封闭N-cadherin解整合素金属蛋白酶水解位点的单链抗体的制备及鉴定  

Preparation and identification of a single chain antibody against the shedding site of N-cadherin cleaved by ADAM10

作　　者：李小鸥[1] 黄巍[2] 李莉[1] 周丽荣[2] 

机构地区：[1]武汉大学人民医院儿科 [2]武汉市医学科学研究所,湖北武汉430030

出　　处：《细胞与分子免疫学杂志》2013年第9期949-952,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81000094);武汉大学自主青年基金(4101016)

摘　　要：目的制备封闭神经型钙黏素(N-cadherin)解整合素金属蛋白酶水解位点(ADAM)的单链抗体(scFv),并进行鉴定。方法首先利用RT-PCR技术从分泌抗N-cadherin的ADAM水解位点单克隆抗体(mAb)细胞株中扩增出重链(V_H)和轻链(V_L)可变区基因片段,然后通过重叠延伸PCR法(SOE-PCR),构建成scFv基因片段。再将其克隆入原核表达载体pET-28a中,在大肠杆菌中诱导表达,通过镍柱纯化和复性后,用SDS-PAGE、ELISA和Western blot法等测定重组蛋白的生物学活性。结果PER、酶切和测序表明scFv片段长744 bp,编码248个氨基酸。scFv基因表达载体转化E.coli BL21(DE3),经IPTG诱导表达出相对分子质量(M_r)约29 000的目的蛋白,主要为包涵体形式,经变性,纯化和复性后,获得纯度达90%以上的scFv蛋白,ELISA和Western blot法检测表明可溶性scFv可以与N-cadherin的ADAM水解位点序列多抗原短肽和全长N-cadherin结合。结论成功构建并表达封闭N-cadherin的ADAM加工位点单链抗体。Objective To construct and express a specific single chain Fv antibody （scFv） against the shedding site of N-cadherin cleaved by ADAM10 and identify its biological activity. Methods The VH and V, genes were amplified by RT-PCR from a hybridoma cell line 2B3 which produced the monoclonal antibody （mAb） against the shedding site of N-cadherin cleaved by ADAM10. SOE-PCR was used to splice the V, and V, genes to construct ScFv, which was subcloned into the prokaryotic expression vector pET-28a. The positive clones were transformed into E. coil BL21 （ DE3 ） and induced with IPTG. The target protein was purified and refolded via Ni-NTA column, and was analyzed by SDS-PAGE, ELISA and Western blotting. Results The VH and VL genes of mAb were cloned successfully and the prokaryotic expression vector of scFv was constructed. The analysis of DNA sequencing showed that the full-length of the constructed scFv gene was 744 bp, and coded 248 amino acids. ScFv was expressed in E. coil BL21 （DE3） as inclusion body under the induciton of IPTG, and showed relative molecular mass （M,） of 29 000 as analyzed by SDS-PAGE and Western blotting. About 90% purity of scFv was obtained following denaturing, purifying and renaturing via Ni-NTA, and ELISA and Western blotting revealed that the soluble scFv exhibited the binding activity to the shedding site of N-cadherin cleaved by ADAM10. Conclusion The scFv against the shedding site of N-cadherin cleaved by ADAM10 has been successfully constructed, which lays the foundation for its diagnostic and therapeutic application.

关 键 词：ADAM10 N-CADHERIN 单链抗体 基因表达 

分 类 号：R392.11[医药卫生—免疫学] R392-33[医药卫生—基础医学]