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Porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as preparation method and application thereof

A technology of recombinant baculovirus and foot-and-mouth disease virus, which is applied in the fields of biotechnology and virology, can solve the problem of high cost and poor quality; although live carrier vaccines such as poxvirus and adenovirus have certain protection, inactivated vaccines cannot provide protection, etc. question

Inactive Publication Date: 2013-05-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to its own reasons, inactivated vaccines also have many defects: 1. There is no cross protection for immunized animals, so an inactivated vaccine cannot provide comprehensive protection; 2. There is a risk of virus escape during the preparation process; 3. Adverse reactions due to immunity with tissue or cellular components
However, these vaccines still have some deficiencies and are not accepted by people, such as subunit vaccines and polypeptide vaccines, which are expensive and have poor immune effects; although vaccines with live vectors such as poxvirus and adenovirus have certain immune effects, due to the carrier Due to the limitation of the virus, people would rather choose the conventional vaccine than the genetically engineered vaccine to prevent foot-and-mouth disease

Method used

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  • Porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as preparation method and application thereof
  • Porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as preparation method and application thereof
  • Porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1: a kind of porcine O-type foot-and-mouth disease virus recombinant baculovirus, its preparation steps are as follows:

[0070] (1) Construction of recombinant baculovirus QP-Ac-FVLP

[0071] 1. Artificial modification and synthesis of immunogenic genes VP0, VP1, VP3 of porcine O-type FMDV

[0072] Referring to the O / HLJOC12 / 03 gene sequence (DQ119643.2) of the FMDV O-type epidemic strain, artificially synthesized VP0 (SEQ ID NO.1), VP1 (SEQ ID NO.2) and VP3 (SEQ ID NO. .3) The sequence of the gene.

[0073] 2. Construction of baculovirus transfer vector pFBDPHmVP013

[0074] 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 CN200910063217.6) as the backbone, digest pFBDPHmHNM1P10eGFP with NcoI and KpnI, and recover The large fragment obtained pFBDPHmHNM1. The VPO gene and pFBDPHmHNM1 were digested with XhoI...

Embodiment 2

[0101] Example 2: Preparation and Application of Recombinant Baculovirus QP-Ac-FVLP Subunit Vaccine

[0102] 1. Preparation of recombinant baculovirus QP-Ac-FVLP subunit vaccine

[0103] In order to evaluate the immune effect of the recombinant baculovirus QP-Ac-FVLP subunit vaccine in preventing type O foot-and-mouth disease virus infection, the obtained recombinant baculovirus QP-Ac-FVLP was inoculated into suspension culture at a multiplicity of infection of 3-5 pfu / cell Insect Cell High Five TM (purchased from Invitrogen Company), the virus liquid was collected after 72 hours, and the cells were collected by centrifugation at 2000rpm for 10 minutes and counted with a cell counting plate, and the number of cells was adjusted to 2×10 with PBS. 7 / ml and 2×10 6 Two gradients per ml were emulsified with an equal volume of complete Freund's adjuvant (Sigma) after repeated freezing and thawing for 3 times, and temporarily stored at 4°C.

[0104] 2. Immune efficacy test in mic...

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Abstract

The invention discloses porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as a preparation method and application thereof. Sequences of VP0, VP1 and VP3 genes are artificially synthesized by referring to an FMDV (Foot And Mouth Disease Virus) O-type epidemic strain gene sequence; the VP0, VP1 and VP3 genes are connected to pFBDPHmHNM1P10eGFP plasmid by adopting the plasmid as a framework vector, so that a baculovirus transfer vector pFBDPHmVP013 is obtained. The baculovirus transfer vector pFBDPHmVP013 is mixed with DH10Bac escherichia coli competent cells, and the positive bacterial colony is selected to obtain a recombinant shuttle vector Bacmid; the shuttle vetcor Bacmid is transferred with a sf9 cell, and the recombinant baculovirus QP-Ac-FVLP is obtained by collecting the cell supernatant. The recombinant baculovirus can be used for efficiently expressing FMDVVP0, Vp1 and Vp3 proteins and forming virus-like particles. And the virus-like particles are used for preparing subunit vaccine, so that the organism is induced to generate specific immunity response after the mouse is immunized.

Description

technical field [0001] The invention relates to the fields of biotechnology and virology. More specifically, the present invention relates to a recombinant baculovirus QP-Ac-FVLP of porcine O-type foot-and-mouth disease virus, a method for preparing the recombinant baculovirus QP-Ac-FVLP of porcine O-type foot-and-mouth disease virus, and a porcine Application of recombinant baculovirus QP-Ac-FVLP of O-type foot-and-mouth disease virus in preparing a subunit vaccine for preventing porcine O-type foot-and-mouth disease virus infection. The recombinant virus of the invention is used to express artificially modified O-type VPO, VP1 and VP3 genes of porcine foot-and-mouth disease virus and assemble them into porcine foot-and-mouth disease virus-like virus particle VLP, which can be used to prepare O-type foot-and-mouth disease virus safe vaccine. Background technique [0002] Foot and Mouth Disease (FMD) is an acute, febrile, highly contact zoonotic disease caused by Foot and M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/42C12N15/866A61K48/00A61P31/14
Inventor 李祥敏钱平杨应立陈焕春
Owner HUAZHONG AGRI UNIV
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