摘要
目的构建抗人精浆蛋白单链抗体（γＳｍＳｃＦｖ），为进一步应用基因工程抗体研究打下基础。方法从分泌抗人精浆蛋白单克隆抗体γＳｍＭｃＡｂ的杂交瘤细胞系Ｅ４Ｂ７中提取总ＲＮＡ，经ＲＴＰＣＲ分离获得了γＳｍＭｃＡｂ轻、重链可变区（ＶＬ、ＶＨ）基因，用连接肽将其构建成具有ＶＨＬｉｎｋｅｒＶＬ结构的单链抗体基因，并将构建的单链抗体基因克隆到融合蛋白表达载体ＰＧＥＸ４Ｔ１中进行表达。结果经测序表明，ＶＨ、ＶＬ及Ｌｉｎｋｅｒ的序列均正确。ＳｃＦｖ在大肠杆菌中的表达量约占菌体总蛋白的４０％。结论构建的抗人精浆蛋白单链抗体。
Objective To clone the V L gene and V H gene from murine McAb against γ seminoprotein,and to construct the single chain Fv gene. Methods Total RNA was extracted from the hybridoma cell line E 4B 7,which secretes McAb against γ seminoprotein,and a set of oligonucleotide primers were designed and used to amplify the cDNAs with RT PCR.The products were cloned into pUC19 vector and their sequences were analysed.The V H and V L fragment chimera tethered by a peptide linker were designa ted as ScFv.The ScFv constructed previusl was inserted into the prokaryotic fusion protien expression vector pGEX 4T 1 and efficiently expressed in E.coli.The expression of GST ScFv fusion protein was induced by IPTG. Results E 4B 7V H consists of 358 bp encodig 119 amino acid residues and E 4B 7V L contains 326 bp encodig 108 amino acid residues.The deduced amino acid sequences of E 4B 7V H/V L did not agree with the characterization of the amino acid present in the mose Ig variable region.The sequences of V H,V L and linker peptide were all correct,SDS PAGE showed a novel protein band with an apparent molecular mass of 52 000Mr,which was about 40% of total bacterial protein. Conclusions Anti human γ seminoprotein ScFv has been successfully constructed for the use in clinical studies.
出处
《中华泌尿外科杂志》
CAS
CSCD
北大核心
1999年第1期5-8,共4页
Chinese Journal of Urology
基金
全军重点实验室研究基金
