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 1 770 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 95 900. 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.
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)
