| 一株H9(N2)型禽流感病毒HA基因的克隆与序列分析 |
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| 引用本文： | 袁建琴,高斌战.一株H9(N2)型禽流感病毒HA基因的克隆与序列分析[J].山西农业大学学报(自然科学版),2008,28(1):60-63. |
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| 作者姓名： | 袁建琴 高斌战 |
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| 作者单位： | 1. 山西农业大学,生命科学学院,山西,太谷,030801
2. 山西隆克尔股份有限公司,山西,太谷,030800 |
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| 基金项目： | 山西农业大学校科研和教改项目 |
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| 摘 要： | 利用反转录-聚合酶链式反应(RT-PCR)技术扩增出禽流感病毒(H9亚型)血凝素的cDNA。通过T-A连接将已加A尾的PCR产物连接到线状克隆载体pGEM-Teasy vector,转化J M109感受态细胞,在含氨苄青霉素的LB平板上筛选阳性克隆。经PCR扩增,进一步确证为目的片段。对目的片段进行测序,结果获得该毒株的HA基因全长。与已知序列进行同源性比较,同源性最高的可达98%,表明这些分离株的亲缘关系较近;系统发育分析表明,该毒株属欧亚种系;通过血凝素裂解位点的氨基酸序列分析可知,该分离株的HA切割位点上未见到典型的高致病力毒株H5、H7所具有的一系列碱性氨基酸,其排列顺序为-PARSSRGLF-。所克隆的基因为禽流感病毒HA基因芯片的研究奠定了基础,同时为诊断和预防AI提供了理论依据。
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| 关 键 词： | 禽流感病毒 HA基因 克隆 |
| 文章编号： | 1671-8151(2008)01-0060-04 |
| 修稿时间： | 2006年12月15 |
Cloning of HA Gene of H9(N2)Avian Influenza Virus |
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| YUAN Jian-qin.Cloning of HA Gene of H9(N2)Avian Influenza Virus[J].Journal of Shanxi Agricultural University,2008,28(1):60-63. |
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| Authors: | YUAN Jian-qin |
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| Abstract: | In this experiment,avian influenza isolate(H9)was propagated in SPF chicken embryo.Virus RNA was extracted from allantoic fluid by means of guandidinium isothiocyanate.The full-length HA cDNA was amplified using RT-PCR.Then the product was cloned by adding A-tail into pGEM-T easy vector by T-A ligation.The JM109 competent cells were transformed with the recombinant plasimid.Consequently,the recombinant plasimid was amplified and extracted from the transformants and tested the inserting of DNA fragment.Sequencing the HA fragment of the positive clone showed that we had cloned one strain whole length of HA gene.Sequencing the HA fragment of the positive clone showed that we had cloned the strain whole length of HA gene(about).Compared with the published HA sequence,the highest homology was 98%,showing that the virus was closely related;Phylogenetic analysis of the virus revealed cluster with Eurasian origin;none of the AIVs had multiple basic amino acids at the cleavage site that confered high pathogeicity to some H5 and H7 AIVs.The product provided a good foundation for the study of gene chips of AIV and theory of diagnosis and prophylaxis of AI. |
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| Keywords: | Avian influenza virus HA gene Cloning |
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