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Construction and Identification of Recombinant Adenovirus Vector Carrying Double Genes Namely Mutant Human VEGF121 and BMP2 and Transfection the Adenovirus into HEK293A Cells

Author: ZhongSheng
Tutor: LiuDanPing
School: Liaoning Medical
Course: Surgery
Keywords: Vascular endothelial growth factor 121 Bone morphogenetic protein 2 Adenovirus Coexpression Angiogenic
CLC: R346
Type: Master's thesis
Year: 2011
Downloads: 72
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Abstract

Objective To construct eukaryotic cells simultaneously in the high expression of vascular endothelial growth factor 121 (Vascular Endothelial Growth Factor, VEGF121) protein and bone morphogenetic protein-2 (Bone Morphogenetic Protein, BMP2) novel recombinant eukaryotic expression vector pAd-CMV -VEGF121-IRES-BMP2, and HEK293A cells packed into adenovirus Ad-CMV-VEGF121-IRES-BMP2. While building its control vector Ad-CMV-VEGF121-IRES-hrGFP-1. For subsequent transfection of rabbit bone marrow stromal cells and in vivo experimental basis. Methods for the gene of plasmid pShuttle-CMV-BMP2 gene carrying human BMP2 sequences were sequenced and its internal restriction endonuclease recognition sites were analyzed using PCR (polymerase chain reaction, PCR) to amplify gene fragments BMP2 and remove its stop codon in the gene before and after adding a new restriction sites Kpn I and Xba I, restriction enzyme digestion, sequencing to detect sequence fragments situation. The pShuttle-CMV-VEGF121-IRES-hrGFP-1 was sequenced, use the Kpn I / Xba I digested, cut hrGFP-1 fragment, agarose gel electrophoresis, recovered pShuttle-CMV-VEGF121-IRES. Then correct sequence BMP2 Gene targeting incoming adenovirus shuttle vector pShuttle-CMV-VEGF121-IRES in. Positive clones were identified by sequencing, Pme Ⅰ digestion and dephosphorylation of linearized and transformed into BJ5183-AD-1 electrocompetent cells using bacterial homologous recombination mechanism VEGF 121 and BMP2 gene expression elements together with their homeopathic recombined into pAdEasy- an adenovirus system, through gene sequencing, PCR and Pac Ⅰ restriction endonuclease to obtain recombinant adenovirus vector pAd-VEGF121-IRES-BMP2. To extract the endotoxin ultrapure recombinant plasmid was digested with Pac Ⅰ after recycling large fragment gel electrophoresis, purification and liposome-mediated transfection Lipofectamine 2000 HEK293A cells, RT-PCR and Western blot detection of transfected cells and VEGF121mRNA of BMP2 gene and protein expression, complete the Ad-CMV-VEGF121-IRES-BMP2 recombinant eukaryotic expression vector. At the same time restructuring the control Ad-CMV-VEGF121-IRES-hrGFP-1. Results 1 through gene sequencing and restriction enzyme digestion showed that recombinant eukaryotic expression vector pShuttle-CMV-VEGF121-IRES-BMP2 constructed successfully. (2) by gene sequencing, PCR testing and restriction analysis showed that recombinant eukaryotic expression vector pAd-VEGF121-IRES-BMP2 was successfully constructed and successfully build their control viral vector pAd-VEGF121-IRES-hrGFP-1. 3. After transfection of HEK293 cells identified by PCR showed that: transfected Ad-CMV-VEGF121-IRES-BMP2 group (A) and transfected with Ad-CMV-VEGF121-IRES-hrGFP-1 control group (B) cells VEGF121mRNA There was a significant expression differences (P lt; 0.01), A group was higher than the B group. 4 HEK293 cells transfected with Western blot results showed that: A group of cells VEGF121 protein expression was significantly higher than that in group B, there was a significant difference (P lt; 0.01). Conclusions can be successfully constructed a new recombinant eukaryotic expression vector Ad-CMV-VEGF121-IRES-BMP2, for subsequent transfection of rabbit bone marrow stromal cells and in vivo experimental basis.

Construction and Identification of Recombinant Adenovirus Vector Carrying Double Genes Namely Mutant Human VEGF121 and BMP2 and Transfection the Adenovirus into HEK293A Cells

Abstract

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