人胰腺癌hedgehog信号转导途径中PTCH基因的克隆与鉴定

目的 克隆人胰腺癌hedgehog信号通路中PTCH基因,构建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R-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-CodonPlusTM-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.结果 从人胰腺癌细胞株SW1990克隆出长为789 bp PTCH目的片段,成功构建重组质粒PET22b/PTCH,并诱导表达目的蛋白.结论 构建重组质粒PET22b/PTCH,并表达PTCH融合蛋白,为制备PTCH多克隆抗体打下良好基础.

Cloning and identification of PTCH gene involved in hedgehog signal pathway in human pancreatic cancer

Objective To clone human pancreatic cancer gene PTCH, construct the recombinant expression plasmid PET22b/PTCH and express the fusion protein. Methods The PTCH gene was amplified by RT-PCR from the total RNA extracted from human pancreatic cancer strain SW1990. The amplified product was inserted into the vector PET22b to construct the recombinant expression plasmid PET22b/PTCH, which was transformed into E. coliBL21-CodonPlus^TM-Rp and then identified by sequence analysis. The expression of fusion protein was induced with IPTG and verified by Western blot method. Results A human pancreatic cancer gene with a reading frame of 789 bp was successfully cloned from human pancreatic cancer strain SW1990, which had the same sequence as that of PTCH gene in Genbank. The expression of PET22b/PTCH was proved by Western blot. Conclusion Human pancreatic cancer gene PTCH was successfully cloned and constructed with PET22b plasmid. The prepared fusion protein lays the basis for further study.