H9N2亚型禽流感病毒M1基因的克隆及其原核表达

Cloning and prokaryotic expression of M1 gene of avian influenza virus H9N2 subtype

为克隆H9N2亚型禽流感病毒M1基因并研究其原核表达产物的反应原性,从H9N2亚型禽流感病毒中提取病毒基因组RNA,采用RT-PCR方法克隆了M1全长基因。把M1基因克隆至pMD18-T载体中之后进行测序。M1基因亚克隆至pET-28a(+)表达载体中,构建重组表达质粒pET-28a-M1。该重组质粒转化至大肠杆菌BL21(DE3)并经IPTG诱导表达,对表达产物进行纯化和鉴定。测序分析结果表明,本研究克隆的M1基因与数株甲型流感病毒的M1基因的核苷酸同源性为89.7%~99.1%。SDS-PAGE分析表明,构建的重组蛋白以可溶性形式存在。Western-blot鉴定结果表明,它具有良好的反应原性。M1的成功表达为进一步研制新型流感疫苗奠定了基础。

To clone an M1gene of avian influenza virus(AIV)H9N2subtype and to investigate reactionogenicity of the prokaryotic expressed product,the viral RNA was extracted from AIV H9N2and the M1gene was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR),and cloned into pMD18-T vector confirmed by DNA sequencing.The M1gene was subsequently subcloned into the expression vector pET-28a(+)to construct a recombinant plasmid named pET-28a-M1.The recombinant plasmid pET-28a-M1was transformed into EscherichiacoliBL21(DE3).The expressed M1protein under induction with IPTG was purified by His affinity chromatography and identified by Western-blot.Compared with the other published cDNA sequences,the homology of M1gene was from 89.7%to 99.1%.SDS-PAGE result indicated that the recombinant was expressed in the soluble form.Western-blot analysis showed that the purified M1protein had strong reactive responses with H9N2and H1N1positive sera,respectively. The recombinant M1protein expressed in the prokaryotic system might laid a foundation for further study on the development of novel avian influenza vaccines.