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

A technology of avian influenza virus and hemagglutinin, applied in application, genetic engineering, virus antigen components, etc., can solve problems such as increased use cost and lack of competitiveness in economic feasibility

Active Publication Date: 2005-06-29
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
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Effect test

Embodiment 1

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

[0054]Using the codon usage frequency table (see Supplementary 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 positi...

Embodiment 2

[0077] Example 2. Synthesis of oligonucleotide fragments and primers for OE-PCR

[0078] 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

[0172] Example 3. OE-PCR amplification, cloning and sequencing identification of 11 fragments of optiHA gene optiHAF1-optiHAF11

[0173] The 11 fragments optiHAF1-optiHAF11 were used in Platihum  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 extension,...

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Abstract

The present invention relates to an artificially synthesized gene optiHA containing codons for chicken partial tropism. Its reading frame contains 1707 bp nucleotides and encodes a total of 568 amino acids. The gene is compatible with H5 subtype highly pathogenic avian influenza virus A / Goose / GuangDong / 1 / 96(H5N1)[GD / 1 / 96(H5N1)]hemagglutinin (HA) gene has a nucleotide homology rate of 70%, an amino acid homology rate of 100%, and encodes the H5 subtype Hemagglutinin (HA) protein of avian influenza virus GD / 1 / 96 (H5N1). The invention also relates to the application of the gene as an immunogenic gene of H5 subtype influenza DNA vaccine and other genetic engineering vaccines.

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 Applications(China)
IPC IPC(8): A61K39/12A61K39/395C12N5/10C12N15/33C12N15/63C12N15/85
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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