摘要
目的:制备抗人乙酰胆碱受体单链抗体637(scFv637)与人血清白蛋白(HSA)的融合蛋白,提高scFv637的稳定性。方法:用PCR扩增人HSA基因,并克隆到含有抗人乙酰胆碱受体scFv637基因的载体pHEN2中构建重组载体pHEN2-scFv637-HSA。以重组载体转化E.coliHB2151,表达产物用斑点杂交试验检测,并以SDS-PAGE和Westernblot鉴定其融合蛋白的相对分子质量(Mr)。结果:琼脂糖凝胶电泳显示,扩增的人HSA基因和融合基因的大小分别为1770bp和7054bp。构建的scFv637-HSA经测序证实核苷酸序列正确,并且正确克隆至载体的开放读码框架内。表达产物仅存在于pHEN2-scFv637-HSA转化的E.coliHB2151外周质裂解液中。表达的融合蛋白的Mr约为95900。结论:在E.coli中成功地表达scFv637-HSA融合蛋白,为进一步对其进行功能研究和应用奠定了基础。
AIM:To prepare the fusion protein of a single chain variable fragment 637(scFv637)against human acetylcholine receptor(AChR)and human serum albumin(HSA)to increase the stability of scFv637.METHODS:HSA gene was amplified by PCR and cloned into vector pHEN2 containing a scFv637 gene to construct recombinant vector pHEN2-scFv637-HSA E.coli HB2151 for expression.tein in periplasm of E.coli was and then transformed into The expression of fusion prodetected by dot hybridization.Relative molecular mass(Mr)of fusion protein was checked by SDS-PAGE and Western blot.RESULTS:Agarose gel electrophoresis analysis showed that the size of HSA gene and fusion gene was about 1770 bp and 7054 bp,respectively.DNA sequencing proved that the nucleotide sequence of constructed scFv637-HSA gene was correct and it was cloned into the open reading frame(ORF)of pHEN2.scFv637-HSA fusion protein only existed in periplasm of E.coli HB2151 transformed with pHEN2-scFv637-HSA.SDS-PAGE and Western blot analysis indicated that Mr of fusion protein was about 95900.CONCLUSION:The scFv637-HSA fusion protein can be successfully expressed in E.coli HB2151,which provides a sound basis for further research into its function and clinical application.
作者
范华英
孟繁平
齐栋
魏晶
李强
李英信
李红花
FAN Hua-ying;MENG Fan-ping;QI Dong;WEIJing;LI Qiang;LI Ying-xin;LI Hong-hua(School of Pharmacy,Yantai University,Yantai 264005,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第4期507-509,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30360100)
