Gene vaccine against SARS virogene and its construction and use

A SARS virus and genetic vaccine technology, applied in antiviral agents, applications, genetic engineering and other directions, can solve the problems of aggravating the disease, inappropriate immunization and prevention of SARS virus, etc., and achieve highly specific effects

Inactive Publication Date: 2009-05-06
INST OF ZOOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some inactivated vaccines and attenuated vaccines are being studied. Among them, the inactivated vaccines do not solve the problems of immunopathology. Usually some inactivated viruses enter the human body and will aggravate the disease when the virus invades; while the attenuated vaccines will take a long time. Time allows the virus to reproduce for many generations, and then gradually reduces most of the activity of the virus, which cannot meet the needs. This vaccine is also not suitable for immunization and prevention of SARS virus

Method used

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  • Gene vaccine against SARS virogene and its construction and use
  • Gene vaccine against SARS virogene and its construction and use
  • Gene vaccine against SARS virogene and its construction and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1. the construction of the genetic vaccine of anti-SARS virus of the present invention---pVAX1-SARS-MNRna vaccine

[0035] The construction steps of the pVAX1-SARS-MNRna gene vaccine provided by the invention are as follows:

[0036] 1) Introduce the plasmids containing SARS virus N protein, M protein, SARS RNA polymerase cDNA fragment and pGEM-tEasyvector vector into DH5a strain, and select the cDNA fragment clone containing SARS virus N protein, M protein and RNA polymerase gene according to conventional methods The single colonies and shaking bacteria of the carrier are extracted to contain the cloning vector of SARS virus N protein, M protein and RNA polymerase gene cDNA fragment; the described SARS virus N protein, M protein and SARS RNA polymerase cDNA fragment are obtained from the gene library respectively 26386-227054 with accession number AY278487 ( figure 1 The sequence shown 1), 28120-29388 ( figure 2 The sequence shown 2) and 2486-2935 ( ima...

Embodiment 2

[0067] The identification of the in vitro expression and expression content of the exogenous mRNA of embodiment 2.pVAX1-SARS-MNRna vaccine

[0068] Establish the in vitro transient expression system of Hela cells and CHO cells, transfect the pVAX1-SARS-MNRna expression plasmid constructed in Example 1 through liposomes, collect the culture fluid at 24 hours, 48 ​​hours, and 72 hours respectively, and use RT-PCR , laser confocal indirect immunofluorescence detection and other methods to identify the transient expression and content of recombinant eukaryotic expression vector pVAX1-SARS-MNRna vaccine in vitro. It can be highly expressed at the mRNA level, and the expression at the mRNA level is 151% higher than that of the control group (empty plasmid).

Embodiment 3

[0069] The identification of the in vitro protein level expression of embodiment 3.pVAX1-SARS-MNRna vaccine

[0070] Establish the in vitro transient expression system of Hela cells and CHO cells, transfect the eukaryotic expression plasmid pVAX1-SARS-MNRna constructed in Example 1 through liposomes, and add it at 24 hours, 48 ​​hours, and 72 hours respectively, pVAX1-SARS-MNRna The serum of BALB / C mice after MNRna immunization was detected by laser confocal indirect immunofluorescence (immunofluorescence), RT-PCR, ELISA (enzyme-linked immunosorbent assay) and other methods to identify recombinant eukaryotic expression vector pVAX1-SARS-MNRna vaccine in vitro Transient expression and content, it can be seen that the pVAX1-SARS-MNRna eukaryotic expression plasmid can be efficiently expressed at the protein level in Hela cells and CHO cell in vitro transient expression systems, and the expression at the protein level is 168% higher than that of the control group (empty plasmid). ...

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Abstract

The present invention relates to a gene vaccine against SARS virus, which is composed of SARS virus N albumen of 26386-227054 fragment code having login number is AY278487 in gene library, M albumen of 28120-29388 fragment code and RNA polyase gene cDNA of 2486-2935 fragment code and mammalian expression carrier pVAX1. The construction of the vaccine includes: choosing single bacterial colony, swinging bacterium, extracting clone carrier plasmid containing N albumen containing SARS virus, M albumen and RNA polyase gene cDNA fragment; designing primer, cloning the N albumen, M albumen and RNA polyase gene cDNA fragment by polyase chain manner expand method; purifying them and recombining them to mammalian special expression carrier pVAX1 to construct gene vaccine against SARS virus-pVAX1-SARS-MNRna vaccine. Compared with existing vaccine, the vaccine needs smaller amount for achieving the same immune effect, and can excite the humoral immunity and cell immunity and stronger immune effect than other immune means, and it is safety, long effect, stable and convenient in operation and can be used for preparing the medicine for defending and curing SARS.

Description

technical field [0001] The present invention relates to a kind of gene vaccine, more particularly, the present invention relates to a kind of anti-SARS gene that contains N protein (Nucleocapsidphosphoprotein nucleoprotein capsid phosphoprotein), M protein (Membrane glycoprotein membrane glycoprotein) and RNA polymerase gene fragment cDNA vaccine. technical background [0002] The prevention of human diseases is accomplished by non-specific and specific immune systems. With the rapid development of gene recombination technology and gene carrier delivery system, in 1992, four laboratories in the United States (Robinson, Johoston, Li, David) developed and researched the prototype of DNA vaccine (gene vaccine) at the same time, and in 1992 A report was also made at the Conference on New Advances in Vaccinology held in September. Since then, the advent of the new technology of DNA vaccine has attracted great attention from all over the world, and it is regarded as a revolution...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/215A61K38/16C12N15/50A61P31/14A61P11/00
Inventor 彭景楩孙泉红常建军石树群徐丽杨颖王金玲夏红飞
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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