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H1a genotype chimeric measles virus attenuated strain as well as preparation method and application thereof

A technology of measles virus and attenuated strains, applied in chemical instruments and methods, methods based on microorganisms, biochemical equipment and methods, etc., can solve the problem of lack of vaccines for H1a genotype measles virus, and achieve optimal protection effect and excellent The effect of protection

Active Publication Date: 2022-02-01
BEIJING CELL FUSION BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently a lack of vaccines against H1a genotype measles virus

Method used

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  • H1a genotype chimeric measles virus attenuated strain as well as preparation method and application thereof
  • H1a genotype chimeric measles virus attenuated strain as well as preparation method and application thereof
  • H1a genotype chimeric measles virus attenuated strain as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] The construction of the plasmid of embodiment 1 H1a genotype chimeric measles virus full-length cDNA

[0083] This embodiment provides the construction method of the plasmid of the full-length cDNA of chimeric measles virus of H1a genotype, comprising the following steps:

[0084] (1) The nucleotide sequence comprising the gene F encoding the fusion protein of the H1a genotype measles virus and the gene H encoding the hemagglutinin protein (the nucleotide sequence is shown in SEQ ID NO.1, referred to as the FH gene , synthesized by Nanjing GenScript) as a template, using the specific primers pSC-H1A-1-F and pSC-H1A-1-R in Table 1, using Phanta Max Super-Fidelity DNA Polymerase from Novizyme Enzyme (product number: P505-d1) uses PCR method to amplify FH gene.

[0085] (2) Using the plasmid pMV as a template, use the specific primer pairs in Table 1 (pSC-2-F, pSC-2-R), (pSC-3-F, pSC-3-R), (pSC -4-F, pSC-4-R) or (pSC-5-F, pSC-H1A-5-R), using Phanta Max Super-Fidelity DNA...

Embodiment 2

[0090] Example 2 Chimeric virus rescue

[0091] A large number of pMV, pMV / FH(H1a) and helper plasmids were extracted and co-transfected into cells for virus rescue. The specific process is as follows:

[0092] (1) BSR-T7 / 5 cells were seeded in a six-well plate and cultured overnight at 37°C, 5% CO 2 , the medium is DMEM medium (containing 10% Gibco's FBS serum by volume and 1% gentamicin sulfate by volume), and the cell wells with a cell confluence of 80-90% are selected for virus rescue;

[0093] (2), the full-length plasmid pMV / FH(H1a) (4 μg) (or pMV (4 μg)), pcDNA3.1-N (1.5 μg), pcDNA3.1-P (0.2 μg), pcDNA3.1-L (1.0 μg) was mixed with LipofectamineTM 2000 transfection reagent (Invitrogen, catalog number: 11668019) (12 μL) in 500 μL DMEM medium, and incubated at 37°C for 20 min to obtain a plasmid transfection reagent mixture;

[0094] (3) Select cell wells with a cell confluence of 80-90% in step (1), wash the cells 3 times with PBS, then add the plasmid transfection reage...

Embodiment 3

[0095] Example 3 MMR combined live attenuated vaccine

[0096] This embodiment provides a combined live attenuated measles-mumps vaccine (MuV+rMV / FH(H1a)+RuV), each dose comprising:

[0097] H1a genotype chimeric measles virus attenuated strain rMV / FH (H1a) in embodiment 2, live virus is not less than 3.0lgCCID 50 ;

[0098] Mumps virus attenuated strain QS-F-SH2 (preserved in the China Center for Type Culture Collection, the preservation number is CCTCCNo:V201950, recorded in the Chinese patent document CN111019910A) live virus is not less than 3.7lgCCID 50 ;

[0099] Attenuated strain RuV of rubella virus (purchased from ATCC, No. VR-1359), the live virus is not less than 3.0lgCCID 50 .

[0100] The preparation method of the above-mentioned measles-mumps combined with attenuated live vaccine comprises the following steps:

[0101] The rMV / FH (H1a) virus solution obtained in Example 2, the RuV virus (purchased from ATCC, No. : V201950, recorded in Chinese patent documen...

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Abstract

The invention relates to the technical field of virus gene engineering, in particular to an H1a genotype chimeric measles virus attenuated strain and a preparation method and application of the H1a genotype chimeric measles virus attenuated strain. The method comprises the following steps: by taking a measles virus attenuated strain as a skeleton, and replacing the fusion protein coding gene F and the hemagglutinin protein coding gene H in the measles virus attenuated strain with nucleotide sequences of the fusion protein coding gene F and the hemagglutinin protein coding gene H containing the H1a genotype measles virus; and according to the present invention, the existing measles vaccine attenuated strain (such as Schwarz) is adopted as the skeleton, the immunogenic proteins F and H are respectively replaced with the H1a genotype related proteins F and H, and the constructed H1a genotype chimeric measles virus attenuated strain has the excellent protection effect on the H1a genotype measles virus.

Description

technical field [0001] The invention relates to the technical field of viral genetic engineering, in particular to an H1a genotype chimeric measles virus attenuated strain, a preparation method and use thereof. Background technique [0002] Measles is an acute viral infectious disease caused by measles virus (MV), characterized by fever, respiratory catarrhal symptoms, and systemic maculopapular rash. The widespread use of vaccines has greatly reduced the morbidity and mortality. However, according to the World Health Organization (WHO), measles is still one of the public health problems that seriously threaten children's health. There is only one serotype of measles virus, but it is divided into 8 genomes (A-H) and 24 genotypes. These genotypes were all prevalent in the world. [0003] The measles vaccine is the A genotype measles virus in the combined attenuated live measles-mumps vaccine at home and abroad. After immunization with A genotype measles virus, the protecti...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/62A61K39/295A61K39/165A61K39/20A61P31/14C12R1/93
CPCC12N7/00C07K14/005A61K39/12A61P31/14C12N2760/18421C12N2760/18422C07K2319/00C12N2760/18434C12N2760/18734C12N2770/36234A61K2039/5254A61K2039/70Y02A50/30
Inventor 安祺田大勇解丽霞朱凤才
Owner BEIJING CELL FUSION BIOTECHNOLOGY CO LTD
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