原核分泌型表达载体的构建及癌胚抗原单链抗体的表达  

Construction of the prodkaryotic secretory expression vector and the expression of anti-CEA signle-chain antibody gene

作　　者：章美云[1,2] 杨青[1,2] 赵莉[1,2] 孔健[1,2] 

机构地区：[1]卫生部北京生物制品研究所 [2]中国科学院遗传研究所

出　　处：《中华微生物学和免疫学杂志》1998年第1期72-75,共4页Chinese Journal of Microbiology and Immunology

摘　　要：目的构建分泌型载体并表达癌胚抗原单链抗体。方法以ｐＧＥＭ－１１ｚｆ（＋）为基础，插入化学合成的１４个寡核苷酸片段（共１７６ｂｐ），构建成含核糖体结合位点（ＲＢＳ）、翻译起始点（ＡＴＧ）、果胶酶信号肽基因（ｐｅｌＢ）和翻译终止点（ＴＡＡ）的分泌型表达载体（ＲＰＹ）。化学合成带有抗体重、轻链可变区基因插入位点和惰性连接序列的８２ｂｐ片段，并克隆到ＲＰＹｐｅｌＢ信号肽的下游，构建成抗体分泌型表达载体ＲＰＹＡ。将ＣＥＡ单抗重、轻链可变区基因（ＶＨ、ＶＬ）插入到ＲＰＹＡ的相应位点，转化大肠杆菌ＨＢ２１５１，ＩＰＴＧ诱导表达。结果序列测定表明ＲＰＹＡ的序列与原设计序列一致，免疫印迹实验表明表达产物具有ＣＥＡ结合活性，分子量为２．６×１０３，胞浆内结合有ｐｅｌＢ信号肽的表达产物分子量为３２×１０３。Objective\ To construct a prokaryotic secretory expression vector and express anti CEA single chain antibody gene.Method\ Fourteen oligonucleiotides which contained ribosome binding site(RBS),translation start codon(ATG),pectate lyase B(pel B) leader peptide gene and translation stop codon(TAA) were synthesized and inserted into pGEM 11zf(+),then the secretory vectory RPY was constructed.In order to express single chain antibody (ScFv),an 82bp fragment with V H and V L cloning sites and linker peptide sequence was synthesized and clone into the downstream of RPY pel B leader peptide gene,the other vector(RPYA) was obtained.After the V H,V L gene of mouse anti CEA monoclonal were inserted into RPYA,the recombinant plasmid was transformed into E.coli HB2151,anti CEA single chain Fv(ScFv) fragment was expressed when transformants were induced with IPTG.Western blot was used to test the antigen binding activity of CEA ScFv.Result\ The result of DNA sequencing demonstrated that the RPYA was consistent with the original design.The molecular weight of CEA ScFv in periplasm was about 26KD,and the CEA ScFv with pel B signal peptide in the inner cytoplasm was about 32KD.Western blot demonstrated that the CEA ScFv was able to bind CEA.Conclusion\ Prokaryotic secretory expression vector(RPYA) can be used in single chain antibody expression.

关 键 词：表达载体 癌胚抗原 单链抗体 基因表达 寡核苷酸 

分 类 号：R392.12[医药卫生—免疫学]