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Construction of Human B-type Natriuretic Peptide in the Lentiviral Vector

Author: LiuXiangYang
Tutor: LuYongZuo
School: Huazhong University of Science and Technology
Course: Cardiology
Keywords: Lentiviral vectors Vector construction B-type natriuretic peptide Myoblasts Gene expression
CLC: R346
Type: Master's thesis
Year: 2006
Downloads: 29
Quote: 0
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Abstract


Objective: To construct the expression of human B -type natriuretic peptide ( hBNP ) 's , pLenti6/V5-hBNP carrier hBNP gene in skeletal myoblasts to achieve long - term , stable expression . Methods : In vitro culture SD ( Sprague Dawley ) rats skeletal myoblasts , and identification of cells obtained by immunohistochemistry using trypan blue staining to determine the activity of the cultured cells and growth curve . The target gene hBNP subcloned into eukaryotic cells the expression vector pLenti6/V5-D-TOPO carrier , the recombinant plasmid pLenti6/V5-hBNP . The the plasmid pLenti6/V5-EGFP on pLenti6/V5-hBNP and positive control respectively Lipofectamin 2000 referral guide transfected 293FT cells , virus particles ; transfection in vitro with virus particles SD neonatal rat skeletal myoblasts . Counted under the fluorescence microscope to determine the positive control plasmid transient transfection number , in order to estimate the transient transfection efficiency of the gene . Join Filter reagent to obtain stable expression of heterologous B-type natriuretic peptide (BNP) myoblasts . Collecting the cell culture medium after transient transfection and screening by enzyme linked immunosorbent assay (ELISA) testing kit to detect the expression of hBNP level . Results: the polymerase chain reaction method , double restriction enzyme digestion and DNA sequencing showed the the hBNP gene is inserted into in a pLenti6/V5-D-TOPO support ; Immunohistochemistry showed this method to develop into muscle cytoplasm purity of 94% , and trypan blue staining cells survival rate reached more than 94% ; positive control plasmid virus particles infected cells after 12 h was observed under a fluorescence microscope , the transfection efficiency of more than 60% . The supernatant was collected by detection results of the negative control was a significant difference ( P lt ; 0.01 ) was observed to # 4 w hBNP continued stable expression . Conclusion: This study successfully constructed and expressed in eukaryotic cells the recombinant plasmid pLenti6/V5-hBNP of B-type natriuretic peptide , and achieved a higher transfection efficiency , lentiviral vector -mediated gene therapy in cardiovascular disease optimistic about the prospects for the application .

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CLC: > Medicine, health > Basic Medical > Human biochemistry, molecular biology
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