摘要
目的研究 HCV核心蛋白在大肠杆菌中的非融合表达及重组蛋白的免疫学特性。方法用 DNA重组技术在两种表达质粒 (p QE3O和 p Trc His A)、4种宿主菌 (DH5 a,TOP10 ,BL2 1和 M15 )中表达 HCV全长及羧基端部分缺失的核心蛋白 (aa1～ 191,aa1～ 15 4和 aa1～ 6 9) ,表达蛋白经 SDS- PAGE检测 ,免疫印迹分析 ,亲和层析法纯化后免疫 BAL B/C小鼠 ,EL ISA法检测免疫小鼠血清中抗 HCV抗体水平。结果 HCV核心蛋白 C15 4,C191在 p QE30 /M15中获得了表达 ,表达量分别占菌体总蛋白的 18.2 %和 9.3%。免疫印迹分析的结果显示在 C15 4和 C191相应位置处出现杂交条带。 EL ISA结果显示C15 4和 C191诱导小鼠产生了抗 HCV抗体。结论表达的重组蛋白具有抗原特异性和免疫原性。“截断”型
ObjectiveTo express HCV core proteins in E coli and investigate the antigenicity and immunogenicity of expressed proteins MethodsThree cDNA sequences encoding full length of HCV core protein (C191) and two truncated forms (C154 and C69) which lack parts of the carboxyl terminal were amplified by PCR and inserted into plasmids pQE30 and pTrcHisA The resulting expression vectors were transformed into 4 strains of E coli (DH5a, TOP10, BL21, and M15) The proteins produced were detected by SDS PAGE and western blot and purified by affinity chromatograph Immunization of BALB/C mice was carried out and the antibodies to HCV in mice serum were tested by ELISA ResultspQE30 C191, C154/M15 produced C191 and C154 with yields of 18 2% and 9 3% of total proteins respectively The expressed C154 and C191 showed specific immunoreactivity with serum from hepatitis C patient in western blot assay The antibodies to HCV were detectable in serum of mice immunized with C154 and C191 ConclusionThe expressed HCV core proteins exhibited antigenicity and immunogenicity The absence of parts of carboxyl terminal didn't weaken the antigenicity and immunogenicity of 'truncated' forms of HCV core proteins
出处
《免疫学杂志》
CAS
CSCD
北大核心
2001年第6期425-429,共5页
Immunological Journal
基金
云南省科委青年基金资助项目 (98C0 2 4Q)
