highly conserved N-glycosylation sites and highly hydrophobic Therefore, the expression in Escherichia coli and other expression systems has not been ideal, and it is difficult to express the complete GP5 protein. In previous studies, the signal peptide sequence or transmembrane region at the N-terminal of the gene was mostly removed. Or through codon optimization and other means to obtain the expression of GP5 protein, but most of them exist in the form of inclusion bodies, which may affect the biological function of GP5 protein, especially in the later stage, protein renaturation is required to obtain soluble protein, and renaturation is very complicated process, and the renaturation efficiency is often related to the properties of specific proteins, which method to choose also requires trial and error, and the operation is cumbersome; (2) GP5 protein antigen presentation is relatively weak

Method used

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  • Novel porcine reproductive and respiratory syndrome virus variant GP5 recombinant protein and preparation method and application thereof
  • Novel porcine reproductive and respiratory syndrome virus variant GP5 recombinant protein and preparation method and application thereof
  • Novel porcine reproductive and respiratory syndrome virus variant GP5 recombinant protein and preparation method and application thereof

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Effect test

Embodiment 1

[0047] Example 1 Design, expression and purification of GP5 recombinant protein

[0048] 1.1 Cloning of the target gene

[0049] 1.1.1 Design and synthesis of primers

[0050] According to the nucleotide sequence of the American PRRSV mutant strain LX13(2), two pairs of specific primers that can amplify the TAT-ORF5(V) and ORF5(V) genes were designed using Primer 5.0 software, and the upstream and downstream 5 EcoR I and Kpn I restriction sites were introduced into the 'ends respectively, and were synthesized by Bao Biological Engineering (Dalian) Co., Ltd. The lengths of the expected amplified fragments were about 660bp and 618bp respectively. with ddH 2 O was diluted to a final concentration of 20pmol / μL, and stored at -20°C. See Table 1 for specific primer information, as shown in SEQ ID NO: 3-6.

[0051] Table 1 PRRSV TAT-ORF5(V) and ORF5(V) gene amplification primer sequences

[0052]

[0053] Note: The bold part is the TAT sequence, TAT is introduced into the ups...

Embodiment 2

[0114] Example 2 Guinea pig immunization test

[0115] 2.1 Immunization and serum preparation of guinea pigs

[0116] 2.1.1 Pretreatment / immunogen preparation of purified protein TF-TAT-GP5(V) and TF-GP5(V)

[0117] Aspirate the eluate containing the purified protein TF-TAT-GP5(V) and TF-GP5(V) collected from the purification and recovery of the recombinant protein, and transfer them to the buffer-treated dialysis bags respectively (the dialysis bags should be pre-treated). Leave a space to prevent excessive water and buffer outside the bag from entering the bag during dialysis and the bag will burst), clamp the two ends of the dialysis bag with sealing clips, and place them respectively in a large container filled with PBS buffer. In the beaker, put a magnetic bar in the beaker, place the beaker on a heating magnetic stirrer, only turn on the stirring power (not heating), and adjust the stirring speed. The PBS buffer was changed every 12 hours and the dialyzed for 72 hours....

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Abstract

The invention discloses a novel porcine reproductive and respiratory syndrome virus variant GP5 recombinant protein and a preparation method and application thereof. The novel porcine reproductive and respiratory syndrome virus variant GP5 recombinant protein is a soluble complete protein expressing PRRSV GP5 or soluble protein expressing PRRSV GP5 fused with TAT. A pCold 1F DNA is employed for efficient soluble expression of the PRRSV mutant GP5 protein and TAT-GP5 fusion protein, so as to ensure the biological function of the recombinant protein and solve the problem that the existing technology is difficult to express the full GP5 protein; and a guinea pig immunization inoculation test shows that TAT-GP5 fusion protein can significantly improve the level of humoral immunity and cellular immunity of the body.

Description

technical field [0001] The invention relates to a GP5 recombinant protein of porcine reproductive and respiratory syndrome virus variant strain, in particular to a new GP5 recombinant protein of porcine reproductive and respiratory syndrome virus variant strain and its preparation method and application. Background technique [0002] Porcine reproductive and respiratory syndrome is a viral infectious disease of pigs caused by PRRSV, which is characterized by reproductive disorders in pregnant sows, respiratory symptoms and high mortality in piglets. The disease has caused huge economic losses to the pig industry in various countries. . Since the virus was isolated in my country, the infectious disease has been found in at least 22 provinces in the country, and the infectious disease has even been prevalent in some provinces. With the inheritance and variation of PRRSV, especially after 2006, the emergence of HP-PRRSV mutant strains has caused huge economic losses to the pig...

Claims

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Application Information

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IPC IPC(8): C07K14/08C12N15/70A61K38/00A61K39/12A61P31/14
CPCA61K38/00A61K39/00C07K14/005C12N15/70C12N2770/10022
Inventor 黄娟单虎任颖超
Owner QINGDAO AGRI UNIV
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