白喉毒素氨基端389个氨基酸-人碱性成纤维细胞生长因子靶向毒素的克隆表达及对人晶状体上皮细胞毒性研究

目的 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12周胎脑皮质RNA,应用聚合酶链反应(PCR)技术分别扩增出编码白喉毒素氨基端389个氨基酸(DT389)的基因片段及编码18kd人碱性成纤维细胞生长因子(hbFGF)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,纯化鉴定表达产物,噻唑蓝(MTT)试验检测其对体外培养人晶状体上皮细胞(HLECs)的毒性作用,流式细胞术鉴定不同剂量融合毒素所致HLECs成活率及细胞死亡形式.结果 扩增得到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;表达的融合蛋白对HLECs有明显的毒性作用并在一定范围内呈明显剂量依赖性,半数致死量为3.8×10-11mol/L,所致细胞死亡方式主要以凋亡为主.结论 DT389-hbFGF免疫毒素克隆表达的成功为药物促晶状体上皮细胞的凋亡、抑制后发障的研究奠定了物质基础.

Cloning, expression and cytotoxin to human lens epithelial cells of targeting toxin DT389-hbFGF

OBJECTIVE: To construct pGEX-DT(389)-hbFGF plasmid, express and identify the cytotoxicity to human lens epithelial cells (HLECs). METHODS: Extracting DNA of dead diphtheria bacillus and RNA of 12-week fetal brain cortex. The fragments of truncated diphtheria toxin (containing 389 amino acids of N-terminus, DT(389)) and full length human bFGF gene (encoding 18kd protein) were amplified by PCR technique respectively. The two fragments were inserted into prokaryotic expression vector pGEX-4T-1. After testing sequence, the expressing plasmid was transformed into E.Coli BL21 strain and induced expression under IPTG. The expressed fusion protein was purified and identified. MTT experiment tested cytotoxicity of the fusion protein to HLECs in vitro. The way of HLECs death under different dosage was identified by flow cytometry. RESULTS: The gene fragments of DT(389) and human bFGF were accurately amplified. The expression vector including DT(389)-hbFGF fused gene was constructed and expressed successfully. DT(389)-hbFGF fusion protein can induce HLECs apoptosis in a dosage dependence manner during certain range. The LD(50) was about 3.8 x 10(-11) mol/L. CONCLUSION: The successful cloning and expression of DT(389)-hbFGF immunotoxin lay a foundation for accelerating lens epithelial cells apoptosis and the targeting therapy toward posterior capsule opacification.