Preparation method and application of recombinant cold-adaptation attenuated influenza vaccine strain

A vaccine strain and cold adaptation technology, applied in the field of recombinant influenza virus rescued by multi-plasmid system and its preparation, can solve the problems of inability to organize cell culture, poor immune protection effect of whole virus inactivated vaccine, and recovery of virulence of influenza attenuated vaccine, etc. question

Inactive Publication Date: 2016-08-24
CHANGCHUN HAIJIYA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, some virus strains suitable for preparing vaccines, such as temperature-sensitive strains, cannot be cultured in tissue cells by existing methods
[0007] 3. The immune protection effect of whole virus...

Method used

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  • Preparation method and application of recombinant cold-adaptation attenuated influenza vaccine strain
  • Preparation method and application of recombinant cold-adaptation attenuated influenza vaccine strain
  • Preparation method and application of recombinant cold-adaptation attenuated influenza vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1, the preparation of recombinant A-type H1N1, H3N2 influenza attenuated virus and recombinant B-type Yamagata strain influenza attenuated virus, recombinant B-type Victoria strain influenza attenuated virus

[0088] 1. Construction of 9-plasmid rescue system for influenza virus

[0089] 1. Construction of pIW3000 vector

[0090] Using the basic method of molecular biology, construct pIW3000 on the basis of vector PHW2000. That is, the poly-A signal sequence BGH (bovine growth hormone) in pHW2000 was transformed into SV40 (simian virus 40). Specific steps are as follows:

[0091] Using the vector pcDNA3.1 (Invitrogen Company) as a template, PCR amplification was performed using primers 1 and 2 to obtain PCR amplification products. The primer sequences are as follows: Primer 1: 5'-AACAATTGAGATCTCGGTCACCTCAGACATGATAAGATACATTGATGAGT-3'; Primer 2: 5'-TATAACTGCAGACTAGTGATATCCTTGTTTATTGCAGCTTATAATGGTTA-3'.

[0092] The procedure of PCR amplification was: first ...

Embodiment 2

[0219] Example 2, Preparation of Recombinant Influenza Quadrivalent Live Attenuated Vaccine Virus

[0220] The recombinant A-type H1N1 subtype influenza attenuated virus in this embodiment, the recombinant A-type H3N2 subtype influenza attenuated virus, the recombinant B-type Yamagata lineage strain influenza attenuated virus and the recombinant B-type Victoria lineage strain influenza attenuated virus are all The recombinant virus obtained by transfecting COS-1 / MDCK cells.

[0221] 1. Preparation of recombinant influenza tetravalent live attenuated vaccine virus

[0222] 1. Preparation of virus inoculum

[0223] Use PBS solution (0.01mol / L, pH7.2) respectively with the recombinant A-type H1N1 subtype influenza attenuated virus, the recombinant A-type H3N2 subtype influenza attenuated virus and the recombinant B-type Yamagata lineage strain influenza in the above-mentioned embodiment 1 Attenuated virus and recombinant type B Victoria lineage influenza attenuated virus were c...

Embodiment 3

[0237] Example 3, Preparation and Effect Evaluation of Recombinant Influenza Quadrivalent Live Attenuated Vaccine

[0238] 1. Recombinant influenza quadrivalent live attenuated vaccine

[0239] The purified recombinant A-type HIN1 subtype influenza attenuated virus, the recombinant A-type H3N2 subtype influenza attenuated virus, the recombinant B-type Yamagata lineage strain influenza attenuated virus and the recombinant B-type Victorialineage strain obtained in step 1 of Example 2 TCID 50 The titer is mixed in equal amounts at 1:1:1:1 to obtain a recombinant influenza quadrivalent live attenuated vaccine; a protective agent (mass fraction of 0.1-0.5% trehalose or chitosan) is added to the recombinant influenza quadrivalent live attenuated vaccine Sugar, the mass fraction is 0.1-0.3% gelatin, the mass fraction is 0.2-0.8% sucrose, the mass fraction is 0.1-0.3% latex protein and the mass fraction is 0.1-0.8% albumin), and the recombinant influenza tetravalent attenuated activi...

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Abstract

The invention discloses a preparation method and an application of a recombinant cold-adaptation attenuated influenza vaccine strain. The method comprises the following steps: importing an influenza virus hemagglutinin HA gene, an influenza virus neuraminidase NA gene as well as seven genes, namely PB2, PB1, PA, NP, M, NS1 and NS2, in an influenza virus to a host cell, and conducting cultivating, so that a recombinant virus is obtained. Tests prove that an attenuated influenza tetravalent vaccine, which is prepared by virtue of a 7+2 plasmid rescuing system, can achieve an excellent immune effect through nasal immunization; the influenza virus, which is recombined by the 7+2 plasmid rescuing system, has a property of being time-efficient; in accordance with the epidemic characteristics of influenza viruses in various years, effective targeted influenza vaccines can be prepared; and the preparation method is rapid, obvious in immunogenicity and immune protection effect, and good in safety; therefore, the problems on safety and stability of attenuated influenza vaccines which are prepared by virtue of a conventional method are solved, and a protection spectrum of the influenza vaccines is more comprehensive.

Description

technical field [0001] The invention belongs to the field of vaccine production, in particular to a method and application of a recombinant cold-adapted attenuated influenza vaccine strain, in particular to a recombinant influenza virus rescued by a multi-plasmid system and its preparation method and application. Background technique [0002] Influenza is an acute febrile respiratory infectious disease caused by influenza virus, which can cause serious complications. The antigens of influenza virus are constantly changing, and they are highly contagious, often causing large-scale epidemics. In normal epidemic seasons, about 10% of the world's population, or more than 600 million people, suffer from influenza. At present, no ideal prevention and treatment drugs have been found. Influenza vaccination is still an effective means of preventing influenza today. Influenza vaccination to healthy people, 70-80% of people will have the effect of preventing the disease (when the circ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N7/01A61K39/145A61P31/16
CPCC12N15/85A61K39/12C07K14/005C07K14/745C12N7/00C12N9/2402C12N2760/16121C12N2760/16122C12N2760/16134C12N2760/16221C12N2760/16222C12N2760/16234C12N2800/107C12Y302/01018
Inventor 杨鹏辉程晋霞刘晓峰刘洪许振国王鋮
Owner CHANGCHUN HAIJIYA BIOTECH CO LTD
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