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目的构建HER2靶向免疫促凋亡蛋白e23sFv-Fdt-tBid的原核表达载体并鉴定其基因工程菌种稳定性。方法应用PCR技术以pCMV-e23sFv-Fdt-tBid为模板扩增e23sFv-Fdt-tBid片段,并将其克隆入pET-22b原核表达载体;将该表达载体转化BL21大肠杆菌并挑取表达蛋白较高的克隆菌,划线接种传代50次;通过革兰氏染色方法、扫描电镜形态观察、菌种质粒酶切鉴定及蛋白诱导表达的Western blot分析鉴定e23sFv-Fdt-tBid免疫促凋亡蛋白表达工程菌的稳定性。结果成功构建pET-22b-e23sFv-Fdt-tBid原核表达载体,并在BL21大肠杆菌中诱导表达;革兰氏染色方法、扫描电镜形态观察、菌种质粒酶切鉴定及Western blot法鉴定结果表明基因工程菌种稳定性良好,可以稳定表达免疫促凋亡蛋白e23sFv-Fdt-tBid。结论成功构建了HER2靶向免疫促凋亡蛋白原核表达载体,鉴定了表达该免疫促凋亡蛋白的基因工程菌种稳定性,为HER2靶向免疫促凋亡蛋白e23sFv-Fdt-tBid的应用研究奠定了基础。
Objective To construct a prokaryotic expression vector for HER2-targeting pro-apoptotic protein e23sFv-Fdt-tBid and identify the stability of its genetically engineered strains. Methods The e23sFv-Fdt-tBid fragment was amplified by PCR from pCMV-e23sFv-Fdt-tBid and cloned into pET-22b prokaryotic expression vector. The recombinant plasmid was transformed into BL21 E.coli and the expression of protein was high Were subcultured 50 times. The expression of e23sFv-Fdt-tBid protein was identified by Gram staining, scanning electron microscopy, bacterial plasmid restriction enzyme digestion and protein-induced Western blot analysis Strain stability. Results The prokaryotic expression vector pET-22b-e23sFv-Fdt-tBid was constructed and expressed in E.coli BL21. The results of Gram staining, scanning electron microscopy, bacterial plasmid restriction enzyme digestion and Western blot showed that the gene The strain of engineering bacteria has good stability and can stably express the immune pro-apoptotic protein e23sFv-Fdt-tBid. Conclusion The prokaryotic expression vector of HER2-targeting pro-apoptotic protein was successfully constructed and the stability of the genetically engineered strain expressing the pro-apoptotic protein was identified. The application of HER2 as a target prokaryotic expression vector e23sFv-Fdt-tBid Foundation.