Construct Eukaryotic Expression Vector and Study the Function of BMP7 Gene

Objective To clone BMP7 gene and to construct eukaryotic expression vector.And observe the changes of cell proliferation of the H22 transfected of BMP7 gene.Methods Use RT-PCR technique successfully amplified BMP7 gene 1293bp cDNA sequence coding region from 293T cells total RNA and in the upstream and downstream of BMP7 gene adding BamHI and Hind Ⅲ restriction sites,then connected with pEGFP-C1 expression plasmid which contains BamH I and Hind Ⅲ restriction enzyme digestion sites.The transfection of BMP7 gene into HL-7702 cells was done by liposome-mediated method.RT-PCR was used to detect the expression of mRNA,and western-blot was used to detect protein expression levels of BMP7.We injected H22 which has transfected of BMP7 in mice subcutaneous.Results Sequencing analysis revealed that the orientation of the ligations and the reading frame were correct.Digested by BamH I and Hind III,two fragments of 4.7kb and 1.3 kb respectively were formed in eukaryotic expressing vector.Electrophoretic results were completely coincident with theoretical calculation.After transfected,the tumor volume increased significantly.Conclusion BMP7 were successfully cloned and eukaryotic expressing vector were successfully constructed.Over expression of BMP7 gene maybe is one of the reasons of carcinogenesis