人骨桥蛋白表达载体的构建及其在大肠杆菌中的表达

Construction of pGEX-6P- human osteopontin and its expression in E. coli XL1-blue

目的 构建人骨桥蛋白 (hOPN)表达载体 ,体外表达及表达产物的纯化。方法 培养人脐静脉内皮细胞 ,提取总RNA ,反转录合成cDNA第一链 ,以此cDNA为模板经PCR合成插入片段 ,连接至载体 pGEX 6P 1,转化到XL1 blue中进行诱导表达。表达产物经SDS 聚丙烯酰胺凝胶电泳后 ,切下目的蛋白进行洗脱纯化。结果 构建的表达载体经限制性内切酶酶切图谱分析和DNA测序 ,证明所构建的质粒是 pGEX 6P OPN。SDS 聚丙烯酰胺凝胶电泳发现该重组质粒经IPTG诱导后表达一种新的蛋白 (表达量为 16 7% ) ,这种蛋白不存在于诱导后的pGEX 6P 1表达产物中 ,纯化后蛋白纯度为 99%。结论 成功构建了hOPN表达载体 ,并进行体外表达。

Objective To construct the recombinant vector pGEX-6P-human osteopontin and express the recombinant protein in Escherichia coli. Methods First, cell culture was used to prepare total RNA; Second, the first chain of cDNA was synthesized using RT-PCR; Third,PCR was used to amplify human osteopontin segment, which was inserted into pGEX-6P-1 to construct expression vector; Then the recombinant vector was transformed into XL1-blue and expressed by adding IPTG; and finally, the expression product was purified from the gel after SDS-PAGE. Results Restriction endonucleases mapping analysis and DNA sequencing demonstrated that the constructed plasmid was the recombinant pGEX-6P-opn. The recombinant vector expressed a new protein which didn't present in pGEX-6P-1. Conclusion Construction and expression of hOPN were successful.