H5N1型禽流感病毒HA基因克隆及序列分析

doi:10.11924/j.issn.1000-6850.casb14120128

中国农学通报 ›› 2015, Vol. 31 ›› Issue (14): 32-38.doi: 10.11924/j.issn.1000-6850.casb14120128

所属专题：生物技术；畜牧兽医

H5N1型禽流感病毒HA基因克隆及序列分析

申燕¹,高冬妮¹,安琦¹,刘颖¹,葛菁萍^1,2,平文祥^1,2

(¹黑龙江大学生命科学学院/微生物省高校重点实验室,哈尔滨 150080；²农业微生物技术教育工程研究中心,哈尔滨 150080）

收稿日期:2014-12-18 修回日期:2015-02-14 接受日期:2015-03-03 出版日期:2015-06-02 发布日期:2015-06-02
通讯作者: 平文祥
基金资助:
国家自然科学基金“表达IBDV主要保护性抗原的重组杆状病毒构建及其作为疫苗的免疫效果研究”(31270143)；国家自然科学基金“菌群演替与温水沤麻系统关键酶产生菌代谢组学特征的耦合机制”(31270534)；黑龙江省高等学校科技创新团队“农业微生物发酵技术”(2012td009)；黑龙江大学高层次人才（创新团队）支持计划“功能微生物选育及其产物应用”(Hdtd2010-17)。

Cloning and Sequence Analysis of HA Gene of H5N1 Avian Influenza Virus

Shen Yan¹, Gao Dongni¹, An Qi¹, Liu Ying¹, Ge Jingping^1,2, Ping Wenxiang^1,2

(¹College of Life Science, Heilongjiang University/Key Laboratory of Microbiology, Harbin 150080;²Agricultural Microbe Technology of Education Engineering Research Center, Harbin 150080)

Received:2014-12-18 Revised:2015-02-14 Accepted:2015-03-03 Online:2015-06-02 Published:2015-06-02

摘要/Abstract

摘要： 为从分子生物学角度进一步研究H5N1型禽流感主要抗原HA基因，探究H5N1 HA基因的遗传变异情况及其蛋白结构，根据GenBank中登录的H5N1型禽流感HA基因的序列利用Primer 5.0设计引物扩增HA基因，将其克隆到pMD18-T载体后进行鉴定，对鉴定正确的重组质粒pMD-H5N1-HA进行测序分析。应用生物信息学软件对禽流感病毒HA基因进行系统进化树分析、氨基酸序列同源性分析、保守区分析，磷酸化位点和序列重复区分析及HA蛋白二级、三级结构的预测。核苷酸序列测定结果表明：HA基因长1661 bp，共编码326个氨基酸；生物信息学分析结果表明：HA基因在种间具有较高保守性，HA基因的氨基酸序列与其他地方性禽流感H5N1病毒株HA基因的序列同源性最高可达99.7%，且为高致病性毒株；HA蛋白的二级、三级结构预测结果显示：HA蛋白多为α螺旋和β转角结构，无规则卷曲，是亲水性蛋白。该结果为禽流感病毒的分子病毒学研究奠定了基础。

关键词: 蒸散量, 蒸散量, 非线性主成分分析, RBF神经网络, 估算

Abstract: This research aimed to study HA gene of H5N1 avian influenza virus pathogen from the perspective of molecular biology and explore its genetic variation and protein structure. Primer 5.0 was used to design a pair of primers according to the sequence of HA gene of H5N1 avian influenza virus published in GenBank. HA gene was amplified, cloned to pMD18-T vector and then identified. The recombinant plasmid pMD-AIV-HA, which was identified to be correct, was then analyzed by nucleotide sequence analysis. Bioinformatics software was used to do phylogenetic analysis of HA gene, amino acid sequence homology analysis, conserved sequence analysis, phosphorylation sites analysis, protein repeated sequence analysis and the secondary and tertiary structure prediction of HA protein. The sequencing results revealed that HA gene was 1661 bp and encoded 326 amino acids. The analysis of bioinformatics indicated that HA gene was conserved in species, and amino acid sequence homology between HA gene of H5N1 avian influenza virus and of other different AIV strains could reach 99.7%. It was also a highly pathogenic strain. The secondary and tertiary structure prediction results manifested that HA protein mostly consisted of α-helix, β-turn and random coil, and was a hydrophilic protein. The results would lay a foundation for the molecular virology research of AIV.

申燕,高冬妮,安琦,刘颖,葛菁萍,平文祥. H5N1型禽流感病毒HA基因克隆及序列分析[J]. 中国农学通报, 2015, 31(14): 32-38.

Shen Yan,Gao Dongni,An Qi,Liu Ying,Ge Jingping and Ping Wenxiang. Cloning and Sequence Analysis of HA Gene of H5N1 Avian Influenza Virus[J]. Chinese Agricultural Science Bulletin, 2015, 31(14): 32-38.

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H5N1型禽流感病毒HA基因克隆及序列分析

Cloning and Sequence Analysis of HA Gene of H5N1 Avian Influenza Virus

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