Mfn2基因克隆及pEGFPmfn2真核表达质粒的构建

Cloning mfn2 gene and constructing pEGFP/mfn2 expression vector

目的利用基因工程技术克隆线粒体融合蛋白2(mitofusin-2,mfn2)基因并构建一种无细胞毒性、不激活原癌基因的真核表达的pEGFPmfn2质粒载体。方法采用高效Trizol试剂快速提取大鼠血管平滑肌细胞株A7r5总RNA,经RT-PCR获得mfn2cDNA,扩增、纯化、回收mfn2基因片段并将质粒pEGFP-N1和mfn2基因片段分别双酶切,前者纯化回收大片段,后者纯化回收2.2 kb片段,再将回收的pEGFP-N1大片段与mfn2基因片段(2.2 kb)重组。对重组pEGFPmfn2质粒进行PCR和双酶切鉴定并测序。结果成功地从A7r5中克隆了mfn2基因,并构建了以pEGFP-N1为载体的真核表达质粒。结论含mfn2基因新型表达质粒构建的成功将为研究mfn2功能、进一步研究该基因异常表达与心血管疾病发生的关系奠定基础。

Objective To clone mitofusin-2（mfn2） gene and construct a new kind of recombinant vector containing rat mitofusin-2（mfn2） cDNA neither cytotoxicity nor activating prot-oncogenes.Methods The RNA was extracted from rat vascular smooth muscle cell（A7r5） with efficient Trizol reagent.The mfn2 cDNA was obtained from it by reverse transcription polymerase chain reaction（RT-PCR） and was amplified,purified and retrieved.To digest pEGFP-N1 plasmid and mfn2 gene with dual-endonuclease,we retrieved a big fragment from pEGFP-N1 and a 2.2 kb fragment from mfn2 gene.The big fragment and the 2.2 kb fragment were recombined.The recombination pEGFPmfn2 plasmid was identified and sequenced by mono-endonuclease and dual-endonuclease.Results We have cloned mfn2 gene from A7r5 and constructed a sort of eukaryon expression plasmid vector with pEGFPmfn2.A 6.9kb fragment was found in the recombination pEGFP-N1/mfn2 after digesting with mono-endonuclease.By digesting the 6.9kb fragment with dual-endonuclease,we obtained two fragments,one is 4.7 kb,the other is 2.2 kb.It was identified by sequencing that the 2.2 kb fragment was same as rats＇.Conclusion A new eukaryon expression plasmid with mfn2 gene has been constructed.It is the basis for studying mfn2 function and the relationship with cardiovascular diseases.