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目的 定点突变法扩增制备完整hHGF αcDNA ,克隆并构建其原核表达载体。方法与结果 以含有人HGFcDNA全序列的质粒 pRC/CMV hHGF为模板 ,设计合成一对特异引物进行聚合酶链反应 (PCR) ,扩增hHGF αcDNA基因 ,琼脂糖凝胶电泳检测 ,结果显示扩增得到了 1 34kb长的目的基因产物。将扩增产物以限制性内切酶XhoⅠ酶将其切为 386bp、95 4bp两个片段 ,分别以EcoRⅠ /XhoⅠ、XhoⅠ /BamHⅠ与 pBSKS载体连接重组 ,对目的基因分段克隆后进行序列测定 ,结果表明除通过定点突变引入的起始密码ATG、终止密码子TAA、TGA及EcoRⅠ、BamHⅠ识别位点外 ,扩增得到的PCR产物序列与Nakamura等报道的HGFcDNA中不包括前体序列的α链部分完全相同。将以EcoRⅠ /XhoⅠ、XhoⅠ /BamHⅠ从pBSKS HGF α 386、pBSKS HGF α 95 4中切出的 386bp、95 4bp两个片段以EcoRⅠ /BamHⅠ与pBV2 2 0连接构建重组表达质粒 pBV2 2 0 HGF α ,限制性内切酶图谱分析显示HGF αcDNA插入到载体pBV2 2 0的EcoRⅠ /BamHⅠ位点 ,插入方向正确。温度诱导表达 ,SDS PAGE显示一分子量与单体hHGF α链吻合的蛋白带 ,证明表达质粒构建成功。结论 设计制备了hHGF α链cDNA ,克隆建立了hHGF α链原核表达质粒。
Objective To amplify hHGF α cDNA by site-directed mutagenesis and clone and construct its prokaryotic expression vector. Methods and Results A pair of specific primers was designed and synthesized by PCR using a plasmid pRC / CMV hHGF containing the full sequence of human HGF cDNA as a template. Amplification of hHGF α cDNA gene and agarose gel electrophoresis were carried out. The results showed that amplification A 34kb long gene product of interest was obtained. The amplified product was cut into 386 bp and 954 bp fragments by restriction endonuclease Xho Ⅰ enzyme, respectively, and ligated with pBSKS vector with EcoRⅠ / XhoⅠ, XhoⅠ / BamHⅠ. The sequence of the target gene was cloned and sequenced. The results showed that except for the ATG, ATG, stop codon TAA, TGA and EcoRI, BamHI recognition sites introduced by site-directed mutagenesis, the amplified PCR product sequence was identical to the α-chain of the HGF cDNA excluding the precursor sequence reported by Nakamura et al Partially identical. The two fragments of 386bp and 95 4bp cut out from pBSKS HGF α 386 and pBSKS HGF α 95 4 with EcoRⅠ / XhoⅠ, XhoⅠ / BamHⅠ were ligated with pBV20 2 with EcoRI / BamHI to construct recombinant expression plasmid pBV2200HGFα, Restriction endonuclease analysis revealed that the HGF α cDNA was inserted into the EcoRI / BamHI site of vector pBV220 in the correct orientation. Temperature induced expression, SDS PAGE showed a molecular weight and monomeric hHGF α chain anastomosis protein bands, the successful construction of the expression plasmid. Conclusion The hHGF α chain cDNA was designed and prepared, and the prokaryotic expression plasmid of hHGF α chain was established.