大鼠肝细胞生长因子与肝再生增强因子融合基因真核表达质粒的构建及鉴定

目的 构建大鼠肝细胞生长因子(rHGF)与大鼠肝再生增强因子(rALR)融合基因的真核表达质粒并进行鉴定,为新的肝纤维化治疗方法奠定实验基础.方法 分别以重组质粒pUC18-rHGF和pUC18-rALR为模板,进行PCR扩增,获得rHGF和rALR的基因片段;利用重叠延伸PCR方法将获得的基因片段通过一个连接序列(linker)进行连接,构建融合基因rHGF-linker-rALR,将融合基因定向插入pcDNA3.1真核表达质粒的Kpn Ⅰ和Xba Ⅰ酶切位点之间,构建重组真核表达质粒pcDNA3.1-rHGF-linker-rALR,并进行双酶切及测序鉴定.结果 rHGF和rALR的PCR扩增产物电泳后分别观察到2200 bp和400 bp的条带,与理论值相符.重叠延伸PCR获得的融合基因产物电泳后观察到2600 bp的条带,与预期值一致.重组真核表达质粒pcDNA3.1-rHGF-linker-rALR经双酶切,电泳后观察到2600 bp和5400 bp的两条DNA条带,与预期值相符,测序鉴定结果表明序列正确.结论 成功构建了rHGF与rALR融合基因的真核表达质粒,为肝纤维化的基因治疗奠定了实验基础.

Construction and further identification of eukaryotlc expression plasmid containing rat hepatocyte growth factor gone and augmenter of liver regeneration gene

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.