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Assembling method and application of PPV (porcine parvovirus)-like particles with chimeric SAT2 FMD (Southern African Territories 2 foot and mouth disease) virus epitopes

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

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

The detection rate in pig herds is as high as 85%, which has caused huge losses to my country's pig industry

Method used

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  • Assembling method and application of PPV (porcine parvovirus)-like particles with chimeric SAT2 FMD (Southern African Territories 2 foot and mouth disease) virus epitopes
  • Assembling method and application of PPV (porcine parvovirus)-like particles with chimeric SAT2 FMD (Southern African Territories 2 foot and mouth disease) virus epitopes
  • Assembling method and application of PPV (porcine parvovirus)-like particles with chimeric SAT2 FMD (Southern African Territories 2 foot and mouth disease) virus epitopes

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Experimental program
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Embodiment 1

[0065] Recombinant baculovirus rBacNT 1 L 2 B 4 The preparation method of B, comprises the following steps:

[0066] (1)NT 1 L 2 B 4 Amplification of B gene and construction of recombinant donor plasmid

[0067] According to the optimized synthesis of the plasmid GS1925OP containing the chimeric sequence, primers P1 / P2 were designed using the bioinformatics software Primer 5.0, and the chimeric gene NT was obtained by amplification 1 L 2 B 4 B; recovery of purified target band NT 1 L 2 B 4 B, the target fragment and pFastBac TM The Dual vector was digested with BamH I and Pst I respectively, recovered and cleaned, then ligated, chemically transformed into DH5α competent cells, screened for positive clones, and the recombinant donor plasmid pFastNT was extracted 1 L 2 B 4 B, and carry out enzyme digestion and sequencing identification;

[0068] Table 1 Primer sequences and restriction sites

[0069]

[0070] Note: Underlined are protected bases (black) and re...

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Abstract

According to simulation results of computer software, an SAT2 FMDV B cell epitope (VYTKAAAAIRGDRAALAAKYADTNHTLPPTFNFGYVTVDK) is respectively embedded in a Loop 2 region and a Loop 4 region on the surface of a VP2 protein of PPV and a T cell epitope (NVQEGRRKHTDVAFLLDRST) is embedded in the N end. A chimeric gene GS1925OP is optimized and synthesized according to insect cell tropism and is cloned into a vector pFastBacTMDual to transform escherichia coli DH10Bac to obtain a recombinant bacmid rBacmidNT1L2B4B, and sf9 cells are transfected to obtain a recombinant baculovirus rBacNT1L2B4B, and the recombinant baculovirus rBacNT1L2B4B is used for infecting HF cells. The chimeric epitope and VP2 protein are detected by indirect immunofluorescence IFA and have specific fluorescence. Western Blotdetects the B and T cell epitopes and the VP2 protein, and a specific band appears at 70KDa. The recombinant protein NT1L2B4B can be self-assembled into VLPs (virus-like particles) under observationby a TEM (transmission electron microscope). The invention can be applied to preparation of a reserve vaccine for SAT2 FMD, and can achieve the purpose of simultaneously preventing SAT2 FMD and PPV onthe basis.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to an assembly method and application of a chimeric South African type 2 foot-and-mouth disease virus antigenic epitope porcine parvovirus-like particle. Background technique [0002] At present, SAT2FMD has not occurred in my country, and the research on foot-and-mouth disease in China is mainly: O, A, C and Asia1 types, and the research on SATFMD is in a blank state. [0003] Porcine Parvovirus (PPV) can cause abortion, infertility, stillbirth, mummified fetus and weak litter in primiparous sows and seronegative multiparous sows. The detection rate in pig herds is as high as 85%, causing huge losses to my country's pig industry. Its genomic DNA of about 5000bp encodes three structural proteins, VP1, VP2, and VP3. The VP2 protein has 4 Loop loops on the surface, which can carry exogenous epitopes and assemble into chimeric virus-like particles (VLPs). Induce the body to si...

Claims

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Application Information

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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 李茜丁耀忠马维民代军飞汪洋侯谦李苗苗马炳刘永生张永光
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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