Objective:To construct and identify recombinant plasmid pUIS3-BLC^+.Methods:The cDNA of B-lymphocyte chemoattractant(BLC) was amplified from the total RNA of spleen tissues by PCR method,and were inserted into plasmid...Objective:To construct and identify recombinant plasmid pUIS3-BLC^+.Methods:The cDNA of B-lymphocyte chemoattractant(BLC) was amplified from the total RNA of spleen tissues by PCR method,and were inserted into plasmid of Plasmodium berghei with U1S3 knockout by digestion of restrictive endonuclease and T7 ligation.The recombinant plasmids were screened,and then underwent restriction enzymatic digestion and DNA sequencing.Then the confirmed plasmid was further transfected into COS-1 cells by lipofectamine and the BLC expression was tested by RT-PCR and Western blotting.Results:The cDNA of BLC gene was correctively amplified by RT-PCR and the recombinant plasmid pUIS3-BLC^+ was constructed successfully,which was confirmed by restriction enzymatic digestion and DNA sequencing.RT-PCR and Western blotting also showed the BLC gene expression in COS-1 cells.Conclusions:The recombinant plasmid pUIS3-BLC^+ has BLC expression in COS-1 cells,and is useful for further study on BLC transgene and UIS3 gene knockout in Plasmodium berghei.展开更多
基金Supported by National Natural Science Fund of China(No.30660177)
文摘Objective:To construct and identify recombinant plasmid pUIS3-BLC^+.Methods:The cDNA of B-lymphocyte chemoattractant(BLC) was amplified from the total RNA of spleen tissues by PCR method,and were inserted into plasmid of Plasmodium berghei with U1S3 knockout by digestion of restrictive endonuclease and T7 ligation.The recombinant plasmids were screened,and then underwent restriction enzymatic digestion and DNA sequencing.Then the confirmed plasmid was further transfected into COS-1 cells by lipofectamine and the BLC expression was tested by RT-PCR and Western blotting.Results:The cDNA of BLC gene was correctively amplified by RT-PCR and the recombinant plasmid pUIS3-BLC^+ was constructed successfully,which was confirmed by restriction enzymatic digestion and DNA sequencing.RT-PCR and Western blotting also showed the BLC gene expression in COS-1 cells.Conclusions:The recombinant plasmid pUIS3-BLC^+ has BLC expression in COS-1 cells,and is useful for further study on BLC transgene and UIS3 gene knockout in Plasmodium berghei.
