cbfa1、satb2双基因共表达载体的构建及鉴定

Construction and identification of cbfa1 and satb2 co-expression vector

目的:构建携带cbfa1和satb2双基因的慢病毒真核表达载体。方法:采用PCR技术,从质粒中扩增基因cbfa1和satb2,经TA克隆、酶切筛选后,对阳性重组子进行商业测序鉴定。将测序正确的cbfa1和satb2分别插入pIRES上、下游的相应酶切位点中,构建pIRES-cbfa1-satb2。然后通过双酶切,得到cbfa1-Ires-satb2片段,将其插到含有neo/kana基因的慢病毒载体pLentinTrident1-CMV的相应酶切位点上,构建慢病毒真核表达载体pLentinTrident1-CMV-cbfa1-Ires-satb2。结果:通过PCR技术成功扩增了cbfa1和satb2基因,借助中间载体pIRES中的内部核糖体进入位点,将cbfa1和satb2同时连到了慢病毒载体pLentinTrident1-CMV上,经PCR、酶切及测序验证,质粒构建成功。结论:本实验成功构建了携带cbfa1和satb2双基因的慢病毒真核表达载体,为进一步研究2种基因的功能奠定了基础。

PURPOSE： To construct a lentiviral eukaryotic expression vector containing two genes of cbfa1 and satb2.METHODS：The aim genes of cbfa1 and satb2 were amplified from plasmids by PCR.After TA cloning,the positive clones were identified by restrictive enzyme digestion and commercial DNA sequencing.Cbfa1 and satb2 with correct sequences were ligated upstream and downstream to pIRES,respectively,to construct pIRES-cbfa1-satb2.Then cbfa1-Ires-satb2 fragment was obtained by double digestion,and inserted into corresponding enzyme cut sites of pLentinTrident1-CMV which had been added resistance gene neo/kana to construct the lentiviral eukaryotic expression vector pLentinTrident1-CMV-cbfa1-Ires-satb2.RESULTS：We amplified the genes cbfa1 and satb2 by PCR and connected them with pLentinTrident1-CMV by internal ribosomal entry site of mediator pIRES successfully.The result was identified by PCR,restrictive enzyme digestion and sequencing.CONCLUSIONS： Recombinant lentiviral eukaryotic expression vector containing both cbfa1 and satb2 genes is successfully constructed.This provides a foundation for further studies on their functions.