H9(N2)型禽流感病毒HA基因的克隆与序列分析

Cloning and Sequence Analysis of HA Gene of H9(N2)Avian Influenza Virus

研究采用异硫氰酸胍法提取禽流感病毒分离株的SPF鸡胚尿囊毒RNA,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。用UNIQ-10柱式DNA胶回收试剂盒回收纯化目的片段,后连接到线状克隆载体pGEM-T easy vector,转化JM109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,并对目的片段进行测序,结果获得3个毒株的HA基因全长。与已知序列进行同源性比较,同源性最高的都可达98％,表明这些分离株的亲缘关系较近;系统发育分析表明,这三个毒株均属欧亚种系;通过血凝素裂解位点的氨基酸序列分析可知,三分离株的HA切割位点上均为-PARSSR-GLF-。所克隆的基因为珠海地区禽流感病毒HA基因芯片的研究奠定了基础。

In this study,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 Ⅰ purified and extracted the target fragment by using UNIQ-10 pillar purification kit and cloned the product into pGEM-T easy vector by T-A ligation.The JM109 competent cells were transformed with the recombinant plasmid. Consequently, the recombinant plasmid 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 have cloned the three strains whole length of HA gene (about).Compared with the published HA sequence,the highest homology is 98%,showing that these viruses were closely related;Phylogenetic analysis of the three viruses revealed that Zhuhai isolates cluster with Eurasian origin; none of the Zhuhai AIVs had multiple basic amino acids at the cleavage site.The product provided a good foundation for the study of gene chips of AIV.