丙型肝炎病毒C区部分基因的克隆表达及抗原性

目的 研究丙型肝炎病毒（ｈｅｐａｔｉｔｉｓ Ｃ ｖｉｒｕｓ，ＨＣＶ）不同长度Ｃ蛋白片段在大肠杆菌中的表达和纯化及其抗原性。方法 应用核酸及蛋白分析软件对Ｃ蛋白的抗原决定簇预测分析，以ＰＣＲ方法扩增３段长度分别为２０１ｂｐ，４０２ｂｐ及５９１ｂ；ｐ的Ｃ基因片段，通过基因体外重组技术克隆到ｐＧＥＸ－３Ｘ表达质粒中，用所获重组蛋白免疫ＢＡＬＢ／Ｃ鼠并应用ＥＬＩＳＡ法对银川市动物系列稀释血清进行抗体测定和

Expression and immunological reactivity of recombinant HCV-core protein

OBJECTIVE: To express HCV-core proteins in E.coli and to develop effective HCV-core DNA-based vaccine. METHODS: The vector that expresses the highly conserved HCV core genes were constructed. The pGEX-3X HCVCore constructs contained the 1-201 ncls (1-67aa, C201), 1-402 ncls (1-134aa, C402) and 1-591ncls ( 1-197aa, C591), then expressed in E.coli cells. RESULTS: The products of HCV C201 and C402 genes were expressed as a fusion protein with glutathione-S-transferase (GST, 26kDa) whose molecular weight were 3.1 x 10(4) and 3.9 x 10(4) separately. C591 gene was not effectively expressed in E.coli. The expressed proteins were sequestered within inclusion bodies (IB) and a variety of procedures designed to minimize IB formation proved unsuccessful. The method finally adopted involved the purification of inclusion bodies followed by the solubilization, purification, and refolding of the expressed protein. The purified C402 protein was antigenically reactive with serum from chronically infected HCV patients. BALB/C mice were immunized by a subcutaneous injection of C402 protein together with Freund's complete adjuvant which produced strong anti-HCV core humoral immune responses. CONCLUSION: It is important for the study of gene vaccine to construct a certain length of HCV core gene.