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MRNA methyltransferase-deficient mumps virus and preparation method and application thereof

A mumps virus, methyltransferase technology, applied in the field of reverse genetics, to achieve the effect of good safety and stability

Active Publication Date: 2019-04-16
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after the application of mumps vaccine containing Urabe or LZagreb vaccine strain or measles-mumps-rubella (MMR) combined vaccine, more adverse reactions such as aseptic meningitis occurred

Method used

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  • MRNA methyltransferase-deficient mumps virus and preparation method and application thereof
  • MRNA methyltransferase-deficient mumps virus and preparation method and application thereof
  • MRNA methyltransferase-deficient mumps virus and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] 1. Extract viral genome RNA

[0087] The mumps virus vaccine S79 strain was provided by the Zhejiang Provincial Center for Disease Control and Prevention, and the genomic RNA of the vaccine strain was extracted according to the instructions of TRIzol cell lysate.

[0088] 2. PCR amplification to prepare DNA fragments of the whole genome of the S79 vaccine strain

[0089] Using the extracted mumps virus RNA as a template, according to III Reverse transcription kit instructions, use Random Primer Mix to reverse-transcribe mumps virus RNA into cDNA, and store at minus 20°C for later use.

[0090] There are 8 DNA fragments in the full-length genome of the S79 vaccine strain, and the 8 fragments are NP, P, M, F, SH, HN, L1 and L2 respectively. Using cDNA as a template, 5 pairs of overlapping primers are used to amplify and prepare Covering the full-length genome of the S79 vaccine strain, the primer sequences are shown in Table 1. The original fragment MuV-SH-HN was muta...

Embodiment 2

[0116] Construction of full-length plasmid of mRNA methyltransferase-deficient mumps virus

[0117] According to the plasmid pYES-MuV-S79 that sequence correctly contains the full-length genome sequence of mumps vaccine strain S79 in Example 1 as template, the S-adenosylmethionine (SAM) on the L gene of mumps virus L gene is combined by PCR site-directed mutation site and 2'-O-methyltransferase (MTase) KDKE tetrad, thereby the full-length plasmid of the mRNA methyltransferase-deficient mumps virus vaccine strain. The specific operation is as follows:

[0118] 1. Amplify the full-length plasmid of the mRNA methyltransferase-deficient mutant strain: use Pfu enzyme (Pfu Turbo HotstartDNA polymerase) to use the pYES-MuV-S79 full-length plasmid as a template, and use the primers shown in Table 3 to perform PCR amplification according to the product manual A 25ul system PCR reaction was carried out according to the procedure, and the plasmid carrying the mutant mRNA methyltransfera...

Embodiment 3

[0130] Rescue of mRNA methyltransferase-deficient mumps vaccine strains

[0131] 1. Plasmid extraction

[0132] Use Plasmid DNA purification to extract high-concentration plasmids according to the instructions for rescue. Finally, use sterilized EB (2mM Tris grains to extract, and use aseptic operation to elute the plasmid. Use NANO DROP 2000 (Thermo, USA) to measure the content and purity of the plasmid. The content is more than 100ug / ml, and the OD260 / OD280 is 1.70-2.00. ready for transfection.

[0133] 2. Preparation of recombinant mumps virus by reverse genetic manipulation technology

[0134] (1) The helper plasmid and the full-length mumps plasmid are mixed according to a certain ratio (N:P:L:Full=3:3:1:10)

[0135] Mix 5 μg of the full-length plasmid of the recombinant parotid virus vaccine strain constructed above with 1.5 μg of helper plasmids pT7-MuV-NP, 1.55 μg of pT7-MuV-P and 0.55 μg of pT7-MuV-L, and divide them into the following nine groups: rMuV- S79, rMuV...

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Abstract

The invention discloses an mRNA methyltransferase-deficient mumps virus and a preparation method and application thereof, and belongs to the technical field of reverse genetics. The mRNA methyltransferase-deficient mumps virus is prepared through site-directed mutation of a mumps virus vaccine S79 strain, wherein the amino acid at the site 1792 of the L protein is mutated from K to A, or the aminoacid at the site 1814 of the L protein is mutated from A to G, or the amino acid at the site 1816 is mutated from G to A, or the amino acid at the site 1818 is mutated from G to A, or the amino acidat the site 1892 is mutated from D to A, or the amino acid at the site 1917 is mutated from D to A, or the amino acid at the site 1953 is mutated from K to A, or the amino acid at the site 1990 is mutated from E to A. According to the invention, through site-directed mutation, the rescued recombinant mumps virus is weakened in virulence and good in immunogenicity, so that the immunogenicity of themumps virus attenuated vaccine is retained, and the safety of the mumps virus attenuated vaccine is improved.

Description

technical field [0001] The invention relates to the technical field of reverse genetics, in particular to an mRNA methyltransferase-deficient mumps virus and its preparation method and application. Background technique [0002] Vaccines refer to biological products made from various pathogenic microorganisms for vaccination. Vaccination is the most economical and effective public health intervention to prevent and control infectious diseases, and it is also an effective means for families to reduce the incidence of diseases among members and reduce medical expenses. [0003] The development of a vaccine is a long, complex process and can be costly. Whole virus inactivated vaccines, split mumps vaccines and subunit vaccines are far less effective than attenuated vaccines, and the attenuated mumps vaccines prepared by traditional methods have defects such as time-consuming, labor-intensive and virulence recovery. With the rapid development of reverse genetics technology, it ...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/85C12N15/10A61K39/165A61P31/14C12R1/93
CPCA61K39/12A61K2039/5254A61P31/14C12N7/00C12N15/1031C12N15/85C12N2760/18721C12N2760/18734C12N2760/18752C12Q2531/113
Inventor 赵正言汪一龙
Owner ZHEJIANG UNIV
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