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《Acta Academiae Medicinae Militaris Tertiae》 2007-01
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Construction of recombinant lentivirol expression vector carrying mouse B7-H1 gene and its expression and identification in B16F10 cells

ZHANG Li-qun,DUAN Wen-yuan,XU Xue-qing,HUANG Gang,CHEN Xue-dan,BAI Yun(Department of Medical Genetics,College of Medicine,Third Military Medical University,Chongqing 400038,China)  
Objective To construct lentivirol expression vector of mouse B7-H1,and identify its expression in B16F10 cells.Methods Mouse B7-H1 encoding sequence was amplified,purified,ligated with pLenti6/V5 lentivirol vector and verified by sequencing.The verified recombinant was transfected into 293FT cells by Lipofectamine~(TM) 2000 reagent.The supernatant of the cultured 293FT cells was collected and used to infect B16F10 cells.B16F10 cell line stably expressing B7-H1 protein was selected in the presence of Blasticidin(4.0 μg/ml).Results The mouse B7-H1 gene was successfully amplified by PCR.The recombinant lentivirol vector carrying B7-H1 gene for expression was constructed and its length was about 877 bp,very close to the expected value.By transfecting package cell line 293FT,the recombinant of pLenti6/V5-D-TOPO lentivirol vector carrying B7-H1 infected B16F10 cells in selective medium.B16F10 transgenic cells could stably express mouse B7-H1 protein on cell membrane.Conclusion Successful construction of mouse B7-H1 lentivirol expression vector and expression of its functional fusion protein based further investigation of the role of B7-H1 in immune tolerance,autoimmune disease and tumor immune escape.
【Fund】: 国家自然科学基金(30271246)~~
【CateGory Index】: R392
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