禽流感病毒(H9亚型)重组CHO细胞258株的构建及鉴定

Construction and Identification of Recombinant CHO Cell 258 Strains of Avian Influenza Virus(H9 Subtype)

为构建禽流感病毒(AIV)重组CHO细胞株,本研究以AIV(H9亚型)258株为模板,以AIV的HA基因胞外域及3个重复串联的M2蛋白胞外域为靶基因,根据CHO细胞密码子偏爱性对其密码子进行优化,将HAM2e基因插入真核表达载体pCI-GS中,构建了含有HA-M2e基因的重组质粒,并将该重组质粒转染CHO细胞,经加压筛选获得了高效表达HA-M2e蛋白的重组CHO细胞株。采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)和蛋白质免疫印迹(Western Blot)法对重组CHO细胞株进行鉴定,并用琼扩试验及酶联免疫吸附试验(ELISA)对表达蛋白进行效价及蛋白含量测定。结果显示,经SDS-PAGE、Western Blot检测,表达蛋白大小约66 ku,与预期大小相符;琼扩效价为1􀏑512,蛋白质含量为1.13 mg·mL^(-1)。表明通过基因工程技术构建的含AIV HA-Mze基因的重组CHO细胞258株可作为疫苗研制用毒株。

In order to construct recombinant CHO cell line of avian influenza virus(H9 subtype),258 strains of avian influenza virus(H9 subtype)were used as the template,and HA gene extracellular domain of avian influenza vincs and three repeated tandem M2 protein extracellular domain were used as the target genes.The codon of CHO cell was optimized according to codon preference.Recombinant plasmid containing HA-M2e gene was constructed by inserting HA-M2e gene into eukaryotic expression vector pCI-GS.The recombinant plasmid was transfected into CHO cells,and the recombinant CHO cell line expressing HA-M2e protein was obtained by pressure screening.The recombinant CHO cell lines were identified by SDS-PAGE and Western Blot,and the titer and protein contents of the expressed proteins were determined by agonal expansion test and enzyme linded immunosorbent assay(ELISA).The results showed that the expressed protein was about 66 ku in size by sodium dodecyl sulfate polyacryla mide gel electrophoresis(SDS-PAGE)and Western Blot,which was consistent with the expected size.AGAR titer was 1􀏑512 and the protein content was 1.13 mg·mL^(-1).The results showed that the recombinant CHO cell 258 strain containing AIV HA-M2e gene constructed by genetic engineering technology could be used as a vaccine strain.