庚型肝炎病毒NS5cDNA片段的表达及其免疫原性的研究

Expression in E.Coli and Antigenicity of HGV NS5 Protein

一段长度为 880bp的庚型肝炎病毒cDNA在大肠杆菌BL2 1(DE3)菌株中得到表达。此cDNA被插入到表达质粒 pGEX 5X 1中 ,位于编码日本血吸虫谷胱甘肽硫转移酶 (GST)的DNA序列下游 ,并与GST处于同一阅读框。用乳糖在 37℃下诱导表达出以包涵体形式存在的GST NS5 3融合蛋白 ,并用脲溶法提取了该蛋白 ;在 2 0℃诱导时 ,表达出的蛋白大部分可溶 ,用谷胱甘肽Sepharose 4B亲和层析柱对可溶性的融合蛋白进行了纯化。免疫印迹实验证明 ,此融合蛋白能被庚型肝炎病人的血清和自制的抗GST血清特异性地识别。用PCgene软件对NS5 3氨基酸序列的亲水性和抗原决定簇进行了分析。本研究为庚型肝炎ELISA诊断试剂研制打下了基础。

A 880 bp cDNA localized to the putative NS5 region of HGV genome was expressed in E.coli BL21(DE3). The cDNA fragment was inserted into a plasmid pGEX 5X 1,at the downstream of the DNA sequence encoding Schistosoma japonicum glutathione S transferase(GST),in the same reading frame with the gene of GST. A 60KD GST NS53 fusion protein was expressed at 37℃ in a form of inclusion bodies amounting to 30 percent of total host protein whereas at 20℃ mainly in a form of soluble protein . The fusion protein was extracted and purified to homologue. The purified GST NS53 fusion protein could be specifically recognized with either the sera from the patient infected by HGV or the antisera directed against GST.