庚型肝炎病毒NS5 cDNA片段的表达及其免疫原性的研究

一段长度为880bp的庚型肝炎病毒cDNA在大肠杆菌BL21(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-NS53融合蛋白，并用脲溶法提取了该蛋白；在20℃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NS53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。

Expression in E.Coli andAntigenicity of HGV NS5 Protein

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.