人β细胞素基因原核表达载体的构建

Construction of human BTC prokaryotic expression vector

目的构建人BTC基因的原核表达载体。方法以人胰岛细胞瘤cDNA为模板,采用聚合酶链式反应(PCR)扩增BTC基因成熟蛋白编码区的全部序列,克隆入原核细胞表达载体PET32a(+)中,经限制性内切酶双酶切及DNA序列分析鉴定目的基因。结果PCR扩增的特异性片段长度为240bp,以此构建的重组质粒PET32a(+)-BTC,经KpnⅠ和XhoⅠ双酶切后显示5.9kb和250bp左右的两条片段,测序结果与Gen-bank中的人BTC基因cDNA(Genbank序列号NM_001729)序列一致。证明人BTC基因已成功克隆到了原核细胞表达载体PET32a(+)中。结论成功构建了PET32a(+)-BTC重组原核表达载体。

[Objective] To construct a prokaryotic expression vector of human BTC gene. [Methods] The whole mature protein coding sequence of BTC gene was amplified by polymcrase chain reaction （PCR） method applied to human pancreatic β-cell tumors eDNA. The fragment was inserted into prokaryotic expression vector PET32a （＋） plasmid. The recombinant plasmid was verified by double digestion and DNA sequencing. [Results] The length of specific fragment applied by PCR was 240 bp, and the recombinant plasmid PET32a （＋） -BTC presented two bands： 5.9 kb and 250 bp using respective restriction enzymes Kpn Ⅰ and Xho Ⅰ. The sequence of BTC gene was comformed by blasting to Genbank. It suggested that BTC gene had been cloned into PET32a （＋） vector correctly. [Conclusion] The recombinant prokaryotic expression vector PET32a （＋） -BTC was successfully constructed.