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Gene encoding hemagglutinin protein of H5 avian influenza virus and its application

An avian influenza virus, hemagglutinin technology, applied in applications, antiviral agents, genetic engineering and other directions, can solve the problems of lack of competitiveness in economic feasibility and increased use costs

Active Publication Date: 2007-07-18
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the DNA vaccine plasmid pCIHA expressing the GD / 1 / 96 (H5N1) HA gene not only exhibits good immunogenicity, but also induces effective protective HI antibodies that can last up to one year (H Chen, et al. DNA immunization elicitshigh HI antibody and protects chicken from AIV challenge.ImmuntionalCongress Series 1219(2001)917-921. Jiang Yongping. The optimization of antigen gene codon and its expression vector enhances the immune protection effect of H5 subtype avian influenza DNA vaccine [PhD thesis ]. Harbin, Harbin Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences, 2004.), but due to the high dose dependence of the H5 subtype avian influenza DNA vaccine, the cost of use is increased, and the economic feasibility is not competitive compared with the traditional avian influenza inactivated vaccine

Method used

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  • Gene encoding hemagglutinin protein of H5 avian influenza virus and its application
  • Gene encoding hemagglutinin protein of H5 avian influenza virus and its application
  • Gene encoding hemagglutinin protein of H5 avian influenza virus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1. Rewriting and Sequence Analysis of Codons of H5 Subtype HA Gene Chicken Preference

[0054]Using the codon usage frequency table (see attached table 1, National Science Digital Library, Life Science Discipline Information Portal, Codon Usage Database, http: / / www.kazusa.or.jp / codon / ), the H5 subgroup All the codons of the HA gene of type avian influenza virus A / Goose / Guangdong / 1 / 96(H5N1)[GD / 1 / 96(H5N1)] were converted into codons for chicken preference, and the codon-optimized codons were written The sequence of the HA gene (named as optiHA), 1779bp in length (SEQ ID NO: 1, see Figure 1), and the DNASTAR software (DNASTAR.Inc.) was used to analyze the sequence and restriction site of the optiHA gene sequence, according to the optiHA gene The optiHA gene can be divided into 11 fragments with a length of about 200bp, and the 11 fragments are named optiHAF1 (fragment length 88bp, position 1-88 on the gene optiHA), optiHAF2 (fragment 162bp long, the position on th...

Embodiment 2

[0058] Example 2. Synthesis of oligonucleotide fragments and primers for OE-PCR

[0059] The oligonucleotide fragment optiHAF1-Forward (101) of fragment optiHAF1 is directly sent to be synthesized by Sigma Company; the two oligonucleotide fragments of fragment optiHAF2 are optiHAF2-Forward (90) and optiHAF2-Reverse (101); the two fragments of optiHAF3 Two oligonucleotide fragments are optiHAF3-Forward (110) and optiHAF3-Reverse (110); two oligonucleotide fragments of fragment optiHAF4 are optiHAF4-Forward (99) and optiHAF4-Reverse (100); two fragments of optiHAF5 The two oligonucleotide fragments are optiHAF5-Forward (97) and optiHAF5-Reverse (99); the two oligonucleotide fragments of fragment optiHAF6 are optiHAF6-Forward (110) and optiHAF6-Reverse (110); the two fragments of optiHAF7 The two oligonucleotide fragments are optiHAF7-Forward (110) and optiHAF7-Reverse (110); the two oligonucleotide fragments of fragment optiHAF8 are optiHAF8-Forward (91) and optiHAF8-Reverse (10...

Embodiment 3

[0069] Example 3. OE-PCR amplification, cloning and sequencing identification of 11 fragments of optiHA gene optiHAF1-optiHAF11

[0070] 11 fragments optiHAF1-optiHAF11 were used Platinum  Pfx Taq DNA polymerase amplification. First, add 10ul of 10X Pfx Taq polymerase buffer, 4ul of 2.5mmoldNTPs, 1ul of Pfx Taq DNA polymerase (1-2U) and the corresponding forward and reverse oligonucleotide fragments (for optiHAF1 only add forward oligonucleotides) to the PCR tube Nucleotide fragments) each 10ul, sterilized deionized water to supplement 100ul, 95°C for 5min, 48°C to 55°C for 1min, 72°C for 5min, and then add 20pmol corresponding to each oligonucleotide fragment after 5 cycles 1 ul each of downstream primers (attached table 3) was amplified on the PTC-100 gene amplification instrument (M.J.Research.inc) (for optiHAF1, only upstream primers were added). The reaction conditions of OE-PCR are 48°C for 1min, 72°C for 1.5min, 30 cycles, and finally 72°C for 5min, take 5ul of PCR p...

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Abstract

The present invention relates to a artificial synthetic chicken bias codons containing gene optiHA which contains 1707bp nucleotide at its reading frame, encodes totally 568 amino acids, wherein said has nucleotide consanguinity rate of 70% and amino acid consanguinity rate of 100% with H5 subtype high-virulence avian influenza virus A / Goose / GuangDong / 1 / 96(H5N1)[GD / 1 / 96(H5N1)] hemagglutinin (HA) gene and encodes the H5 subtype avian influenza virus GD / 1 / 96(H5N1) hemagglutinin (HA) protein. At the same time, the present invention relates to applications of the gene when it is used as immunogen gene of H5subtype influenza DNA immunomodulators for vaccines and vaccines in other genetic engineerings.

Description

technical field [0001] The present invention relates to a kind of artificially synthesized gene optiHA containing codons of chicken partial tropism. 96(H5N1)] The nucleotide homology rate of the hemagglutinin (HA) gene is 70%, the amino acid homology rate is 100%, and the blood of the H5 subtype avian influenza virus GD / 1 / 96 (H5N1) is encoded and expressed lectin (HA) protein. The gene can be used as the immunogenic gene of H5 subtype influenza DNA vaccine and other genetic engineering vaccines. Background technique [0002] Avian Influenza (Avian Influenza, AI) is an avian infection and / or disease syndrome caused by Avian Influenza Virus (AIV), which belongs to: Virus Kingdom (Vira) ---- Orthomyxovirus in taxonomy Section (Orthomyxoviridae) ---- Influenza Virus (Influenza Virus A and B) ---- Avian Influenza Virus (Avian Influenza Virus). [0003] Highly Pathogenic Avian Influenza (HPAI) is listed as a Class A severe infectious disease by the International Veterinary Bure...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/33C12N15/63C12N15/85A61K39/12A61K39/395A61P31/16C12N5/06C12N5/08C12N5/10
Inventor 陈化兰姜永萍步志高于康震
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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