LMP-1与LMP-3真核双表达质粒的构建及体外表达

Construction of co-expression plasmid pBudCE4.1-LMP-1-LMP-3 and its expression in vitro

目的:构建真核双表达质粒pBudCE4.1-LMP-1-LMP-3,并检测其在体外的表达。方法:采用人工设计合成人LIM矿化蛋白-1(LIM mineralization protein-1,LMP-1)和LIM矿化蛋白-3(LIM mineralization protein-3,LMP-3)基因片段,分别连接于中介质粒Puc57,经酶切、测序鉴定后,LMP-1和pBudCE4.1先连接,经酶切鉴定,再连接LMP-3,重组双表达质粒行酶切、测序鉴定。用脂质体包裹转染骨髓间充质干细胞(Mesenchymal stem cells,MSCs),按转染情况分为5组:未转染组(A组)、转染空载体组(B组)、转染LMP-1基因组(C组)、转染LMP-3基因组(D组)、转染LMP-1和LMP-3双基因组(E组)。采用RT-PCR和Western blot检测LMP-1和LMP-3的表达。结果:酶切及测序证实质粒Puc57-LMP-1、Puc57-LMP-3及其pBudCE4.1-LMP-1-LMP-3真核双表达质粒构建成功。该双表达质粒在体外转染骨髓MSCs后可表达LMP-1和LMP-3分子。对RT-PCR及Western blot检测结果行灰度值测量显示:LMP-1 mRNA及蛋白水平的表达,A、B组与C、D、E组间差异有统计学意义(P<0.05),C组与E组差异无统计学意义(P>0.05);LMP-3 mRNA及蛋白水平的表达,A、B组与C、D、E组间差异有统计学意义(P<0.001),D组与E组差异有统计学意义(P<0.05),C组及D组在LMP-1及LMP-3的表达上的差异无统计学意义(P>0.05)。结论:成功构建真核双表达质粒pBudCE4.1-LMP-1-LMP-3,证实转染的骨髓MSCs能在体外同时表达LMP-1和-LMP-3分子。

Objective：To construct the mammalian CO-expression plasmid pBudCE4.1-LMP-1-LMP-3,and to detect the expression of the plasmid in vitro.Methods：Fragments of LMP-1 gene and Fragments of LMP-3 gene were gained from the Company,and were constructed respectively into the plasmid vector Puc57,The inserted target genes in plasmid were verified by nucleotide sequencing and enzymes.fragments of LMP-1 gene were constructed firstly into the plasmid vector pBudCE4.1,fragments of LMP-3 gene were constructed into the plasmid vector pBudCE4.1-LMP-1 after iden tification with nucleotide sequencing and enzymes.MSCs cell line was transfected with this Co-expression plasmid using lipofectin reagent.according to the transfect situation,the MSCs were divided into 5 groups,the non-transfected group（Group A）,the group transfected by empty vector（Group B）,the group transfected by LMP-1（Group C）,the group transfected by LMP-3（Group D）and the group transfected both LMP-1 and LMP-3（Group E）.the expression of LMP-1 and LMP-3 were detected by RT-PCR and Western blot technique.Results：The plasmids Puc57-LMP-1、Puc57-LMP-3 and pBudCE4.1-LMP-1-LMP-3 were obtained successfully and verified by nucleotide sequencing and enzymes.After transfection with this mammalian Co-expression plasmid,the LMP-1 and LMP-3 molecules were expressed in MSCs cells.The results of RT-PCR and Western Blot were measured with the grey value.To the expression of mRNA and protein of LMP-1,the diferences between groups A、B and groups C、D、E were significant（P0.05）.the diferences between groups C and E were not significant（P0.05）;To the expression of mRNA and protein of LMP-3,the diferences between groups A、B and groups C、D、E were significant（P0.001）.the diferences between groups D and E were significant（P0.05）;the expression of LMP-1 or LMP-3 between groups C and D were not significant（P0.05）.Conclusion：the constructed mammalian Co-expression plasmid pBudCE4.1-LMP-1-LMP-3 can express LMP-1 and LMP-3 molecules in vitro at the same time.