DT_(389)-hbFGF免疫毒素的克隆与表达(英文)  

Cloning and expression of immunotoxin DT_(389)-hbFGF

作　　者：尹连荣[1] 袁佳琴[2] 

机构地区：[1]中国中医科学院眼科医院眼科,中国北京市100040 [2]天津医科大学眼科中心,中国天津市300070

出　　处：《国际眼科杂志》2007年第1期15-18,共4页International Eye Science

摘　　要：目的:构建含有白喉毒素氨基端389个氨基酸的片断(DT389)人碱性成纤维细胞生长因子(hbFGF)的融合蛋白(DT389-hbFGF)表达质粒并表达该免疫毒素,为阻止白内障术后囊膜混浊寻找物质基础。方法:提取灭活的白喉杆菌DNA和12wk胎脑皮质RNA,应用PCR技术分别扩增出编码DT389的基因片段及编码18kDahbFGF的基因全序列,将两基因先后插入表达载体中,构建含有DT389-hbFGF融合基因的表达质粒,测序后,转化大肠杆菌,IPTG诱导表达,纯化并鉴定表达产物。结果:扩增后得到DT389基因片段及hbFGF全基因序列;构建了DT389-hbFGF融合基因的原核表达载体并成功表达。结论:DT389-hbFGF免疫毒素克隆表达的成功为药物抑制后发性白内障的研究奠定了物质基础。AIM： To express the DT389-hbF-GF （389 amino acid residues of the N-terminus of diphtheria toxin （human basic fibroblast growth factor） fusion protein for potential targeting therapy towards posterior capsule opacification （PCO） after cataract surgery. METHODS： The DNA of inactivated diphtheria bacillus and RNA of 12-week fetal brain cortex were extracted, respectively. The fragments of truncated diphtheria toxin （containing 389 amino acids of N-terminus, DT389 ）and full-length human basic fibroblast growth factor （hbFGF） sequence （encoding 18kDa protein） were amplified by PCR. The two fragments were inserted into pGEX-4T-1 prokaryotic expression vector to obtain pGEX-DT389-hbFGF prokaryotic expression plasmid. After sequence analysis, the expressing plasmid was transformed into Escherichia Coli BL21 strain and expression was induced under TPTG. The expressed fusion protein was purified and identified. RESULTS： The gene fragments encoding DT389 and hbF-GF were amplified and their gene sequences were confirmed. Hybrid gene expression plasmid pGEX-DT389 （hbFGF was constructed. The fusion protein DT389-hbFGF was expressed and purified. CONCLUSIONS： The successful cloning and expression of DT389-hbFGF immunotoxin provides a foundation for targeting therapy towards posterior capsule opacification.

关 键 词：免疫毒素 白喉毒素 碱性成纤维细胞生长因子 

分 类 号：R776.1[医药卫生—眼科]