O-type foot and mouth disease virus strain with improved replication titer as well as construction method and application of O-type foot and mouth disease virus strain

A technology of foot-and-mouth disease virus and construction method, which is applied in the field of O-type foot-and-mouth disease virus strain and its construction, can solve the problems of long time for virus collection, low replication titer, unsuitability for large-scale production of FMD vaccine, etc.

Active Publication Date: 2022-08-05
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

However, the O / XJ / CHA / 2017 strain isolated in my country (even other strains of the same lineage popular in China) has a low replication titer on BHK-21 susceptible cells (TCID 50 about 10 5.2 or so), the collection time is too long (more than 40h), it is not suitable for the large-scale production of FMD vaccine

Method used

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  • O-type foot and mouth disease virus strain with improved replication titer as well as construction method and application of O-type foot and mouth disease virus strain
  • O-type foot and mouth disease virus strain with improved replication titer as well as construction method and application of O-type foot and mouth disease virus strain
  • O-type foot and mouth disease virus strain with improved replication titer as well as construction method and application of O-type foot and mouth disease virus strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] 1. Construction method of full-length clone containing FMD epidemic strain O / XJ / CHA / 2017L+P1 gene

[0057] Taking the FMD vaccine strain O / HN / CHA / 93 half-length plasmid pSK-Z123 as the backbone, according to the L and The nucleotide sequence of P1 (SEQ ID NO: 2), the recombinant half-length plasmid pSK-Z123XJLP1 (synthesized by Jinweizhi Biotechnology Co., Ltd.) containing the L+P1 gene of the virus was designed and synthesized. The plasmid was digested with SpeI and BglII restriction endonucleases, and the target fragment of about 5400 bp was recovered, and inserted into the pOFS plasmid digested with the same enzyme to obtain the L+P1 gene of the chimeric O / XJ / CHA / 2017 virus. Full-length plasmid PQSA (see figure 1 ). Wherein, the nucleotide sequence of the L+P1 gene of the plasmid PQSA is shown in SEQ ID NO:2, the nucleotide sequence of the G-H loop is shown in SEQ ID NO:3, and the amino acid sequence of the G-H loop is shown in SEQ ID NO:6. The plasmid was identif...

Embodiment 2

[0062] Rescue of recombinant virus

[0063] Plasmids pQSA, pQSE and PQTM were prepared by QIAGEN Plasmid Midi Kits. After Not I line, they were purified and recovered by DNA fragment recovery kit as transfection templates. When the conventionally cultured monolayer BSR / T7 cells grow to 70% to 80%, liposome LipofectamineTM2000 is used to mediate transfection (see the operation instructions for the specific operation method). 5h after transfection, 2 mL of DMEM medium containing 8% fetal bovine serum was added, and set at 37°C with 5% CO. 2 The incubator continued to grow and the cells were observed for cytopathic development.

[0064] The results showed that the three plasmids showed typical cytopathogenic effect (CPE) 60h after transfection into BSR / T7 cells, that is, the cells with fibrous distribution became larger and rounded ( Figure 4 ). The cells were harvested 72 h after transfection, and after repeated freezing and thawing 3 times, the cells were serially passaged ...

Embodiment 3

[0066] Identification of recombinant viruses

[0067] 1. RT-PCR identification

[0068] The transfected supernatant was taken to extract the total cytotoxic RNA with RNAasyMini Kit, and the primer pair OZ1490(+) / OZ3980(-) in the table (OZ1490(+): gacaagaccacgccgtatt (SEQ ID NO: 9), OZ3980(-) : tgcatctggttgatggtgtc (SEQ ID NO: 10)) RT-PCR to amplify the P1 gene fragments of the transfected supernatant respectively, purify and recover, and send them to Shanghai Sonny Co., Ltd. for sequencing to verify the correctness of the recombinant virus.

[0069] The sequencing results show that the rHN / XJ, rHN / XJ / NXGH and rHN / XJ / MSGH recombinant FMDVs all contain the expected substitutions, indicating that the present invention has successfully constructed a recombinant FMDV containing target gene substitutions.

[0070] 2. Indirect immunofluorescence

[0071] When BHK-21 monolayer cells grew to 70% to 80% full, rHN / XJ, rHN / XJ / NXGH and rHN / XJ / MSGH recombinant viruses were inoculated resp...

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Abstract

The invention provides an O-type foot and mouth disease virus strain with improved replication titer as well as a construction method and application thereof, and belongs to the technical field of biological products. Through a reverse genetic manipulation technology of the foot and mouth disease virus, a G-H ring gene of the foot and mouth disease virus O / NXYCh / CHA / 2018 strain is further used for replacing a corresponding gene on a recombinant virus skeleton of a main immune gene of the chimeric foot and mouth disease virus O / XJ / CHA / 2017 strain, the time of causing 100% infected cell lesion by the constructed recombinant virus rHN / XJ / NXGH is remarkably shortened to 12 h, and the time of causing 100% infected cell lesion by the recombinant virus rHN / XJ / NXGH is remarkably shortened to 12 h. And the replication titer of infected cells for 12 hours is improved by more than 5 times. The recombinant virus is continuous in passage and good in hereditary stability, and replacement of the G-H ring does not affect immunogenicity of the vaccine. Therefore, the recombinant foot-and-mouth disease virus strain rHN / XJ / NXGH provided by the invention has the potential to be used as a vaccine candidate strain for effectively preventing and controlling the O-type foot-and-mouth disease in China.

Description

technical field [0001] The invention belongs to the technical field of biological products, and in particular relates to an O-type foot-and-mouth disease virus strain with improved replication titer and a construction method and application thereof. Background technique [0002] Foot-and-Mouth Disease (FMD) is a severe infectious disease caused by Foot-and-Mouth Disease Virus (FMDV), which infects more than 70 kinds of cloven-hoofed animals such as pigs, cattle and sheep. The disease spreads rapidly, is highly contagious, and has a very high incidence rate. The World Organization for Animal Health (OIE) has listed it as a must-reportable animal disease. The outbreak and prevalence of FMD seriously endanger the productivity of livestock and the quality of livestock products, affect the international trade of livestock and their products, and cause huge economic losses to the livestock breeding industry in the affected areas. Therefore, the effective prevention and control of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12N15/66A61K39/135A61P31/14C12R1/93
CPCC12N7/00C12N15/85C12N15/66A61K39/12A61P31/14C12N2770/32121C12N2770/32134C12N2800/107A61K2039/552A61K2039/5252Y02A50/30Y02A40/70
Inventor 李平花卢曾军查晶晶马雪青孙普李冬白兴文曹轶梅李坤袁红付元芳包慧芳刘在新赵志荀王健张克强陈应理张强
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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