Recombinant baculovirus expressing porcine parvovirus VP2 protein as well as preparation method and application

A technology of recombinant baculovirus and parvovirus, which can be used in the fields of biotechnology and virology to solve problems such as economic losses

Inactive Publication Date: 2015-04-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PPV-related diseases have brought huge economic losses to my country's pig industry. At present, the control of PPV infection is mainly through strengthening feeding management and biosecurity measures, but the infection rate of PPV in pigs is high and the resistance of PPV to the external environment is relatively low. Therefore, it is very important to prevent PPV infection through safe and effective vaccine immunization

Method used

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  • Recombinant baculovirus expressing porcine parvovirus VP2 protein as well as preparation method and application
  • Recombinant baculovirus expressing porcine parvovirus VP2 protein as well as preparation method and application
  • Recombinant baculovirus expressing porcine parvovirus VP2 protein as well as preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Construction of a recombinant baculovirus Ac-PPVP2 expressing artificially modified and synthesized PPV2 immunogenic gene VP2

[0063] (1) Construction of recombinant baculovirus Ac-PPVP2

[0064] 1. Artificial modification and synthesis of PPV main immunogenic gene VP2

[0065] Referring to the VP2 gene sequence of the Chinese strain of porcine parvovirus (GenBank: AY583318), the VP2 gene of PPV was artificially synthesized according to the codon preference. Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded protein is SEQ ID Shown in NO.2.

[0066] 2. Construction of baculovirus transfer vector pFBDPHm3VP2

[0067] Using the pFBDPHmHNM1P10eGFP plasmid (Qian Ping, a recombinant baculovirus expressing the HA-NA-M1 gene of the artificially modified and synthesized influenza A H1N1 virus, patent publication number CN101624580A, patent number ZL200910063217.6) as the backbone, through BamHI and EcoRI enzymes pFBDPHmHNM1P10eGFP was cut, and the large fragme...

Embodiment 2

[0093] Safety and Immunity Efficacy of Recombinant Baculovirus Ac-PPVP2 Subunit Vaccine

[0094] (1), safety test of the invented recombinant baculovirus Ac-PPVP2 subunit vaccine

[0095] 1. Preparation of recombinant baculovirus Ac-PPVP2 subunit vaccine

[0096] The recombinant baculovirus obtained in Example 1 was inoculated with a multiplicity of infection of 3-5pfu / cell (the insect cell High Five of suspension culture TM , purchased from Invitrogen Company) sf9 cells, collected cells and supernatant after 72 hours, processed by a cell disruptor, centrifuged to remove cell debris, inactivated by 7mM binary ethyleneimine (BEI) for 36-48 hours, added an equal amount of sulfur Sodium sulfite was neutralized and emulsified with oil adjuvant to prepare a subunit vaccine, which was stored at 4°C for later use. The exogenous protein PPV VP2 protein in the finally obtained vaccine is 20 micrograms per milliliter (μg / ml), which is used for the safety test.

[0097] 2. Newborn pig...

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Abstract

The invention discloses recombinant baculovirus expressing porcine parvovirus VP2 protein as well as a preparation method and an application. The method comprises the following steps: artificially synthesizing VP2 gene by referring to the VP2 gene sequence of a porcine parvovirus (PPV) isolate; with pFBDPHmHNM1P10eGFP plasmid as a skeleton, connecting the synthesized VP2 gene to the plasmid to obtain a baculovirus transfer vector pFBDPHm3VP2 and then obtain recombinant bacmid rBacmid-PPVP2; and transfecting the bacmid with sf9 cell to obtain recombinant baculovirus Ac-PPVP2. The recombinant baculovirus Ac-PPVP2 efficiently expresses PPV VP2 protein and successfully forms virus-like particles. The protein expressed by the recombinant baculovirus disclosed by the invention is used for preparing a subunit vaccine; and after the subunit vaccine immunizes an animal, the body can be induced to generate a specific immunoreaction, and the porcine body can be fully protected from the attack of strong poison of parvovirus.

Description

technical field [0001] The invention relates to the fields of biotechnology and virology. Specifically relates to a recombinant baculovirus Ac-PPVP2 of porcine parvovirus, also relates to a preparation method of recombinant baculovirus Ac-PPVP2 of porcine parvovirus, and also relates to the preparation of a recombinant baculovirus Ac-PPVP2 subunit vaccine of porcine parvovirus application. The recombinant baculovirus of the invention is used to express the artificially modified porcine parvovirus VP2 gene and assemble into porcine parvovirus-like virus particle VLP, which can be used to prepare a safe and effective porcine parvovirus subunit vaccine. Background technique [0002] In 1996, Mary and Mahnel discovered porcine parvovirus (Porcine parvovirus, PPV) (Mary et al., 1968) during tissue culture of swine fever virus, and then in 1967, Cartwright isolated and identified pigs from infertile and aborted sow disease feed parvovirus and demonstrated its pathogenicity (Cart...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/35C12N15/866A61K48/00A61K39/23A61P31/20
Inventor 钱平李祥敏钱苏红石林陈焕春
Owner HUAZHONG AGRI UNIV
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