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Measles virus mRNA transmethylase defective attenuated vaccine strain and application thereof

A technology of methyltransferase and measles virus, which is applied in the field of bioengineering, can solve problems such as side reactions and achieve good immune effects

Active Publication Date: 2018-06-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem that certain side effects will still be caused after inoculation of measles vaccine, the present invention provides a measles virus mRNA methyltransferase-deficient attenuated vaccine strain with further weakened pathogenicity. The genome of the strain (S191) is used as the backbone, and the recombinant vaccine obtained by mutating the methyltransferase region and combining with the helper plasmid rescue

Method used

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  • Measles virus mRNA transmethylase defective attenuated vaccine strain and application thereof
  • Measles virus mRNA transmethylase defective attenuated vaccine strain and application thereof
  • Measles virus mRNA transmethylase defective attenuated vaccine strain and application thereof

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Experimental program
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Effect test

Embodiment 1

[0074] Example 1 extracts viral genome RNA

[0075] The Shanghai 191 (S191) measles attenuated vaccine strain was provided by the Zhejiang Provincial Center for Disease Control and Prevention, and the experiment was performed according to the instructions of TRIzolReagent cell lysate.

Embodiment 2

[0076] Embodiment 2PCR amplification prepares the DNA fragment of whole genome of S191 vaccine strain

[0077] according to Instructions for III Reverse Transcription Kit, use the extracted measles virus RNA as a template, use Random Primer Mix to reverse transcribe measles virus RNA into cDNA, and store it at minus 20°C for future use.

[0078]Using cDNA as a template, 8 pairs of overlapping primers were used to amplify and prepare 8 DNA fragments covering the full-length genome of the S191 vaccine strain. The 8 fragments are N, P, M1, M2, F, H, L1 and L2, respectively. Schematic diagram of segmented amplification of measles virus figure 1 shown. The reaction program was: pre-denaturation at 98°C for 40 s, denaturation at 98°C for 8 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, and after 35 cycles, extension at 72°C for 2 min. The results of 1.0% agarose gel electrophoresis were identified as figure 2 As shown, 8 target DNA fragments were successfully amp...

Embodiment 3

[0082] The construction of embodiment 3 recombinant plasmids

[0083] Use the pEASY-Blunt Cloning Kit to connect the target fragments N, P, M1, M2, F, H, L1 and L2 with the pEASY-Blunt Cloning vector to obtain pEASY-N, pEASY-P, pEASY-M1, pEASY- M2, pEASY-F, pEASY-H, pEASY-L1, and pEASY-L2, among them, it was found that there was a T-C point mutation at position 8762 on the H fragment of the virus strain preserved in this experiment, and the mutation position was changed back by in vitro site-directed mutagenesis technique, and finally The recombinant plasmids pEASY-N, pEASY-P, pEASY-M1, pEASY-M2, pEASY-F, pEASY-H-M, pEASY-L1 and pEASY-L2 were constructed. The mutation position is used as a genetic marker to distinguish it from the original strain in the laboratory.

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Abstract

The invention relates to the field of bioengineering, and in particular discloses a measles virus mRNA transmethylase defective attenuated vaccine strain and an application thereof. The measles virusmRNA transmethylase defective attenuated vaccine strain plasmid provided by the invention is obtained through site-specific mutagenesis of a transmethylase conserved sequence on measles virus L protein in a recombinant vector containing measles virus attenuated vaccine strain S191 genome, and the vaccine strain can reduce a replication capacity of rescued measles viruses, so that a purpose of attenuating the viruses is achieved, without changing immunogenicity of the viruses. The measles virus mRNA transmethylase defective attenuated vaccine strain provided by the invention, which is obtainedthrough rescuing of helper plasmid and the measles virus mRNA transmethylase defective attenuated vaccine strain plasmid, is relatively low, and even highly reduced, in pathogenicity, and meanwhile, the vaccine strain can stimulate a human body to generate and maintain a high-titer neutralizing antibody, so that a better immune effect can be achieved.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a measles virus mRNA methyltransferase-deficient attenuated vaccine strain and application thereof. Background technique [0002] Measles virus (MV) belongs to the Paramyxoviridae family (Paramyxoviridae), and the genus Morbillivirus (Morbillivirus). It is a single-stranded negative-sense RNA virus without segments. pack. Measles is an acute infectious disease caused by measles virus that seriously threatens public health. It can cause severe clinical symptoms and even cause death. It belongs to the national statutory category B infectious disease. [0003] The measles vaccine strain used in China is the Shanghai 191 (S191) strain. The measles vaccine produced by the Shanghai 191 strain has good stability, good clinical safety and immunogenicity, and its effectiveness and safety have been clearly affirmed. It is a Chinese measles vaccine. The main production strain of the vaccine....

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/54C12N7/04A61K39/165A61P31/14C12R1/93
CPCA61K39/12A61K2039/5254C12N7/00C12N9/1007C12N15/85C12N2760/18421C12N2760/18434C12N2760/18452C12N2760/18462C12N2760/18471
Inventor 赵正言黄耀伟汪一龙李建荣
Owner ZHEJIANG UNIV
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