H5N1亚型禽流感病HA1基因的克隆测序与表达

提取禽流感病毒液中RNA,根据GenBank中收录的禽流感病毒(AIV)H5N1亚型的血凝素(HA)基因序列设计并合成了1对引物。RT-PCR扩增出HA全基因,并克隆到pMD18-T载体,对重组质粒进行酶切鉴定及序列测定。与GenBank收录的其他AIV H5N1亚型的HA基因进行序列比较分析,同源性达96%~99%。从HA基因序列中截取HA1基因,亚克隆到表达质粒pET-28a中构建HA1基因原核表达质粒,转化E.coliBL21(DE3)细胞,在IPTG诱导下表达出HA1蛋白,所表达的蛋白质分子质量约为37~40ku。该研究为进一步开发用于禽流感抗体检测的抗原蛋白打下了基础。

Cloning Sequencing and Expression of Hemagglutinin 1 (HA1) Gene of the H5N1 Subtype Avian Influenza Virus

Specimens of avian influenza virus(AIV)were collected from a chicken farm and were inoculated into chicken embryo and the allantoid fluid containing virus was collected.The RNA of the virus was extracted.Primers were designed and synthesized according to the sequences of hemagglutinin(HA)gene published in GenBank.The whole sequence of HA was amplified with RT-PCR,and inserted into pMD18-T vector,the recombinant plasmid was identified by digesting with restricted endonuclease and sequencing.Compared with other sequences of AIV subtype strain H5N1 in GenBank,the results showed that they shared 96%~99% identity.HA1 gene was obtained from HA sequence and subcloned into pET28a.Therefore an expression plasmid of HA1 pET28-HA1 was constructed.The plasmid was transformed into E.coli BL21(DE3),and then HA1 was expressed after induced with IPTG.The molecular weight of expression protein was about 37~40 ku.It provides a basis of the antigen development for use in the detection of AIV antibody.