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Construction of F gene replaced chimeric measles attenuated strain

A technology of attenuated strains and measles virus, applied in genetic engineering, inactivation/attenuation, plant genetic improvement, etc., can solve problems such as insufficient antibody titer, measles human health threat, and rapid antibody attenuation

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

Even more frightening is that, although the global vaccination rate has reached 70% so far, measles is still a vaccine-preventable infectious disease causing child deaths due to immunization gaps and partial immunization failures, as well as genotype mismatches between vaccines and circulating strains. In short, measles poses a huge threat to human health, even affects the quality of life and induces other diseases
[0005] The possible reasons for the frequent local outbreaks of measles in the world and in my country in the past ten years are: (i) insufficient vaccine coverage; (ii) the antibody titer of some populations after immunization is not high enough or the antibody decays too fast; (iii) despite the There is only one serotype, but complete cross-protection cannot be achieved between viruses of different genotypes, so that when the antibody declines, it may no longer be able to resist infection by other genotypes of measles viruses

Method used

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  • Construction of F gene replaced chimeric measles attenuated strain
  • Construction of F gene replaced chimeric measles attenuated strain
  • Construction of F gene replaced chimeric measles attenuated strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1: Construction and identification of chimeric virus full-length cDNA

[0136] Such as figure 1 Shown is the chimeric measles virus skeleton.

[0137] With the cDNA reverse-transcribed of the MV-1 strain measles virus nucleic acid as a template, use specific primers MV-F-F and MV-F-R in Table 1 to amplify its F gene fragment (comprising the F gene 5'UTR and the F gene 3'UTR), using the S191 full-length plasmid as a template, using specific primers TY-HN-F and FH-ZL-R to amplify its F gene deletion vector by PCR, and then obtain the target gene by one-step homologous recombination plasmid.

[0138] It was confirmed by enzyme digestion that the full-length cDNA of the chimeric virus was successfully cloned, and the results were as follows figure 2 shown.

[0139] Table 1 The specific detection primers of chimeric virus

[0140]

[0141]

Embodiment 2

[0142] Example 2: Chimeric virus rescue

[0143] Experimental method and steps:

[0144] Cells were seeded in 6-well plates, and when the cells grew to about 80%, full-length plasmids PAC-S-191, PAC-S-191-F and related auxiliary plasmids pcDNA3.1-N, pcDNA3.1-P, 293T cells were co-transfected with pcDNA3.1-L and pCAGGS-T7. After 6 hours, the medium was discarded and the medium containing 2% FBS fetal bovine serum was added. After 4 days of culture, the cells were passaged at a ratio of 1:3 and an equal amount of Vero-SLAM cells were added until the cells became confluent to collect the virus supernatant.

[0145] A large number of pMV full-length clones and helper plasmids were extracted; they were co-transfected into cells for virus rescue.

[0146] 293T cells were inoculated in a six-well plate for overnight culture, and cell wells with a cell confluence of 80-90% were selected for virus rescue.

[0147] The specific process is as follows: the full-length plasmid pMV or pM...

Embodiment 3

[0152] Example 3: Analysis of in vitro properties of chimeric viruses

[0153] In order to evaluate the growth characteristics of the chimeric virus in vitro, in this example, the multi-step growth curves of the chimeric virus and its parent virus were studied in Vero and Vero-Slam cell lines, respectively.

[0154] First, make a cell suspension with a cell concentration of 1.0×10 5 -5.0×10 5 Between cells, the cells are divided into 2×10 5 cells / well were seeded in a 12-well plate.

[0155] The next day, when the cells are about 90% full, wash the cells with PBS, inoculate 1ml of virus at 0.01 MOI, incubate at 37°C for 1 hour, discard the virus solution, rinse twice with PBS, and replace the maintenance solution. Sampling was then carried out every 24 hours for 5 consecutive days. Virus titers were then detected by the microcytopathic assay.

[0156] The result is as Figure 5 As shown, in Vero cells, the titer of the chimeric virus rMV / F(H1a) was slightly higher than t...

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Abstract

The invention provides a construction method and application of an F gene replaced chimeric measles attenuated strain. Specifically, the invention provides the chimeric measles virus attenuated strain, and the attenuated strain is a measles virus rMV / F (H1a) with the preservation number of CCTCCNO: V202101. The invention also provides a vaccine composition containing the F gene substituted chimeric measles attenuated strain or the derivative virus strain thereof as an active ingredient and a preparation method of the vaccine composition. Compared with the existing measles vaccine strain in the market, the chimeric virus provided by the invention has stronger replication ability and better immunogenicity.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a chimeric measles attenuated strain replaced by an F gene and a preparation method and application thereof. Background technique [0002] Measles is an acute systemic eruptive infectious disease caused by measles virus (Measles Virus, MV) infection. sickness. The main clinical manifestations of patients are fever, conjunctival hyperemia, runny nose, cough, maculopapular rash on the skin and measles mucous membrane spots on the oral mucosa, and complications may even occur: diarrhea, deafness, blindness, encephalitis, myocarditis and severe malnutrition etc. The patients are basically infants and young children, but adults also have measles, and the symptoms of measles in adults are usually severe. [0003] The measles virus genome is divided into 8 genomes (A~H). According to the latest data released by WHO, the number of genotypes has been increased from 2...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/45C07K14/12A61K39/155A61P31/14C12R1/93
CPCC12N7/00C07K14/005A61K39/12A61P31/14C12N2760/18421C12N2760/18422C12N2760/18423C12N2760/18434A61K2039/5254A61K2039/545Y02A50/30
Inventor 安祺朱凤才田大勇解丽霞
Owner SHANGHAI KING CELL BIOTECHNOLOGY CO LTD
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