Construction method and application of recombinant enterovirus phenotype hybrid system containing cytokine adjuvant

A technology of enteroviruses and cytokines, applied in the direction of cytokines/lymphokines/interferons, medical preparations containing active ingredients, viruses, etc., can solve problems such as interference with proliferation efficiency, interference, and insufficient protection, and achieve a wide range of The effect of applying value

Inactive Publication Date: 2017-05-31
SHANTOU UNIV MEDICAL COLLEGE
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AI Technical Summary

Problems solved by technology

Two or more enteroviruses at the same time will cause interference, which will reduce the proliferation efficiency of the virus, and the replication and proliferation efficiency of different viruses after mixed infection is different, and effective quality control cannot be carried out. However, for the application value of the vaccine Virus strains require high replication efficiency, which is why the current vaccine research field uses single-type viruses to multiply separately, and then mixes the various types of vaccines prepared separately as needed, instead of using multiple viruses to infect cells at the same time to multiply viruses, and not to use Mechanism of phenotypic mixing
[0007] For viruses mixed with "natural" phenotypes, in addition to interference phenomena and bottlenecks of different proliferation efficiencies, if they are sourced from strong virus strains, they need to be inactivated and prepared as inactivated vaccines. On the

Method used

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  • Construction method and application of recombinant enterovirus phenotype hybrid system containing cytokine adjuvant
  • Construction method and application of recombinant enterovirus phenotype hybrid system containing cytokine adjuvant
  • Construction method and application of recombinant enterovirus phenotype hybrid system containing cytokine adjuvant

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Embodiment 1

[0038] 1. Construction of cells stably expressing VP1 of CA16 and EV71

[0039] The specimens of coxsackievirus A group 16 (hereinafter referred to as CA16) and enterovirus 71 (hereinafter referred to as EV71) infected cases confirmed by clinical and laboratory were used to extract the viral RNA of the infected cases respectively by using the virus RNA extraction kit. SuperScript III reverse transcriptase and random primers were used to reverse transcribe the viral cDNA of CA16 and EV71.

[0040] Using CA16 and EV71 viral cDNA as templates, the upstream primer of CA16-VP1 gene: 5'-CCTAAGCTTTCTGGGTACTTTGACTATTACACC, and the downstream primer 5'-CAGCTCGAGTCATGTTGTTATCTTGTCTCTACTAC. The upstream primer of EV71-VP1 gene: 5'-CCTAAGCTTTATGCCCGAGATGGAGTGTTTGAC, the downstream primer 5'-CAGCTCGAGTCATTTCCCAAGAGTGGTGATTGCTG.

[0041] Amplification reaction conditions: pre-denatured at 94°C for 3 minutes, then cycled, denatured at 94°C for 30 seconds, annealed at 52°C for 30 seconds, ex...

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Abstract

The invention provides a construction method of a recombinant enterovirus phenotype hybrid system containing a cytokine adjuvant. The construction method comprises the following steps: firstly respectively constructing a VP1 recombinant cell line and a recombinant attenuated enterovirus strain loaded with cytokine genes; then performing infection amplification on the recombinant attenuated enterovirus strain in the VP1 recombinant cell line; the VP1 recombinant cell line is one of or two of VP1 cell lines for expressing an enterovirus type 71 and a coxsackie virus A group type 16; the recombinant attenuated enterovirus strain loaded with the cytokine genes selects from a coxsackie virus group B type 3, the enterovirus type 71 or a poliovirus type I; a cytokine selects from cholera toxin or interferon gamma; recombinant attenuated enteric viruses loaded with the cytokine genes infect the VP1 recombinant cell line, and after a cytopathic effect, progeny viruses are obtained and can activate host immunity to generate immune protection aiming at a variety of intestinal viruses; therefore, the construction method has application values.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, to a construction method and application of a recombinant enterovirus phenotype mixing system containing a cytokine adjuvant. Background technique [0002] Hand-foot-mouth disease (HFMD) is an infectious disease caused by enterovirus infection. It mainly occurs in infants and young children under 5 years old. Herpes or ulcers appear in the oral mucosa, perianal and other parts. Hand, foot and mouth disease is usually self-limiting, and most children recover by themselves in about a week, but a small number of children will develop complications of the central nervous system, cardiovascular system, and respiratory system such as aseptic meningitis, encephalitis, myocarditis, and pulmonary edema. Rapidly progresses to severe hand, foot and mouth disease, which can cause death in severe cases. Hand, foot and mouth disease is highly prevalent in my country and poses a huge threat to ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N5/10A61K39/12A61K39/39A61P31/14
CPCA61K39/00A61K2039/55522C07K14/28C07K14/57C12N7/00C12N2770/32321C12N2770/32352Y02A50/30
Inventor 李蕊曾俊陈小璇赵颖邓辉雄代剑平王革非李康生
Owner SHANTOU UNIV MEDICAL COLLEGE
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