LMP-1与LMP-3真核双表达质粒的构建及体外表达
发布时间：2018-12-25 09:26
【摘要】：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(P0.05),C组与E组差异无统计学意义(P0.05);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(P0.001),D组与E组差异有统计学意义(P0.05),C组及D组在LMP-1及LMP-3的表达上的差异无统计学意义(P0.05)。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。
[Abstract]:Aim: to construct eukaryotic double expression plasmid pBudCE4.1-LMP-1-LMP-3, and detect its expression in vitro. Methods: human LIM mineralization protein-1 (LIM mineralization protein-1,LMP-1) and LIM mineralized protein-3 (LIM mineralization protein-3,LMP-3) gene fragments were synthesized by artificial design. The fragments were ligated to the intermediate plasmid Puc57, by enzyme digestion and sequenced, respectively. LMP-1 and pBudCE4.1 were ligated first, identified by enzyme digestion, then ligated with LMP-3, recombinant double expression plasmid for restriction endonuclease digestion and sequencing. Bone marrow mesenchymal stem cells (Mesenchymal stem cells,MSCs) were transfected with liposome and divided into 5 groups: untransfected group (group A), transfected empty vector group (group B) and transfected LMP-1 genome (group C). The genomes of LMP-3 (group D) and LMP-1 and LMP-3 (group E) were transfected. The expression of LMP-1 and LMP-3 was detected by RT-PCR and Western blot. Results: the eukaryotic expression plasmid Puc57-LMP-1,Puc57-LMP-3 and its pBudCE4.1-LMP-1-LMP-3 were successfully constructed by restriction endonuclease digestion and sequencing. The double expression plasmid can express LMP-1 and LMP-3 molecules after transfection of bone marrow MSCs in vitro. The gray value measurement of RT-PCR and Western blot showed that the expression of LMP-1 mRNA and protein was significantly different between group A (P 0.05) and group C (P 0.05). There was no significant difference between group C and group E (P0.05). The expression of LMP-3 mRNA and protein, there was significant difference between the two groups (P0. 001), D group and E group (P0.05). There was no significant difference in the expression of LMP-1 and LMP-3 between group C and group D (P0.05). Conclusion: the successful construction of eukaryotic double expression plasmid pBudCE4.1-LMP-1-LMP-3, confirmed that the transfected bone marrow MSCs could simultaneously express both LMP-1 and LMP-3 molecules in vitro.
【作者单位】： 重庆医科大学附属第一医院骨科;
【分类号】：R341
[Abstract]:Aim: to construct eukaryotic double expression plasmid pBudCE4.1-LMP-1-LMP-3, and detect its expression in vitro. Methods: human LIM mineralization protein-1 (LIM mineralization protein-1,LMP-1) and LIM mineralized protein-3 (LIM mineralization protein-3,LMP-3) gene fragments were synthesized by artificial design. The fragments were ligated to the intermediate plasmid Puc57, by enzyme digestion and sequenced, respectively. LMP-1 and pBudCE4.1 were ligated first, identified by enzyme digestion, then ligated with LMP-3, recombinant double expression plasmid for restriction endonuclease digestion and sequencing. Bone marrow mesenchymal stem cells (Mesenchymal stem cells,MSCs) were transfected with liposome and divided into 5 groups: untransfected group (group A), transfected empty vector group (group B) and transfected LMP-1 genome (group C). The genomes of LMP-3 (group D) and LMP-1 and LMP-3 (group E) were transfected. The expression of LMP-1 and LMP-3 was detected by RT-PCR and Western blot. Results: the eukaryotic expression plasmid Puc57-LMP-1,Puc57-LMP-3 and its pBudCE4.1-LMP-1-LMP-3 were successfully constructed by restriction endonuclease digestion and sequencing. The double expression plasmid can express LMP-1 and LMP-3 molecules after transfection of bone marrow MSCs in vitro. The gray value measurement of RT-PCR and Western blot showed that the expression of LMP-1 mRNA and protein was significantly different between group A (P 0.05) and group C (P 0.05). There was no significant difference between group C and group E (P0.05). The expression of LMP-3 mRNA and protein, there was significant difference between the two groups (P0. 001), D group and E group (P0.05). There was no significant difference in the expression of LMP-1 and LMP-3 between group C and group D (P0.05). Conclusion: the successful construction of eukaryotic double expression plasmid pBudCE4.1-LMP-1-LMP-3, confirmed that the transfected bone marrow MSCs could simultaneously express both LMP-1 and LMP-3 molecules in vitro.
【作者单位】： 重庆医科大学附属第一医院骨科;
【分类号】：R341
【共引文献】
相关硕士学位论文 前1条
1 唐春晖;LMP-1与LMP-3真核双表达质粒的构建及体外表达[D];重庆医科大学;2010年
【二级参考文献】
相关期刊论文 前1条
1 孙佳,鹿培源,贾弘y，
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