人源化抗肾小球基底膜抗体的单链抗体scFv3的原核表达及鉴定  

Expression and characterization of human single chain Fv antibody to anti-glomerular basement membrane (anti-GBM) antibody in Escherichia coli

作　　者：肖静[1] 刘章锁[1] 王雅楠[1] 赵国强[2] 王沛[1] 

机构地区：[1]郑州大学第一附属医院肾内科,郑州450052 [2]郑州大学微生物学与免疫学教研室,郑州450001

出　　处：《中国免疫学杂志》2011年第1期50-53,共4页Chinese Journal of Immunology

基　　金：国家卫生部资助项目(WKJ2007-2-033);河南省医学科技攻关重大项目(200701006)

摘　　要：目的:在原核细胞中构建并表达抗肾小球基底膜(GBM)抗体单链抗体scFv3,并鉴定其活性。方法:用PCR扩增抗GBM抗体单链抗体scFv3基因,将scFv3 DNA与pGEM-T Easy质粒连接,构建克隆载体pGEM-scFv3。用酶切后电泳鉴定构建载体的正确性;测序检测质粒重组后序列有无改变。再将scFv3亚克隆入表达载体pQE-80L,构建pQE80L-scFv3重组质粒,转化大肠杆菌Rosetta2,酶切鉴定。IPTG诱导表达蛋白后,通过SDS-PAGE和Western blot鉴定表达产物的抗体活性。结果:PCR扩增scFv3 DNA片段约为750 bp,与预期结果相同;克隆载体pGEM-scFv3酶切后显示scFv3成功的克隆入pGEM-T Easy质粒;测序验证插入片段全序列无改变;表达载体pQE80L-scFv3酶切图谱与预期相同;SDS-PAGE显示,在分子量约为27 kD处可见目的蛋白带,Western blot证实scFv3能与抗GBM抗体结合。结论:成功构建并表达了抗GBM抗体的单链抗体scFv3蛋白,为进一步研究scFv3的生物学特性和基因工程抗体的制备奠定了基础。Objective：To construct the prokaryotic expressing plasmid for human scFv3 gene against or of anti-GBM antibody and prepare the scFv3 protein for identifying biological function.Methods：The scFv3 gene of anti-GBM antibody was amplified by PCR and cloned into pGEM-T Easy Vector.The sequence of cloned scFv3 was confirmed by restriction analysis and DNA sequencing.A scFv3 prokaryotic expressing plasmid,pQE80L-scFv3,was then constructed by subclone.pQE80L-scFv3 transformed E.coli Rosetta2 was induced by IPTG.The expression of scFv3 protein was analyzed by SDS-PAGE and Western blot.Results：The amplified DNA fragment was in size of 750 bp as expected.Restriction analysis showed that the amplified gene was inserted in pQE80L correctly.Sequence showed there was no mutation in both ends of the cloned scFv3.Restrict analysis showed that the recombinant pQE80L-scFv3 was right.A Mr 27×103 protein could be seen in Rosetta2 induced by IPTG with SDS-PAGE and was positive while reacting with anti-GBM antibody by Western blot.Conclusion：The construction of the prokaryotic expression plasmid of scFv3 gene and preparation of scFv3 fragment establish a solid basis of further studying the biological function of the human scFv3 to anti-GBM antibody.

关 键 词：抗GBM抗体 单链抗体scFv3 原核表达 鉴定 

分 类 号：R392.11[医药卫生—免疫学]