LMP-1與LMP-3真核雙表達(dá)質(zhì)粒的構(gòu)建及體外表達(dá)
發(fā)布時間：2018-12-25 09:26
【摘要】：目的:構(gòu)建真核雙表達(dá)質(zhì)粒pBudCE4.1-LMP-1-LMP-3,并檢測其在體外的表達(dá)。方法:采用人工設(shè)計合成人LIM礦化蛋白-1(LIM mineralization protein-1,LMP-1)和LIM礦化蛋白-3(LIM mineralization protein-3,LMP-3)基因片段,分別連接于中介質(zhì)粒Puc57,經(jīng)酶切、測序鑒定后,LMP-1和pBudCE4.1先連接,經(jīng)酶切鑒定,再連接LMP-3,重組雙表達(dá)質(zhì)粒行酶切、測序鑒定。用脂質(zhì)體包裹轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞(Mesenchymal stem cells,MSCs),按轉(zhuǎn)染情況分為5組:未轉(zhuǎn)染組(A組)、轉(zhuǎn)染空載體組(B組)、轉(zhuǎn)染LMP-1基因組(C組)、轉(zhuǎn)染LMP-3基因組(D組)、轉(zhuǎn)染LMP-1和LMP-3雙基因組(E組)。采用RT-PCR和Western blot檢測LMP-1和LMP-3的表達(dá)。結(jié)果:酶切及測序證實(shí)質(zhì)粒Puc57-LMP-1、Puc57-LMP-3及其pBudCE4.1-LMP-1-LMP-3真核雙表達(dá)質(zhì)粒構(gòu)建成功。該雙表達(dá)質(zhì)粒在體外轉(zhuǎn)染骨髓MSCs后可表達(dá)LMP-1和LMP-3分子。對RT-PCR及Western blot檢測結(jié)果行灰度值測量顯示:LMP-1 mRNA及蛋白水平的表達(dá),A、B組與C、D、E組間差異有統(tǒng)計學(xué)意義(P0.05),C組與E組差異無統(tǒng)計學(xué)意義(P0.05);LMP-3 mRNA及蛋白水平的表達(dá),A、B組與C、D、E組間差異有統(tǒng)計學(xué)意義(P0.001),D組與E組差異有統(tǒng)計學(xué)意義(P0.05),C組及D組在LMP-1及LMP-3的表達(dá)上的差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論:成功構(gòu)建真核雙表達(dá)質(zhì)粒pBudCE4.1-LMP-1-LMP-3,證實(shí)轉(zhuǎn)染的骨髓MSCs能在體外同時表達(dá)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.
【作者單位】： 重慶醫(yī)科大學(xué)附屬第一醫(yī)院骨科;
【分類號】：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.
【作者單位】： 重慶醫(yī)科大學(xué)附屬第一醫(yī)院骨科;
【分類號】：R341
【共引文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 唐春暉;LMP-1與LMP-3真核雙表達(dá)質(zhì)粒的構(gòu)建及體外表達(dá)[D];重慶醫(yī)科大學(xué);2010年
【二級參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 孫佳,鹿培源,賈弘y，
本文編號：2390993
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