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Thermal-stability foot-and-mouth disease O type recombinant virus as well as preparation method and application thereof

A technology for recombinant virus and foot-and-mouth disease, applied in biochemical equipment and methods, antiviral agents, virus/bacteriophage, etc., can solve the problems of affecting efficiency, low rescue efficiency of recombinant rabies virus, and cell pathology affecting rescue efficiency, etc., to achieve good genetics Stability, improvement of thermal stability, effect of thermal stability improvement

Inactive Publication Date: 2019-05-28
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But because the reverse genetic operation may affect the efficiency, for example, the rescue efficiency of the successfully rescued recombinant rabies virus is low, at 10 7 Only about 1 virion is obtained per transfected cell
In addition, the efficient replication of recombinant virus on animal cells will also affect its rescue efficiency.
At the same time, reverse mutations in the post-passage process of the virus after reverse genetic manipulation cannot maintain beneficial biological characteristics for a long time.
At the same time, the reverse genetic manipulation technology is used in the prior art to modify the biological characteristics such as the virulence of the virus or the yield of the virus. At present, there is no rescue plan for the heat-stable FMD O-marked virus in the prior art.

Method used

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

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Experimental program
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preparation example Construction

[0041] In the present invention, the O / rV virus strain preparation method preferably includes the following steps:

[0042] The half-length plasmids pOMQ-Z123 and pSK-Z4 of FMDV OZK / 93-08 containing the VP1 gene of Myanmar 98 lineage were digested with SpeⅠ and BglⅡ enzymes respectively, and the 5400bp target gene obtained was ligated with linearized pSK-Z4 to obtain a complete Long plasmid pQED, the full-length plasmid pQED is transfected into BSR / T7 cells, cultivated, and the cells are collected and frozen and thawed repeatedly, and the obtained virus is an O / rV virus strain. The pOMQ-Z123 half-length plasmid and the pSK-Z4 half-length plasmid are disclosed in a patent with the publication number CN102614507A and the title of the invention is a type O foot-and-mouth disease molecular marker vaccine and its preparation method.

[0043] In the present invention, the amino acid mutation schematic diagram of the heat-stable foot-and-mouth disease O-type recombinant virus is show...

Embodiment 1

[0073] Construction method of pQEC full-length plasmid

[0074] On the basis of the O / ZK / 93-08 virus strain containing the VP1 gene of the Myanmar 98 lineage, the structural protein VP2 93S and 98Y were mutated to F (VP2S93F+Y98F). A gene sequence (SEQ ID No.5) of about 1134 bp containing FMDVVP2S93F+Y98F was synthesized by gene synthesis technology, and the gene fragment was inserted into a pUC plasmid, and the resulting cloning plasmid containing two amino acid mutations was named pUC / VP2S93F+Y98F . The pUC / VP2S93F+Y98F plasmid was digested with Bam H I and Bss H II endonuclease respectively, and the target fragment of about 1134bp was recovered, and it was connected into the plasmid pOMQ-Z123 digested with the same endonuclease (this plasmid was published in Reported in the patent CN 102614507A), the half-length recombinant plasmid pBG was obtained. Send the pBG half-length plasmid to Jinweizhi Biotechnology Co., Ltd., and use primers OZ1725(+) and OZ3004(-) for sequence ...

Embodiment 2

[0087] The pQEA, pQEB, pQEC and pQED recombinant plasmids prepared above were identified by enzyme digestion with Spe I and PstI, and the result was a target band (838bp, 4000bp and 6528bp) that was consistent with the expected size (see figure 2 ). The correct recombinant plasmid identified by enzyme digestion was sent to Jinweizhi Biotechnology Co., Ltd., and the sequence was determined with primers OZ1725(+) and OZ3004(-) (see Table 1 for the specific sequence) to verify the correctness of the constructed plasmid. The results showed that the constructed recombinant plasmids all contained the expected mutation modification. The VP2 nucleotide sequences of pQEA, pQEB, pQEC and pQED plasmids are shown in SEQ ID No.10, SEQ ID No.11, SEQ ID No.12 and SEQ ID No.13 respectively, and the corresponding amino acid sequences are shown in SEQ ID No.14 , SEQ ID No.15, SEQ ID No.16 and SEQ ID No.17.

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Abstract

The invention provides thermal-stability foot-and-mouth disease O type recombinant virus as well as a preparation method and application thereof and belongs to the technical field of biological products of veterinary drugs. The virus takes an O / rV virus strain as a basic virus strain and a 98th site of structural protein VP2 is mutated into phenylalanine. According to the preparation method of therecombinant virus, point mutation S93F and point mutation Y98F are introduced into the VP2 at the same time; however, the Y98F is stably kept in a cell passage process. The FMDV O / rV-3 containing VP2Y98F has good thermal stability and a good immune effect. A thermal mutation resisting strain O / rV containing the VP2 Y98F, which is constructed by the invention, can be used as a high-quality inactivated vaccine for producing seed virus; the content of effective antigens in the inactivated vaccine is remarkably improved, the immune effect of the inactivated vaccine is increased and the inactivated vaccine is used for effectively preventing and controlling foot-and-mouth diseases of China.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug biological products, and in particular relates to a heat-stable foot-and-mouth disease O-type recombinant virus and a preparation method and application thereof. Background technique [0002] Foot-and-Mouth Disease (FMD) is a major animal disease that seriously threatens the development of animal husbandry in my country. The pathogen of foot-and-mouth disease-foot-and-mouth disease virus (Foot-and-Mouth Disease virus, FMDV) has been resident in my country for a long time, causing the epidemic, which not only seriously affects the healthy development of animal husbandry, but also causes trade obstacles for livestock and their products, and poses a threat to public health and the country. The reputation has a serious negative impact, so my country attaches great importance to the prevention and control of the disease. [0003] FMDV belongs to the Picornaviridae family, and its genome is abou...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/66C12N15/86A61K39/135A61P31/14
Inventor 李平花刘在新卢曾军高雅杜平孙普白兴文李冬曹轶梅付元芳马雪青李坤
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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