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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。
OBJECTIVE: To construct a DT389-hbFGF fusion protein (DT389-hbFGF) expressing the 389-amino-terminal fragment of diphtheria toxin (DT389) and to express the immunotoxin. Turbid looking for the material basis. Methods: The inactivated DNA of C. diphtheriae and the fetal brain cortex of 12wk were extracted. The gene fragment encoding DT389 and the full-length cDNA encoding 18kDahbFGF were amplified by PCR. The two genes were inserted into the expression vector successively to construct the recombinant plasmid containing DT389-hbFGF Fusion gene expression plasmid, sequenced, transformed into E. coli, IPTG induced expression, purification and identification of expression products. Results: The DT389 gene fragment and hbFGF gene sequence were amplified. The prokaryotic expression vector of DT389-hbFGF fusion gene was constructed and successfully expressed. Conclusion: The successful cloning and expression of DT389-hbFGF immunotoxin has laid the material foundation for the study of drugs inhibiting the development of post-cataract.