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Preparation and application of porcine parvovirus-like particles displaying B cell epitope and T cell epitope of South African foot-and-mouth disease virus type 2

A foot-and-mouth disease virus and parvovirus technology, applied in the field of bioengineering, can solve problems such as the loss of pig farming

Inactive Publication Date: 2020-10-16
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

PPV has strong resistance, especially pH and temperature insensitivity, causing great losses to my country's pig industry

Method used

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  • Preparation and application of porcine parvovirus-like particles displaying B cell epitope and T cell epitope of South African foot-and-mouth disease virus type 2
  • Preparation and application of porcine parvovirus-like particles displaying B cell epitope and T cell epitope of South African foot-and-mouth disease virus type 2
  • Preparation and application of porcine parvovirus-like particles displaying B cell epitope and T cell epitope of South African foot-and-mouth disease virus type 2

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Experimental program
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Embodiment 1

[0066] recombinant baculovirus rBacNT 1 L 2 The preparation method of B comprises the following steps:

[0067] (1)NT 1 L 2 Amplification of B gene and construction of recombinant donor plasmid

[0068] According to the optimized synthesis of the plasmid GS1923OP containing the chimeric sequence, primers P1 / P2 were designed using the bioinformatics software Primer 5.0, and the chimeric gene NT was amplified. 1 L 2 B; Recovery and purification of the target band NT 1 L 2 B, combine the target fragment with pFastBac TM Dual vectors were digested with BamH I and Pst I respectively, recovered and cleaned, ligated, chemically transformed into DH5α competent cells, positive clones were screened, and the recombinant donor plasmid pFastNT was extracted 1 L 2 B, and carried out enzyme digestion, sequencing identification;

[0069] Table 1 Primer sequences and restriction sites

[0070] Table 1 Primer sequence and enzyme cutting site

[0071]

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Abstract

According to simulation results of a computer software, SAT2 FMDV B cell epitope (VYTKAAAAIRGDRAALAAKYADTNHTLPPTFNFGYVTVDK) is embedded in a Loop2 region of porcine parvovirus PPV VP2 protein, and a Tcell epitope (NVQEGRRKHTDVAFLLDRST) is embedded in an N end. A chimeric gene GS1923OP is synthesized in an optimized mode according to insect cell tropism and cloned onto a vector pFastBacTMDual, a competent cell DH10Bac is transformed to obtain recombinant bacmid rBacmidNT1L2B to transfect a sf9 cell, recombinant baculovirus rBacNT1L2B is harvested and used to infect a HF cell, an indirect immunofluorescence IFA needle is used to detect a chimeric epitope and VP2 protein, and specific fluorescence is achieved. A We stern-blot needle is used to detect the B cell epitope, the T cell epitope and the VP2 protein, and a specific band appears at 67KDa. Transmission electron microscope (TEM) observation shows that the recombinant protein NT1L2B can self-assemble into virus-like particles. The preparation and application can be applied to prepare a reserve vaccine for South Africa foot-and-mouth disease type 2, and can achieve the purpose of simultaneously preventing South Africa foot-and-mouth disease type 2 and Porcine parvovirus infection on the basis.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to the preparation and application of porcine parvovirus-like particles displaying B cell epitopes and T cell epitopes of South African type 2 foot-and-mouth disease virus. Background technique [0002] Foot and Mouth Disease Virus (FMDV) is a member of the genus Aphthovirus in the family Picornaviridae, which can cause natural foci diseases of cattle, sheep and other artiodactyl animals. The FMDV nucleic acid is a single-strand positive-strand RNA with a total length of 8300 nucleotides, encoding a polypeptide, which produces different structural proteins (VP4, VP3, VP2 and VP1) and some non-structural proteins (2A, 2B, VP1) under the action of enzymes. 2C, 3A, 3B, 3C, 3D). FMD can cause huge social impact and economic loss, and is one of the zoonotic diseases that OIE legally reports to it. At present, my country's FMD research is mainly: O, A type, and the research of SA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/866C07K19/00A61K39/295A61K39/125A61K39/135A61P31/14A61P31/20C12R1/93
CPCA61K39/12A61K2039/5258A61K2039/552A61K2039/70A61P31/14A61P31/20C07K14/005C07K2319/00C12N7/00C12N15/86C12N2710/14043C12N2710/14051C12N2750/14034C12N2750/14322C12N2750/14323C12N2770/32122C12N2770/32134C12N2800/105C12N2800/22
Inventor 汪洋马维民丁耀忠李茜代军飞马炳侯谦李苗苗刘永生张杰张永光
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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