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Avian influenza vaccine and preparation thereof

A bird flu virus and recombinant gene technology, applied in the field of bird flu vaccine and its preparation, can solve problems such as poor antibody response

Active Publication Date: 2009-07-22
浙江中奇生物药业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Experimental results show that immune-induced antibody responses are suboptimal

Method used

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  • Avian influenza vaccine and preparation thereof
  • Avian influenza vaccine and preparation thereof
  • Avian influenza vaccine and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0037] Experimental Example 1: Construction of recombinant gene SP-HA-TM.

[0038] The main antigenic gene of avian influenza virus is the HA gene, and the 5' and 3' ends of the HA gene are respectively connected with the signal peptide gene sequence encoding the baculovirus envelope protein gp64 and the transmembrane domain gene sequence encoding the baculovirus envelope protein gp64 , to construct the recombinant gene. By primer design, BamH I and Not I endonuclease restriction sites were introduced at the 5' and 3' ends of the recombinant gene, respectively. Three pairs of primers were designed using the signal peptide gene sequence of baculovirus AcNPV envelope protein gp64, the HA gene sequence of avian influenza virus H5N1, and the transmembrane domain gene sequence of baculovirus AcNPV envelope protein gp64 as templates, and first PCR amplified Target fragments: gp64 signal peptide gene sequence (sp), HA gene and gp64 transmembrane domain gene sequence (tm), and then u...

experiment example 2

[0098] Experimental Example 2: Construction of Cloning Plasmid pGEM-HA

[0099] Connect the recombinant gene sp-HA-tm amplified by PCR to the pGEM-T vector:

[0100] Recombinant gene sp-HA-tm 2ul

[0101] pGEM-T vector 0.5ul

[0102] 2×Rapid Ligation buffer 5ul

[0103] T4DNA ligase 1ul

[0104] wxya 2 O 1.5ul

[0105] 10ul

[0106] The above components were mixed and reacted at 25°C for 1h.

[0107] The reaction mixture was transformed into competent cells E.coli DH5α, spread on a plate, and cultured at 37°C. Colonies could appear after 12-16 hours. Pick a single colony, extract the plasmid, carry out enzyme digestion and sequencing, the sequence is completely correct, and the plasmid pGEM-HA is obtained.

experiment example 3

[0108] Experimental Example 3: Acquisition of the Baculovirus Transfer Plasmid pBacHA Containing the Recombinant Gene SP-HA-TM

[0109] Plasmid pGEM-HA was digested by BamH I and Not I, and the fragment of recombinant gene SP-HA-TM was cut out, and cloned into the plasmid pBacPAK8 that was digested by BamH I and Not I, and the recombinant gene SP-HA-TM was placed Under the control of the polyhedrin (ph) gene promoter, a recombinant transfer plasmid pBacHA was constructed, and the sequence of the gene was confirmed to be correct by restriction analysis.

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Abstract

The invention relates to an avian influenza vaccine and a preparation method thereof, in particular to the preparation of a recombinant plasmid and a recombinant virus in the avian influenza vaccine preparation. The invention provides a recombinant gene and a preparation method thereof, and the recombinant gene is formed by fusing a baculovirus envelope protein gp64 signal peptide gene sequence, a major antigen gene HA sequence of an avian influenza virus and a baculovirus envelope protein gp64 transmembrane gene sequence. The invention further provides a recombinant plasmid that is constructed by recombining the recombinant gene and a pBacPAK8 plasmid, the recombinant bombyx mori baculovirus BmHA that contains the recombinant gene and a virus preparation method. The recombinant bombyx mori baculovirus BmHA that contains the recombinant gene is preserved in China General Microbiological Culture Center of Microbial Culture Collection Management Committee with the preservation number of CGMCC No.2316. The invention further provides an expression protein of the recombinant gene. The recombinant bombyx mori baculovirus BmHA and the protein that are provided by the invention can be used for preparing the avian influenza vaccine. The methods of the invention are applicable to large-scale production, and have lowered cost, high yield and large application value of the produced avian influenza vaccine.

Description

technical field [0001] The invention relates to an avian influenza vaccine and its preparation, in particular to the preparation of a recombinant plasmid and a recombinant virus in the preparation of the avian influenza vaccine. Background technique [0002] Avian influenza is an acute respiratory infectious disease caused by some strains of certain subtypes of avian influenza A virus. The large-scale outbreak of animal bird flu will not only bring serious losses to the farmers, but also will bring great losses to the national economy, and at the same time, will also endanger the survival and safety of human beings. [0003] Animal bird flu vaccine is an effective method to prevent and treat animal bird flu. [0004] Existing vaccines are produced using chicken embryos, and manufacturers need to order chicken embryos 12 months in advance. The virus needs to adapt to the chicken embryo before it can grow in the chicken embryo, and the production cycle of the vaccine is long...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/44C12N15/866C12N7/01C07K14/11A61K39/145A61K39/00A61P31/16
Inventor 张耀洲金来荣吕正兵陈琴
Owner 浙江中奇生物药业股份有限公司
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