pGEX载体表达马立克氏病病毒囊膜糖蛋白gI基因的最佳条件

STUDIES ON IN VITRO EXPRESSION FOR gI GENE OF MAREK'S DISEASE IN E.COLI BY pGEX VECTOR

通过聚合酶链式反应 (PCR) ,扩增出马立克氏病病毒特超强毒 (vv +MDV) 648株囊膜糖蛋白gI基因 ,并将该基因按正确的阅读框 (ORF)克隆到表达性载体质粒pGEX 6P 1中谷胱甘肽转移酶 (GST)基因的下游。重组质粒 (pGEX gI)经氯化钙转化宿主菌BL2 1 ;通过建立重组菌生长时间与OD60 0 值间的关系曲线 ,以及对诱导时间、诱导温度、IPTG浓度等条件的摸索 ,根据聚丙烯酰胺凝胶电泳 (SDS PAGE)判定GST gI融合蛋白的最佳表达条件 ,并经蛋白质印迹试验 (WesternBlotting)对表达产物进行了验证。将表达产物免疫小鼠 ,所得抗血清能与MDV感染的鸡胚成纤维细胞 (CEF)在间接免疫荧光试验 (IFA)中 ,呈较强的细胞膜荧光着色。实验结果表明 :IPTG的最佳浓度为 0 2～ 0 5mmol L ;最适的诱导时期为重组菌生长对数中期 ;温度对表达几乎没有影响。

Glycoprotein I Gene(gI) was amplified from genomic DNA of Marek's disease virus(MDV) 648 strain by polymerase chain reaction(PCR).PCR product was cloned into pGEX\|6p\|1 according to the right open reading frame(ORF).The expression of GST\|gI fusion protein was studied in detail on many factors including temperature,timing and IPTG.The curve for OD 600 and the growing time of the recombinant bacteria is also established.,which is helpful to find the optimal inducing time.GST\|gI fusion protein was identified by SDS\|PAGE and Western\|blotting., and the result shows that the best concentration of IPTG is 0.2～0.5mmol/L and inducing time have great effects on expression while temperature has little.The fusion protein was injected into mouse five times to identify its antigenicity and the result is positive in indirect fluorescent assay IFA.