人TAT-PDX-1融合蛋白原核表达载体的构建

摘要

目的 构建人TAT-PDX-1融合蛋白原核表达载体.方法 以人胰腺癌(PANC)-1细胞株cDNA为模板,采用反转录聚合酶链式反应(RT-PCR)扩增胰腺十二指肠同源异型盒基因-1(PDX-1)蛋白编码的全部序列,克隆入原核表达载体pET28a中,经限制性内切酶HindⅢ和BamHI双酶切及DNA序列分析重组质粒的目的基因.结果 RTPCR扩增产物的特异性片段长度为885 bp,以此构建的重组质粒pET28a-TAT-PDX-1经HindⅢ和BamHI双酶切后显示5 900 bp和885 bp左右的2条片段,测序结果与Genbank中的人PDX-1基因cDNA序列一致.结论 成功构建了人TAT-PDX-1融合蛋白原核表达载体.%Objective To construct a prokaryotic expression vector of human TAT-PDX-1 fusion protein. Methods rncDNA of human pancreatic ( PANC)-1 cell line as a template, all coding sequence of PDX-1 protein were amplified by reverse rntranscription polymerase chain reaction ( RT-PCR). The fragment was inserted into prokaryotic expression vector pE128a plasrnmid. Target genes of recombinant plasmid were analyzed by the restriction enzyme HindⅢ and BamHI double digestion and rnDNA sequencing. Results The length of specific fragment applied by RT-PCR was 885 bp, and after the pET28a-TAT-PDX-1 rnof recombinant plasmid digested by HindⅢ and BamHI double digestion displayed two fragments of 5 900 bp and 885 bp. The rnsequencing results consistent with cDNA sequence of human PDX-1 gene in Genhank. Conclusion The prokaryotic expresrnsion vector of human TAT-PDX-1 fusion protein is successfully constructed.