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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。
Objective To clone BMP7 gene and construct eukaryotic expression vector of BMP7 gene and stably transfected into H22 cell line to prepare tumor-bearing mice. Methods The cDNA sequence of 1 293 bp coding BMP7 gene was successfully amplified from total RNA of human embryonic kidney cell line 293T by RT-PCR. After double-digested with BamHI and HindⅢ, The BMP7 gene was cut and inserted into pEGFP-C1 expression plasmid containing BaraH I and Hind III restriction sites to construct recombinant eukaryotic expression plasmid pEGFP-C1-BMP7 of BMP7 gene. The transfected cells were transfected by liposome, the mRNA and protein expression were detected by RT-PCR and western-blot, and the tumor-bearing mice were injected subcutaneously. Results The amplified cDNA was identical to that of the gene library by sequencing. After double digestion with BamHI and Hind Ⅲ, the two plasmids formed about 4.7 kb and about 1.3 kb, which were completely consistent with the theoretical values. After transfection with BMP7 gene, the tumor size of H22 cells in mice increased significantly. Conclusion The successful cloning of BMP7 gene and construction of eukaryotic expression vector of BMP7 gene. BMP7 gene overexpression may be one of the reasons for cell carcinogenesis.