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目的 研究丙型肝炎病毒 (HCV)多表位抗原重组体在大肠杆菌中的非融合表达及其免疫原性分析。方法 利用DNA重组技术将构建的HCV多表位抗原重组体克隆入原核表达载体pBV2 2 0并在BL 2 1中表达 ,非融合表达蛋白经SDS PAGE检测 ,排阻层析纯化 ,利用ELISA和免疫印迹分析其抗原反应性。免疫昆明鼠和猕猴 ,并检测其抗体和特异性CTL(Cyto toxicTlymphocyte) ,在抗体转阴后再次用HCV病人血清攻击以检测其免疫应答能力。结果 HCV多表位重组体在pBV2 2 0 BL 2 1中表达量占菌体总蛋白量的 15 %。Western blot和ELISA分析表明HCV多表位抗原能与HCV病人血清特异性结合 ,抗体水平和特异性CTL检测结果显示该抗原肽能诱导小鼠和猕猴产生较好的抗体水平和特异性CTL效应。用病人血清再次攻击后受试动物的抗体迅速阳转。结论 表达的多表位重组体具有特异的抗原反应性和免疫原性
Objective To study the non-fusion expression and immunogenicity analysis of recombinant hepatitis C virus (HCV) multi-epitope antigen in Escherichia coli. Methods Recombinant HCV multi-epitope antigen was cloned into prokaryotic expression vector pBV220 using DNA recombination technology and expressed in BL21. The non-fusion protein was purified by exclusion chromatography and ELISA. Blots were analyzed for antigen reactivity. Immunization of Kunming mice and macaques, and detection of their antibodies and specific CTL (Cyto toxicTlymphocyte), the antibody negative again after the attack with HCV patient serum to detect its immune response capacity. Results The expression level of HCV polytope was 15% of the total protein in pBV2 2 0 BL 2 1. Western blot and ELISA analysis showed that the HCV multi-epitope antigen could specifically bind to the serum of HCV patients. The antibody level and CTL detection showed that the peptide could induce good antibody level and specific CTL effect in mice and macaques. After the patient’s serum is challenged again, the test animal’s antibody rapidly turns positive. Conclusion The expressed multi-epitope recombinant has specific antigenic reactivity and immunogenicity