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Modified measles virus whole gene cDNA clone and infectious virus preparation

A measles virus, whole gene technology, applied in the field of virology and molecular biology, can solve the problem of measles immunization plan not being strictly implemented and affecting the effect of vaccine immunization.

Inactive Publication Date: 2008-12-17
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The effectiveness and safety of measles vaccine have been clearly affirmed. However, the World Health Organization’s global efforts to eliminate measles have not stopped. There is a relatively consistent understanding of the main reasons in the industry: first, the measles immunization program has not been strictly implemented, Second, the genetic variation of the new epidemic strain of measles virus also partially affects the immune effect of the vaccine
[0004] Prior to 1998, there was never a standard nomenclature to characterize the variation of wild-type measles virus genes

Method used

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  • Modified measles virus whole gene cDNA clone and infectious virus preparation
  • Modified measles virus whole gene cDNA clone and infectious virus preparation

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Construction of embodiment 1 measles virus long-47 full-length cDNA clone

[0037] According to the method described in the document "Hu Kongxin, Li Dexin et al. Construction of full-length positive-strand cDNA of measles virus and research on infectivity. Acta Virus Sinica, 2004, 3: 210-212", the intermediate plasmid pMVfull containing the full gene of measles virus long-47 strain was constructed -2S, specifically:

[0038] The measles virus Chang-47 vaccine strain developed by traditional biological methods is produced by primary chicken embryo fibroblast cells (provided by Changchun Institute of Biological Products, it is a commercial vaccine, the batch number is 9812445). Whole-cell RNA was extracted from cells infected with the vaccine virus, reverse-transcribed into cDNA with random primers, and used as a template to amplify Chang-47 virus genome cDNA by DNA Polymerase Chain Reaction (PCR). Then clone and splice the full-length positive-strand cDNA sequence of me...

Embodiment 2

[0041] Example 2 Obtaining of H Gene Fragment of Measles Virus China93-7 Wild Strain

[0042] The wild strain measles virus China93-7 was cultured with B95a cells (purchased from ATCC, USA) and the virus-infected cells were harvested. Whole-cell RNA was extracted from the infected cells with TRizol (purchased from Invitrogen Corporation, USA), and cDNA was synthesized with random primers, and primed with primer 5 '-GGT TGA TAG GGA TCC CCG CT-3' and the primer 5'-CGG CCG AAC GGG GAACCA CTT GGA CC-3' were combined for PCR amplification, and the PCR product contained the cloned fragment of the virus China93-7 virus H gene. The amplified product was directly cloned into the pGEM-T vector (purchased from Promega, USA), and then digested with BamHI (the site inserted into the measles virus fragment) and NotI (the enzyme cutting site on the vector), and the insert was recovered. A small fragment, which is mainly the H gene fragment.

Embodiment 3

[0043] The construction of embodiment 3 modified measles virus full-length cDNA cloning plasmids

[0044] Using primers D1 (5'-ACG CGT ATC TGG CGC CCT ACA GCT CAA CTT ACC TGC-3') and C2 (5'-CGG CCG AAC GGG GAA CCA CTT GGA CC-3') PCR amplification of long-47 virus cDNA , the product was directly cloned into the pGEM-T vector (Promege Company) to obtain the plasmid pGEM-cc47-SpeI, which was digested with BamHI (site in the insert) and NotI (restriction site on the vector) respectively, and recovered Carrier large fragment, and clone the recovered product of the digested fragment obtained in Example 2 into the BamHI and NotI digested vector of plasmid pGEM-cc47-SpeI to obtain a new fragment containing the China93-7 H gene and measles long-47 Recombinant plasmid pGEM-cc47-937H-SpeI of part of the gene, pGEM-cc47-937H-SpeI was directly digested with SpeI, the insert fragment (large fragment, about 5.8kb) was recovered, and then cloned into pMVfull-2S also digested with SpeI Among ...

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Abstract

The invention provides an entire-gene cDNA clone of modified measles virus, which is obtained from the substitution and transformation of main surface antigen genes (H gene) of main existing epidemic strains (genotype of H1 and representative strain of China93-7) in China to the full-length cDNA clones of measles virus strain CC-47. The invention adopts the RNA virus reverse genetic technique to carry out the rescue of modified CC-47 virus on the cDNA level so as to obtain modified infective virus, thus providing great convenience for the research on the replication, the proliferation and the package of novel virus stains. In addition, the novel produced infective virus has the hemagglutinin antigen of an epidemic strain and has more suitable application value of the hoplivac.

Description

technical field [0001] The invention belongs to the fields of virology and molecular biology, and relates to a preparation method for constructing an infectious clone after replacing the hemagglutinin gene fragment of a wild strain with the genome of a live attenuated measles vaccine strain successfully developed in my country, and for cloning the coding region Sequence replacement methods and their application in vaccine research. Background technique [0002] Measles virus (Measles Virus, MV) is a member of the genus Morbillivirus in the Paramyxoviridae family. It is a non-segmented single-stranded negative-sense RNA virus and one of the viruses that cause highly infectious diseases worldwide. Infection through respiratory droplets, or contact. The incidence rate of measles among susceptible populations is almost 100%. Most infected persons can recover completely, but a few can cause severe respiratory tract or central nervous system complications. [0003] The Chang-47 (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/45C12N7/01
Inventor 李德新胡孔新修梅红梁米芳王晓芳李川张全福
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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