人源His-talin1原核表达载体的构建及其蛋白表达  

Construction and prokaryotic expression of human His-talin 1 gene

作　　者：吴爽[1] 张娟辉 游娟[3] 

机构地区：[1]广东药学院基础学院组织胚胎学教研室,广东广州510006 [2]广东省武警医院妇产科,广州510507 [3]广东药学院基础学院生物化学与分子生物学教研室,广州510006

出　　处：《解剖学研究》2012年第3期212-214,共3页Anatomy Research

摘　　要：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。Objective To recombine human talinl gene in Ecoli cells and purify the His-talinl protein. Methods The talinl cDNA was amplified by PCR. The talinlcDNA 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 prod- ucts were purified in affinity chromatography and identified in SDS-PAGE and Western blot method. Results The 2.4 kb of human tal- in 1 gene was obtained successfully. The recombinant plasmid pET32a （＋）-talin 1 was constructed. By analyzed in SDS-PAGE and West- ern blot methods, the fusion protein His-talinl was purified successfully. Conclusion The prokaryotic expression system of His-talinl was established. The His-talinl protein purified provides to further study the relationship of talinl and P-selectin.

关 键 词：原核表达 Talin基因重组 亲和层析 P—selectin 

分 类 号：R363[医药卫生—病理学]