抗乙酰胆碱受体单链抗体原核表达载体及表达菌株的构建  

Construction of Prokaryotic Vector and Bacteria Expressing Single-chain Fv Antibody Fragments against Acetylcholine Receptor

作　　者：陈辉[1] 李柱一[1] 徐江[1] 张惠中[2] 

机构地区：[1]西安第四军医大学唐都医院神经内科,陕西西安710038 [2]西安第四军医大学唐都医院中心实验室,陕西西安710038

出　　处：《中国神经免疫学和神经病学杂志》2006年第6期319-322,共4页Chinese Journal of Neuroimmunology and Neurology

基　　金：国家自然科学基金资助项目(30570641)

摘　　要：目的将含有抗人乙酰胆碱受体(AChR)单链抗体基因的载体重新构建并建立表达菌株。方法采用聚合酶链反应(PCR)从质粒pHEN1中克隆单链抗体scFv1929#全长基因,将PCR产物再定向克隆至载体pET32a(+)的多克隆位点中,并转入大肠杆菌BL21(D E3)plysS以构建表达菌株。结果PCR产物约730bp,与预期一致,重组阳性克隆菌株JM109+pET32a(+)-scFv1929#经菌液PCR可见730 bp处有特异性条带,所提取质粒pET32a(+)-scFv1929#经PCR及酶切鉴定正确,经测序后证实scFv1929#的核苷酸序列正确并正确地插入载体pET32a(+)的读码框内。将新构建载体转化大肠杆菌BL21(D E3)plysS以获得表达菌株。结论已成功地重新构建含有抗AChR单链抗体基因的载体并建立大肠杆菌表达菌株BL21(D E3)plysS+pET32a(+)-scFv1929#,为今后表达抗AChR单链抗体scFv1929#融合蛋白打下基础。Objective To construct prokaryotic expression vector of single-chain Fv （scFv） antibody fragments against acetylcholine receptor （AChR） and to obtain its expression bacteria. Methods Using pHEN1 as template, the scFv1929 # DNA with the restriction sites of EcoR v and Notl were amplified by polymerase chain reaction （PCR） and cloned into prokaryotic expression plasmid pET-32a（ ＋ ）. Then E. coli BL21（D E3） plysS cells were transformed with the recombinant expression vector pET-32a（＋） -scFv1929 #. Results The size of scFv1929 # PCR products from pHEN1 was 730 bp. The length of the scFv1929 # gene fragment was 730 bp after digesting the recombinant vector with EcoR v and Notl, and it confirmed that the scFv1929 # DNA was inserted into the expression vector correctly. DNA sequences were verified by sequencing. The host for expression was obtained by transforming the recombinant into E. coli BL21 （D E3） plysS. Conclusions The prokaryotic expression vector of anti- AChR scFv fragment is constructed and expression bacteria is obtained successfully. It provides the clue for expressing the scFv1929 # fusion protein.

关 键 词：单链抗体 乙酰胆碱受体 原核表达载体 

分 类 号：R746.102.7[医药卫生—神经病学与精神病学]