携带人VEGF121和BMP2双基因重组腺病毒载体构建、鉴定及其转染HEK293A细胞

发布时间：2018-04-19 09:18

  本文选题：血管内皮生长因子121 + 骨形态蛋白2　； 参考：《辽宁医学院》2011年硕士论文

【摘要】：目的 构建能够同时在真核细胞中高表达人血管内皮生长因子121(Vascular Endothelial Growth Factor,VEGF121)目的蛋白和人骨形态蛋白2(Bone Morphogenetic Protein , BMP2 )的新型重组腺病毒真核表达载体pAd-CMV-VEGF121-IRES-BMP2 ,并在HEK293A细胞中包装成重组腺病毒Ad-CMV-VEGF121-IRES-BMP2。同时构建其对照载体Ad-CMV-VEGF121-IRES-hrGFP- 1。为后续转染兔骨髓基质干细胞及动物体内实验奠定基础。 方法 对目的基因供体质粒pShuttle-CMV-BMP2携带的人BMP2基因进行测序和对其序列内部限制性内切酶识别位点进行分析,利用PCR(polymerase chain reaction,PCR)技术扩增BMP2基因片段以及去掉其终止密码子,之后在基因前后添加新的酶切位点Kpn I和Xba I,酶切、测序检测片段序列情况。将pShuttle-CMV-VEGF121- IRES-hrGFP-1进行测序后,使用Kpn I/Xba I双酶切,切掉hrGFP-1片段,电泳切胶回收pShuttle-CMV-VEGF121-IRES.再将序列正确BMP2基因定向连入腺病毒穿梭载体pShuttle-CMV-VEGF121-IRES中。经测序鉴定筛选阳性克隆,PmeⅠ酶切线性化且去磷酸化后转化BJ5183-AD-1电感受态细胞,利用细菌内同源重组机制将VEGF 121和BMP2基因连同其顺势表达元件重组入pAdEasy-1腺病毒系统,通过基因测序、PCR及PacⅠ酶切鉴定获得重组腺病毒载体pAd-VEGF121-IRES-BMP2。去内毒素超纯提取该重组腺病毒质粒,经PacⅠ酶切电泳后凝胶回收大片段,提纯并通过脂质体Lipofectamine 2000介导转染HEK293A细胞,RT-PCR和Western blot方法检测转染细胞中BMP2基因和VEGF121mRNA和蛋白表达情况,完成Ad-CMV-VEGF121-IRES-BMP2重组腺病毒真核表达载体的构建。同时重组对照Ad-CMV-VEGF121-IRES-hrGFP-1。 结果 1.经基因测序及酶切鉴定表明,重组腺病毒真核表达载体pShuttle-CMV-VEGF121-IRES-BMP2构建成功。 2.经基因测序、PCR检测及酶切鉴定表明,重组腺病毒真核表达载体pAd-VEGF121-IRES-BMP2构建成功,同时成功构建其对照病毒载体pAd-VEGF121-IRES-hrGFP-1。 3.转染HEK293细胞后经PCR检测鉴定表明:转染Ad-CMV-VEGF121-IRES-BMP2组(A组)与转染Ad-CMV-VEGF121-IRES-hrGFP-1对照组(B组)细胞内VEGF121mRNA表达量存在显著性差异(P0.01),A组高于B组。 4.转染HEK293细胞后Western blot结果表明:A组细胞的VEGF121蛋白表达量明显高于B组,存在显著性差异(P0.01)。 结论 成功构建能够新型重组腺病毒真核表达载体Ad-CMV-VEGF121-IRES-BMP2,为后续转染兔骨髓基质干细胞及动物体内实验奠定基础。
[Abstract]:PurposeA novel recombinant adenovirus eukaryotic expression vector pAd-CMV-VEGF121-IRES-BMP2 was constructed, which could express both the target protein of 121(Vascular Endothelial Growth factor-VEGF121 and human bone morphogenetic protein 2(Bone Morphogenetic Protein (BMP2) in eukaryotic cells. The recombinant adenovirus Ad-CMV-VEGF121-IRES-BMP2 was packaged in HEK293A cells.Meanwhile, the control vector Ad-CMV-VEGF121-IRES-hrGFP-1 was constructed.To lay a foundation for the subsequent transfection of rabbit bone marrow stromal cells and in vivo experiments.MethodThe human BMP2 gene carried by the target gene donor plasmid pShuttle-CMV-BMP2 was sequenced and the restriction endonuclease recognition sites within its sequence were analyzed. The BMP2 gene fragment was amplified by PCR(polymerase chain reaction- PCR technique and its terminating codon was removed.After that, new restriction sites Kpn I and Xba I were added before and after the gene, and the sequence of the fragments was detected by enzyme digestion and sequencing.After sequencing pShuttle-CMV-VEGF121- IRES-hrGFP-1, Kpn I/Xba I was used to cut out the hrGFP-1 fragment, and pShuttle-CMV-VEGF121-IRESwas recovered by gel electrophoresis.Then the correct BMP2 gene was inserted into the adenovirus shuttle vector pShuttle-CMV-VEGF121-IRES.The positive clone PME 鈪，

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