抗乙肝病毒表面抗原单链抗体在大肠杆菌中的高效表达

高荣凯　王　琰　刘群英　朱迎春　化　冰　陈宇萍

　　中国图书分类号　R371-33
　　摘　要　目的：在大肠杆菌中高效表达抗乙肝表面抗原的单链抗体。方法：通过PCR将单链抗体基因重组到原核细胞表达载体pT7中，构建单链抗体高效表达载体pT7SC。将pT7SC质粒转化大肠杆菌BL21(DE3)，IPTG诱导进行表达。结果：获得了ScFv的高效表达，表达产物占菌体总蛋白的28%以上。竞争抑制性ELISA检测诱导后的细菌培养上清，证明所表达的ScFv具有抗体活性。进一步将c-Myc十肽连接在ScFv的羧基端，使所表达的ScFv易于检测。结论：在大肠杆菌高效表达抗乙肝ScFv，将促进小分子抗体的应用。
　　关键词　乙肝病毒表面抗原　单链抗体　高效表达

High level expression of anti-HBsAg single-chain Fv in E.coli

GAO Rong-Kai，WANG Yan,LIU Qun-Ying et al.
Central Laboratory,Navy General Hospital,Beijing 100037

　　Abstract Objective：To obtain high level expression of anti-HBsAg ScFv in E.coli.Methods：An anti-HBsAg ScFv expression vector pT7SC was constructed by inserting the ScFv gene obtained by PCR amplification from vector pHBSCS constructed previously into a T7 promoter containing vector. pT7SC was transformed into E.coli BL21(DE3) cells and expression was induced by IPTG.Results:The ScFv product constituted 28% of the total bacterial protein.HBsAg binding activity was detected in the supernatant of the induced bacteria by competitive ELISA.In order to facilitate the detection of ScFv,the vector was further modified attaching sequences encoding c-Myc derived decapeptide to the 3′-end of ScFv gene.ScFv protein thus produced could be easily detected by ELISA using McAb 9E10.Conclusion:Success in high level expression of anti-HBsAg ScFv would facilitate the use of small antibody molecules.
　　Key words HBsAg　Single-chain Fv(ScFv)　High level expression

　　单链抗体是将重链可变区(VH)和轻链可变区(VL)通过一个人工设计的柔性连接肽连接在一起，形成单一肽链的小分子抗体，它以一条多肽链的形式表达存在，能够正确折叠，保持与特异抗原结合的能力［1，2］。用噬菌体抗体库技术克隆了抗HBsAg的人抗体基因［3，4］，并构建了抗HBsAg单链抗体的分泌型表达载体［5］，本文在此基础上构建了ScFv的高效表达载体，并在大肠杆菌中获得高效表达。

1　材料与方法
1.1　大肠杆菌HB101、BL21(DE3)为本室保存菌种。质粒pHBSCS为本室构建的分泌型ScFv表达载体［5］，内含抗HBsAg单链抗体基因片段。质粒pT7为原核细胞表达载体，含有T7 RNA聚合酶启动子和终止子，由美国引进。分泌 9E10 单抗的杂交瘤细胞株由美国引进，9E10单抗所针对的抗原决定基是c-Myc蛋白内的一段十肽［6］。
1.2　为了构建pT7SC,参照ScFv基因序列合成两段特异引物。5′-引物为GCGGCCATATGAAAAAACA
GCG-TA

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