CAJ | 학술논문

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.
목적 구건대서간세포생장인자(rHGF)여대서간재생증강인자(rALR)융합기인적진핵표체질립병진행감정,위신적간섬유화치료방법전정실험기출.방법 분별이중조질립pUC18-rHGF화pUC18-rALR위모판,진행PCR확증,획득rHGF화rALR적기인편단;이용중첩연신PCR방법장획득적기인편단통과일개련접서렬(linker)진행련접,구건융합기인rHGF-linker-rALR,장융합기인정향삽입pcDNA3.1진핵표체질립적Kpn Ⅰ화Xba Ⅰ매절위점지간,구건중조진핵표체질립pcDNA3.1-rHGF-linker-rALR,병진행쌍매절급측서감정.결과 rHGF화rALR적PCR확증산물전영후분별관찰도2200 bp화400 bp적조대,여이론치상부.중첩연신PCR획득적융합기인산물전영후관찰도2600 bp적조대,여예기치일치.중조진핵표체질립pcDNA3.1-rHGF-linker-rALR경쌍매절,전영후관찰도2600 bp화5400 bp적량조DNA조대,여예기치상부,측서감정결과표명서렬정학.결론 성공구건료rHGF여rALR융합기인적진핵표체질립,위간섬유화적기인치료전정료실험기출.
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.