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Construction and Expression of Bait Vector for the Recombinant Plasmid SARP1 Gene and Detection of Their Self-activation in Yeast Two-hybrid Analysis.

Author: ZhangWei
Tutor: HeGuangZhao；MaBing
School: Chongqing Medical University
Course: Surgery
Keywords: SARP1 gene Carrier Transfection Scar
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 32
Quote: 0
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Abstract


Objective: To construct the human SARP1 gene yeast two-hybrid bait vector protein interaction between the identification SARP1 gene , and lay the foundation for to explore SARP1 gene biological functions in the scar tissue . Method: according to the SARP1 the genebank found in mRNA sequence , design are respectively provided with Nde I and Sal I cleavage points on the downstream primer, synthetic the SARP1 gene fragment , select pTA2 as a cloning vector, SARP1 gene fragment was amplified , double digestion with Nde I and Sal I , respectively pTA2-SARP1 PCR product and the pGBKT7 vector and gel recovery after the two are connected to construct a recombinant plasmid containing gene pGBKT7-SARP1 . E.coli DH5α competent cells to recombinant plasmid pGBKT7-SARP1 was transformed kanamycin positive clones were screened positive clones were selected with Nde I and Sal I double digestion , gel electrophoresis and sequencing , the recombinant plasmids of pGBKT7-SARP1 inserted SARP1 sequence integrity and reliability . Based on the the Clontech yeast hybrid operation manual , using the lithium acetate method (Li-Ac) will sequence the correct reorganization plasmid pGBKT7-SARP1 was transformed into AH109 yeast strains of the pGBKT7-SARP1 in AH109 in expression observed in the deficient medium , detection bait vector whether the toxic effects and self- activation effect . Results : digestion , gel electrophoresis and DNA sequencing confirmed the pGBKT7-SARP1 recombinant vector was constructed correctly , the yeast strain AH109 non-toxic , and of no effect of the independent activation of the reporter gene . Conclusion: We successfully constructed the pGBKT7-SARP1 recombinant yeast two-hybrid bait vector for the further use of the yeast two-hybrid method the identification SARP1 genes interacting protein , explore SARP1 gene in scar fibroblasts experimental basis .

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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