人源His-talin1原核表达载体的构建及其蛋白表达

目的克隆人源talin1 cDNA,构建其高效原核表达载体,并纯化得到高纯度His-talin1融合蛋白。方法 PCR法扩增talin1基因并连接到原核表达载体pET32a(+),筛选和测序鉴定阳性克隆。将重组质粒转化大肠杆菌BL21(DE3),经过IPTG诱导表达和亲和层析分离纯化表达产物,对纯化的蛋白进行SDS-PAGE和Western blot鉴定。结果成功扩增了2.4kb的talin1基因,构建了pET32a(+)-talin1重组质粒并在大肠杆菌中诱导表达出His-talin1融合蛋白。经SDS-PAGE和Western blot方法 ,验证了高纯度纯化的融合蛋白。结论建立了高效稳定的His-talin1表达体系,为进一步研究Talin1蛋白的结构及其与P-selectin之间的关系打下基础。

Construction and prokaryotic expression of human His-talin 1 gene

Objective To recombine human talin1 gene in Ecoli cells and purify the His-talin1 protein.Methods The talin1 cDNA was amplified by PCR.The talin1cDNA was inserted into the plasmid pET32a(+).By screening and sequencing the recombinant plasmid,the positive constructs were transformed into the E.coli BL21(DE3) host cells.After induced in IPTG,the recombinant products were purified in affinity chromatography and identified in SDS-PAGE and Western blot method.Results The 2.4 kb of human talin1 gene was obtained successfully.The recombinant plasmid pET32a(+)-talin1 was constructed.By analyzed in SDS-PAGE and Western blot methods,the fusion protein His-talin1 was purified successfully.Conclusion The prokaryotic expression system of His-talin1 was established.The His-talin1 protein purified provides to further study the relationship of talin1 and P-selectin.