人胰腺癌Hedgehog信号通路中膜蛋白受体PTCH的纯化

Purification of membranous receptor PTCH in Hedgehog signal pathway of human pancreatic cancer

目的构建人胰腺癌PTCH基因表达载体并诱导融合蛋白表达。方法从人胰腺癌细胞株SW1990抽提总RNA,经RT-PCR扩增出PTCH基因,经纯化、回收目的基因PTCH,将其插入表达载体pET22b,转化E.ColiBL21-CodonPlus(TM)-RP,构建重组质粒pET22b/PTCH,IPTG诱导表达融合蛋白,免疫印迹进行鉴定,Ni-螯合亲和层析纯化融合蛋白。结果从人胰腺癌细胞株SW1990克隆出长为789bpPTCH目的片段,成功构建重组质粒pET22b/PTCH,并诱导表达目的蛋白;经Ni-螯合亲和层析得到纯化的融合蛋白。结论成功构建了重组质粒pET22b/PTCH,并获得纯化的融合蛋白,为进一步研究Hedgehog信号通路在胰腺癌中的发病机制提供了有效工具。

Objective To clone human pancreatic cancer gene PTCH and construct pET22b/ PTCH expression plasmid. The protein was used to immunize rabbits to prepare anti-PTCH multiclonal antibody, which was then used to detect the expression of PTCH in pancreatic cancer, Methods Total RNA was isolated from human pancreatic cancer cell line SW1990. The PTCH gene was amplified from the total RNA by RT-PCR. The resulting product was cloned into pET22b vector. The pET22h/ PTCH construct was then transformed into E. coliBL21-CodonPlusTM-RP, and the gene was sequenced, The fusion protein was expressed by IPTG and verified by Western blot. The expressed protein was purified with a His. Bind column, Results A human pancreatic cancer gene with a reading frame of 789 hp was successfully cloned from human pancreatic cancer cell SW1990, which had the same sequence as that of PTCH gene in Genehank, The expression of pET22b/PTCH was proved by Western blot, and the fusion protein was also purified by Ni-NTA affinity chromatography. Conclusions Human pancreatic cancer gene PTCH was successfully cloned and expressed in E. coli, and the fusion protein has been purified by Ni-NTA affinity chromatography, which would provide a useful tool in the study of Hedgehog signal in pancreatic cancer.