人癌胚抗原单链抗体基因的构建和筛选  

Construction and Screening of the Anti-CEA Single Chain Antibody Gene

作　　者：章美云[1] 孔健[1] 

机构地区：[1]卫生部北京生物制品研究所

出　　处：《生物化学与生物物理进展》1996年第5期470-474,共5页Progress In Biochemistry and Biophysics

摘　　要：从分泌抗癌胚抗原（ｃａｒｃｉｎｏｅｍｂｒｙｏｎｉａｎｔｉｇｅｎ，ＣＥＡ）单抗的杂交瘤细胞株Ｃ５０中提取总ＲＮＡ，逆转录成ｃＤＮＡ，ＰＣＲ扩增分别得到抗体轻、重链可变区基因，再利用两对ＰＣＲ引物合成和扩增得到全单链抗体基因．将含轻、重链可变区序列的ＤＮＡ片段克隆于含噬菌体基因Ⅲ的噬菌粒ｐＣＡＮＴＡＢ５．重组克隆在噬菌体表面表达基因Ⅲ与单链抗体的融合蛋白．表达具抗原结合活性的单链抗体的重组噬菌体可以通过亲和筛选的方法筛选得到并富集．利用该方法我们可以从许多分泌不同抗体的杂交瘤细胞ＲＮＡ中快速克隆和筛选功能性抗体可变区基因．A strategy based on polymerase chainreaction (PCR) and a phagedisplay system forthe rapid method for constructing and screeningfunctional single chain antibody genes wasreported. Total RNA from a hybridoma (C50 )producing an antibody that reacts with carcinoembryonic antigen (CEA) was used as a template to make the first strand of a cDNA, lightand heavy-chain variable-region genes were separately amplified by PCR. Then whole singlechain antibody gene was synthesized and amplified by using two sets of DNA primers that (1 )hybridized to the ends of the light- and heavychain variable regions, (2) encoded a linker peptide, and (3 ) contained appropriate restrictionenzyme sites for cloning. After 30 cycles ofPCR, the DNA fragments containing sequencesencoding the light- and heavy-chain variableregions were cloned into pCANTAB 5 phagemidcontaining gene Ⅲ of the phage. Clones encoding single-chain antibody was express on the surface of the phage as a fusion with gene Ⅲ protein. Recombinant phage expressing anti-CEAsingle chain antibody which retained its antigenbinding capability was screened by affinity selection and enriched. By using this approach itshould be possible to rapidly clone and screen thefunctional variable region sequences of manydifferent antibodies from hybridoma RNA.

关 键 词：癌胚抗原 抗体基因 制备 

分 类 号：R392－33[医药卫生—免疫学]