H1亚型猪流感病毒广东株HA基因的原核表达

为了研制猪流感检测试剂和流感亚型特异性单抗筛选所需的重组抗原,本研究克隆和原核表达了H1N1亚型猪流感病毒的血凝素(hemagglutinin,HA)基因.首先采用RT-PCR方法扩增了H1亚型猪流感病毒(SIV)广东株的HA基因.测序结果表明,扩增的SIV HA基因cDNA长度为 1 701 个核苷酸,共编码566个氨基酸.BLAST分析表明,H1N1亚型SIV广东株HA基因核苷酸序列与GenBank中已发表的中国香港特别行政区、中国大陆、美国分离的经典H1亚型毒株相近,核苷酸序列同源性在89%以上.然后将HA基因的cDNA片段亚克隆至pET32a( )表达载体中,构建重组质粒pET32a-H1,转化大肠杆菌BL21(DE3)并进行诱导表达.SDS-PAGE和凝胶扫描分析表明,HA基因在大肠杆菌中获得了高效表达,重组融合蛋白的表达量占菌体总蛋白的25.2%.经免疫印迹证实重组蛋白可以被SIV特异性抗体所识别.

Prokaryotic Expression of the HA Gene of Swine Influenza Virus H1N1 Subtype

To obtain a recombinant influenza antigen,the HA gene of H1N1 subtype swine influenza virus(SIV) isolated from Guangdong Province was successfully amplified by RT-PCR and the sequence has been submitted in GenBank.The result showed that the HA gene of this SIV isolate is most closely related to that of SIV strains isolated in China mainland,Hongkong and the United States(with homology rate more than 89%).The PCR product was then cloned into pET-32a( ) to generate a prokaryotic recombinant plasmid pET32a-H1.After induced with IPTG,the HA gene was successfully expressed in the E.coli BL21(DE3).The highest expression content of the target protein added up to 25.2% of the total bacterial protein.Western-blotting analysis revealed that the recombinant protein could be recognized by H1N1 subtype SIV positive serum.