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F genotype mumps virus attenuated strain as well as construction method and application thereof

A mumps virus and genotyping technology, applied in the field of bioengineering, can solve problems such as poor judgment of the end point, reduced immunogenicity, weak virus failure, etc.

Active Publication Date: 2020-04-17
SHANGHAI KING CELL BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are two disadvantages: first, the success rate is low, the virus may fail to be weakened, and it may also cause the immunogenicity to decrease as the virulence weakens; second, the end point of weakening is not easy to judge
Special attention should be paid to the fact that the live attenuated mumps vaccines around the world are all produced in primary chicken embryo fibroblasts. If other cell substrates are used, a lot of research on process and quality control needs to be completed, and the development cycle will be long

Method used

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  • F genotype mumps virus attenuated strain as well as construction method and application thereof
  • F genotype mumps virus attenuated strain as well as construction method and application thereof
  • F genotype mumps virus attenuated strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] Embodiment 1: Construction of rMuV-ΔSH deletion virus

[0138] 1.1 Construction of full-length plasmid of QS-F virus

[0139] According to the sequence information of mumps F genotype provided by NCBI, universal primers were designed for whole genome sequencing of QS-F virus. The sequencing primer information is as follows:

[0140] Table 1 Sequencing primer sequence information

[0141]

[0142]

[0143] Primers were designed according to the sequencing results, and the F genotype QS-F was divided into five segments for cloning. The specific strategy is shown in Figure 1.

[0144]Using pAC as a vector, F1 and F5 were digested with Not I, BstE II and Not I and Rsr II respectively to obtain plasmids pAC-F1 and pAC-F5, and then F2 and Not I, Hind were double digested with BstE II and Rsr II III double digestion of F4 to obtain pAC-F1-F2, and pAC-F4-F5. Again, pAC-F4-F5 and pAC-F1-F2 were digested with Pac I and Rsr II to obtain plasmid pAC-F1-F2-F4-F5. Fragment...

Embodiment 2

[0162] Example 2: Growth characteristics and genetic stability of recombinant viruses

[0163] 2.1 Growth characteristics of recombinant virus

[0164] The three recombinant viruses were respectively inoculated at MOI=0.01 (volume 300 μL) into Vero cells cultured in a monolayer in a 24-well plate. After infection for 1 h, the cells were washed three times with PBS, and cultured in DMEM medium containing 2% fetal bovine serum. On 1d, 2d, 3d, 4d and 5d after virus inoculation, the cell supernatant was collected and the virus titer was determined. And draw multi-step growth curves to analyze the in vitro replication characteristics of the three strains of mumps virus.

[0165] The result is as Figure 5 As shown, the growth kinetic curves of the three recombinant viruses in Vero cells have a certain overlap, and there is no significant difference in growth kinetics.

[0166] 2.2 Passage adaptability of recombinant virus on chicken embryo cells

[0167] The three recombinant ...

Embodiment 3

[0177] Example 3: Immunogenicity detection test in mice

[0178] Select 60 mice of SPF grade 6-8 weeks, and randomly divide them into 4 groups namely A, B, C and D. Group A was inoculated with the commercialized attenuated mumps vaccine JL inoculation dose of 4×10 5 CCID 50 per bird, group B was inoculated with F genotype wild virus at a dose of 4×10 5 CCID 50 per mouse, group C was inoculated with the recombinant vaccine QS-F-SH2 at a dose of 4×10 5 CCID 50 / only, D group was inoculated with blank dilution. The inoculation sites in each group were intramuscularly injected into the hind limbs, 50 μL on each side. After 21 days of immunization, a booster immunization was given with the same dose. 14 days after the second immunization, blood was collected from the mice, blood was collected, and serum was separated. Determine the neutralizing titer of the virus (challenge virus species: F genotype WT), pay attention to clinical observation of mice every day before and a...

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Abstract

The invention provides an F genotype mumps virus attenuated strain as well as a construction method and application thereof. Specifically, the invention provides an F genotype mumps virus attenuated strain and the attenuated strain is a mumps virus QS-F-SH2 with an accession number of CCTCC NO: V201950. The invention also provides a vaccine composition containing the F genotype mumps virus attenuated strain as an active ingredient and a preparation method thereof. The mumps virus attenuated vaccine strain disclosed by the invention can match the F type mumps virus predominantly popular in China, and the level of the mumps virus attenuated vaccine strain is equivalent to that of the current vaccine strain in the aspects of growth characteristics, immunogenicity, neurotoxicity and the like.In addition, the mumps virus genetic engineering attenuated strain screened by the invention can be stably produced in chick embryo cells, and the safety is high.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an attenuated F genotype mumps virus strain and a preparation method and application thereof. Background technique [0002] Mumps is an acute infectious disease caused by the mumps virus. Before the middle of the 20th century, it was one of the infectious diseases that seriously endangered the health of children and adolescents. The clinical symptoms of mumps are mainly parotid gland swelling and pain, often secondary to encephalitis and reproductive system inflammation, etc., and severe patients may die; and the disease has become one of the six major causes of infertility in my country. [0003] Since the 1960s, with the successful development and widespread use of live attenuated mumps vaccines, the incidence of mumps in most countries in the world, including my country, has dropped sharply and remained at a low level for a long time (100,000 less than one-third). Howe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04A61K39/165A61P31/14C12R1/93
CPCC12N7/00A61K39/12A61P31/14C12N2760/18721C12N2760/18734A61K2039/5254A61K2039/54C12N2760/18722C12N2760/18764C07K14/005A61K39/165A61K2039/5252A61K2039/545
Inventor 田大勇张亚静朱凤才傅振芳解丽霞刘元宝阮俊程王金亮安祺
Owner SHANGHAI KING CELL BIOTECHNOLOGY CO LTD
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