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Recombinant F genotype mumps virus live vector measles vaccine

A technology of mumps virus and mumps virus strain, which is applied in the field of biomedicine and can solve problems such as complexity, impact on vaccine effect, interference, etc.

Active Publication Date: 2021-08-24
SHANGHAI KING CELL BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The vaccine is prepared by mixing three virus strains separately; it is more complicated to produce three kinds of vaccines separately, and after mixing the three viruses, different viruses will cause interference and affect the vaccine effect

Method used

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  • Recombinant F genotype mumps virus live vector measles vaccine
  • Recombinant F genotype mumps virus live vector measles vaccine
  • Recombinant F genotype mumps virus live vector measles vaccine

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preparation example Construction

[0087] The preparation method of virus strain of the present invention

[0088] In the present invention, a kind of method for preparing recombinant F genotype mumps virus strain is provided, and described method comprises steps:

[0089] (S1) Constructing the full-length recombinant plasmid of the recombinant F genotype mumps virus inserted into the measles virus H gene sequence in the F gene sequence and the HN gene sequence;

[0090] (S2) obtaining three kinds of helper plasmids comprising N gene, P gene and L gene in mumps virus respectively; and

[0091] (S3) Co-transfect the host cells with the full-length recombinant plasmids obtained in (S1) and the three helper plasmids described in (S2), culture for 3 days, lyse the cells, and centrifuge the cell lysate to obtain the supernatant , to obtain the recombinant F genotype mumps virus strain.

[0092] In another preferred embodiment, the host cell is selected from the group consisting of BSR-T7 cells, 293T cells, Vero ce...

Embodiment 1

[0116] Construction of recombinant virus full-length clone

[0117] The basic idea of ​​the full-length cloning of the recombinant virus is as follows: First, based on the F genotype mumps virus reverse genetic manipulation technology platform, the plasmid pMuV (plasmid map as shown in figure 1Shown) linearized, and obtained fragment A of 16405bp by gel recovery; then using the cDNA of MV as a template, primers MV-H-1-F and MV-H-1-R, MV-H-2-F were used respectively and MV-H-2-R, MV-H-3-F and MV-H-3-R amplified to obtain fragment 1, fragment 2 and fragment 3; with primers MV-H-1-F and MV-H- 3-R used fragment 1, fragment 2 and fragment 3 as templates, obtained fragment 4 (3633bp) by fusion PCR, and then digested with restriction endonucleases PmlI and NaeI to obtain fragment 5 with a size of 3370bp; finally fragment A and Fragment 5 was ligated overnight at 16°C with T4 ligase, and transformed into competent cell XL10; finally, positive clones were obtained by enzyme digestion ...

Embodiment 2

[0121] virus rescue

[0122] First, a large number of full-length clones and helper plasmids are extracted with an endotoxin-removing kit; then the cells are seeded in a six-well plate and cultured overnight for transfection, and the confluence of the cells should be 80-90%. The transfection process was as follows: the full-length plasmid pMuV-MV-H (7 μg), the helper plasmid pcDNA3.1-N (1.5 μg), pcDNA3.1-P (0.2 μg), pcDNA3.1-L (1.0 μg) and Lipofectamine TM 2000 transfection reagent (12 μL) was added to 500 μL DMEM medium and mixed gently, and the mixture was incubated at 37°C for 20 min for later use; cells were washed 3 times with PBS, and the supernatant was discarded after the third wash ; Add the mixed solution into the cell well, incubate at 37°C for 6h; then wash with PBS for 3 times, replace with DMEM medium containing 2% serum and 1% antibiotics, and continue to culture at 37°C for 3-4d; put the cells together with the supernatant Freeze and thaw twice, centrifuge a...

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Abstract

The invention provides a recombinant F genotype mumps virus live vector measles vaccine. Specifically, the invention provides a recombinant F genotype mumps virus strain, the virus strain is a recombinant F genotype mumps virus strain with a preservation number of CCTCC NO: V202102, and a measles virus H gene is integrated in a genome. The invention also provides a vaccine composition containing the recombinant F genotype mumps virus strain as an active ingredient and a preparation method of the vaccine composition. The vaccine provided by the invention can be better matched with predominant epidemic F-type mumps viruses in China regions, can prevent mumps and measles at the same time, and is equivalent to the current vaccine strains in the aspects of growth characteristics, immunogenicity and the like. In addition, only one virus strain is needed during preparation of the vaccine, production is simple, and quality control is easy.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant F genotype mumps virus live carrier measles vaccine. Background technique [0002] Measles is an acute systemic eruptive infectious disease caused by measles virus (MV) infection, and the main route of transmission is droplet transmission of respiratory secretions. Before the advent of the measles vaccine, the vast majority of people would suffer from measles in childhood, so they were once crowned the most powerful "child killers". Adults also have measles, and the symptoms of adult measles are usually severe. With the widespread use of live attenuated measles vaccines, the global incidence of measles has been greatly controlled; however, in the past ten years, when the global vaccination rate exceeds 70%, local measles outbreaks have repeatedly occurred in Europe and Asia. This phenomenon has attracted the attention of governments and scientists in various countries. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/45A61K39/295A61K39/165A61P31/14
CPCC12N7/00C07K14/005A61K39/12A61P31/14C12N2760/18721C12N2760/18722C12N2760/18734C12N2760/18422C12N2760/18434A61K2039/70A61K2039/54A61K2039/5256
Inventor 安祺朱凤才田大勇刘元宝谢丽霞
Owner SHANGHAI KING CELL BIOTECHNOLOGY CO LTD
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