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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18 kd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品状体上皮细胞(HLECs)的毒性作用,流式细胞术鉴定不同剂量融合毒素所致HLZCs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。
Objective To prevent cataract surgery capsule opacity to find the material basis. Methods Inactivated DNA of C. diphtheriae and fetal brain cortex of 12 weeks were extracted. The gene fragment of 389 amino acids (DT389) encoding the amino terminal of diphtheria toxin and the gene encoding 18 kd human alkaline were amplified by polymerase chain reaction (PCR) HbFGF fusion gene was inserted into the expression vector to construct the expression plasmid containing DT389-hbFGF fusion gene. After sequencing, the recombinant plasmid was transformed into E. coli and induced with IPTG to express and identify the expressed product. Thiazole MTT assay was used to detect the cytotoxicity on human lenticular epithelial cells (HLECs). Flow cytometry was used to identify the survival rates and cell death patterns of HLZCs induced by different doses of fusion toxins. Results The DT389 gene fragment and the full-length hbFGF gene sequence were amplified. The prokaryotic expression vector of DT389-hbFGF fusion gene was constructed and successfully expressed. The expressed fusion protein showed obvious toxic effects on HLECs in a dose-dependent manner. The median lethal dose of 3.8 × 10-11mol / L, resulting in the main way of cell death to apoptosis. Conclusion The successful cloning and expression of DT389-hbFGF immunotoxin has laid the material foundation for the study of the apoptosis of drug-induced lens epithelial cells.