Foot and mouth disease virus recombinant virus sample particle as well as preparation method and application thereof

A foot-and-mouth disease virus, foot-and-mouth disease technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of reducing the production performance, economic loss, harm and other problems of infected herds

Active Publication Date: 2017-10-10
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease is particularly harmful to major livestock species such as cattle, pigs, and sheep. It can not only cause the death of infected animals, but also greatly reduce the production performanc

Method used

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  • Foot and mouth disease virus recombinant virus sample particle as well as preparation method and application thereof
  • Foot and mouth disease virus recombinant virus sample particle as well as preparation method and application thereof
  • Foot and mouth disease virus recombinant virus sample particle as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of recombinant vector pFBDM-VPO / VP1 / VP3 / EGFP

[0048] Using O / Mya98 / XJ / 2010 strain genomic cDNA as a template (O / Mya98 / XJ / 2010 strain genomic cDNA can be extracted from commercially available foot-and-mouth disease O-type inactivated vaccine containing O / Mya98 / XJ / 2010 strain and reversed were recorded), and corresponding primers were designed (see Table 1). The O-type foot-and-mouth disease VPO gene was amplified with primers VPOF and VP0R, and the fragment was 909 bp in length. Sequencing showed that its nucleotide sequence was shown in sequence 1 in the sequence list. The O-type foot-and-mouth disease VP1 gene is amplified with primers VP1F and VP1R, and the fragment is 639 bp in length. Sequencing shows that its nucleotide sequence is shown in sequence 2 in the sequence list. The O-type foot-and-mouth disease VP3 gene is amplified with primers VP3F and VP3R, and the fragment is 660 bp in length. Sequencing shows that its nucleotide sequence is...

Embodiment 2

[0055] The preparation of embodiment 2 recombinant baculovirus rBAC-fluoFMDV

[0056] The pFBDM-VP0 / VP1 / VP3 / EGFP recombinant plasmid was transformed into DH10Bac competent cells, cultured in liquid LB medium containing tetracycline and kanamycin for 4 hours, and coated with gentamicin, tetracycline and kanamycin LB plate, through two rounds of blue-white screening, pick white colony, with M13F / VP1F and VPOF / M13R primer pair (M13F / R primer is synthesized according to the sequence provided by Invitrogen company Bac-to-Bac instructions ), carry out PCR detection and identification of bacterial liquid, expand and cultivate the correct bacterial strain, extract recombinant baculovirus expression plasmid, name it Bacmid-VP0 / VP1 / VP3 / EGFP, and save it for future use.

[0057] The sf9 cells in the logarithmic growth phase were spread on a six-well plate, and the recombinant baculovirus expression plasmid Bacmid-VP0 / VP1 / VP3 / EGFP DNA was diluted according to the transfection method requi...

Embodiment 3

[0058] Embodiment 3 Amplification and toxicity determination of recombinant baculovirus rBAC-fluoFMDV

[0059] The rBAC-fluoFMDV P0 generation virus was inoculated into sf9 cells in the logarithmic growth phase according to the ratio of virus to medium 1 / 10 (v / v), cultured in a constant temperature incubator at 27°C for 96 hours, collected the cell culture supernatant, and centrifuged at 8000rpm for 5min , collect the supernatant, and obtain the P1 generation virus. The P1 generation virus is used to amplify to obtain the P2 generation, and the P2 virus is used to amplify to obtain the P3 generation. TCID 50 The method detects that the virus titer of rBAC-fluoFMDV P3 generation is 10 -7.5 / 0.1mL.

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Abstract

The invention discloses a foot and mouth disease virus recombinant virus sample particle as well as a preparation method and application thereof. The recombinant virus sample particle is commonly assembled and expressed by components in a DNA molecule composition. The DNA molecule composition contains an O-type foot and mouth disease VP0 gene, an O-type foot and mouth disease VP1 gene and an O-type foot and mouth disease VP3 gene and further contains a green fluorescent protein gene. By virtue of the property that FMDV VLPs is self-assembled through VP0, VP1 and VP3, a construction method of a baculovirus recombinant vector is improved, a green fluorescent protein label is added into the vector, and FMDV VLPs is successfully prepared by virtue of a pFBDM Bac-to-Bac system, so that a theoretical foundation is laid for the further development of safe and efficient O-type FMDV genetic engineering vaccines.

Description

technical field [0001] The invention relates to foot-and-mouth disease virus recombinant virus-like particles, a preparation method and application thereof, and belongs to the field of genetic engineering vaccines. Background technique [0002] Foot-and-mouth disease (FMD) is caused by foot-and-mouth disease virus (FMDV) of the Picornaviridae genus Foot-and-Mouth Disease Virus (FMDV). An acute, severe infectious disease. The disease is particularly harmful to major livestock species such as cattle, pigs, and sheep. It can not only cause the death of infected animals, but also greatly reduce the production performance of infected herds; it will also have a certain impact on the international trade of animals and their products in countries where the epidemic occurs Negative impact, resulting in huge economic losses. The International Organization for Animal Health (OIE) lists FMD as the first animal disease to be reported legally, and my country also lists it as a first-cla...

Claims

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Application Information

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IPC IPC(8): C12N15/42C12N15/866C12N15/66C12N15/65C12N7/04A61K39/135A61P31/14
CPCA61K39/00C07K14/005C12N7/00C12N15/65C12N15/66C12N15/86C12N2710/14043C12N2770/32122C12N2770/32123C12N2770/32134
Inventor 张晓战张国栋王楠王飞巴利民李玲肖进齐鹏
Owner CHINA ANIMAL HUSBANDRY IND
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