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Recombinant virus of standard attack strain CVS-11 and preparation method thereof

A CVS-11, recombinant virus technology, applied in the biological field, to achieve the effect of high specificity, economical convenience and good consistency

Inactive Publication Date: 2013-01-09
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no real rapid, economical and efficient detection method for rabies virus neutralizing antibody at home and abroad

Method used

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  • Recombinant virus of standard attack strain CVS-11 and preparation method thereof
  • Recombinant virus of standard attack strain CVS-11 and preparation method thereof
  • Recombinant virus of standard attack strain CVS-11 and preparation method thereof

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Embodiment 1

[0042] 1. Construction of CVS-11 Whole Genome cDNA Recombination Plasmid

[0043] According to the instructions of Simple P Total RNA Extraction Kit, the total RNA of CVS-11 cytotoxicity was extracted. According to the manual of AMV reverse transcriptase (Promega Company), the viral genome cDNA was obtained. Use DNAMAN software to analyze the unique restriction site of CVS-11 whole genome, design 4 pairs of primers (as shown in Table 1), in order to facilitate the splicing of segmented amplified fragments into pcDNA3.1, and subsequent laboratory transformation of CVS-11 genome , corresponding enzyme cutting sites were added upstream and downstream of the primers. Using viral cDNA as a template, referring to the instructions of TransStart Fast Pfu DNA Polymerase and the primers and their annealing temperatures in Table 1, perform PCR amplification on the 4 segments of the whole genome, and use the primers to replace G-L with the three restrictions of BssHII, BsiwI and SacII e...

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Abstract

The invention provides a recombinant virus of standard attack strain CVS-11 and a preparation method thereof. The invention establishes a reverse genetic manipulation system for rabies virus international standard attack strain CVS-11, provides an effective platform for further modifying or reconstructing the CVS-11 genome structure, and lays foundation for researching pathopoiesis mechanism, antigen site, virulence determined site and the like of rabies virus on the molecular level. On the basis of the CVS-11 reverse genetic manipulation system, the invention establishes a recombinant virus rCVS-11-eGFP for saving international standard attack strain CVS-11. The rCVS-11-eGFP expresses eGFP in the proliferation process, and can be used for directly observing the proliferation state and diffusion of the virus. By utilizing the characteristics of no toxicity, capability of self-luminescence without dependence on any accessory factor or substrate, and the like, the rCVS-11-eGFP is used as a detection antigen for determining the anti-rabies virus neutralizing antibody titer, and no operations, such as stain fixation and the like, are needed in the result determination process, so that the invention is economic and convenient.

Description

[0001] Technical field: [0002] The invention discloses a recombinant virus of the standard attack strain CVS-11 (abbreviated as: rCVS-11-eGFP) and also provides a preparation method of the recombinant virus, which is used for the detection of anti-rabies virus neutralizing antibodies in animal and human serum. Detecting an antigen belongs to the field of biotechnology. [0003] Background technique: [0004] Rabies is mad dog disease, also known as hydrophobia, which is a zoonotic infectious disease caused by the infection of warm-blooded animals and humans by Rabies Virus, mainly affecting the central nervous system. Dogs and cats account for 85%-95% of the sources of human rabies infection in my country. WHO research shows that a dog herd immunity rate of about 70% can effectively block the chain of rabies transmission. Therefore, it is very important to establish a complete animal rabies prevention system and monitor the antibody level of animals from time to time. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/63G01N33/569C12R1/93
Inventor 夏咸柱杨松涛薛向红郑学星高玉伟冯娜王化磊王承宇黄耕王铁成赵永坤
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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