BMP7基因真核表达载体的构建及荷瘤小鼠制备

目的克隆BMP7基因及构建BMP7基因真核表达载体,稳定转染于H22细胞系,制备荷瘤小鼠。方法应用RT-PCR技术从人胚肾细胞系293T细胞总RNA中成功扩增出1 293 bp包含BMP7基因编码区的cDNA序列,在上下游加上BamHI及HindⅢ双酶切位点后形成带酶切位点的BMP7基因,然后连入含BaraH I及HindⅢ双酶切位点的pEGFP-C1表达质粒上,构建BMP7基因的重组真核表达质粒pEGFP-C1-BMP7。脂质体介导方法转染细胞,RT-PCR、western-blot方法检测其mRNA及蛋白表达,皮下注射制备荷瘤小鼠。结果扩增的cDNA经测序比较与基因文库完全一致;BamHI及HindⅢ双酶切后,质粒形成约4.7 kb和约1.3 kb两条带,与理论计算值完全一致。注射转染BMP7基因后H22细胞小鼠瘤体明显增大。结论成功克隆BMP7基因及构建BMP7基因的真核表达载体。BMP7基因高表达可能是细胞癌变的原因之一。

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