人ING4的基因克隆及真核表达

目的克隆人生长抑制因子家族（inhibitor of growth famility member4，ING4）基因，构建其真核表达载体pEGFP—ING4。方法提取人胎盘总RNA，经RT—PCR扩增出ING4 cDNA，克隆至pEGFP—C2载体，构建的真核表达载体pEGFP—ING4用双酶切、基因测序进行序列鉴定；转染MCF-7细胞用荧光显微镜和免疫组化检测重组质粒的表达。结果RT—PCR产物为750bp的条带，双酶切和基因测序正确，转染可见目的蛋白融合表达。结论从人胎盘组织中成功克隆了ING4基因并构建其真核表达质粒在人MCF-7细胞中表达，为进一步研究1NG4基因的作用及抗肿瘤机制奠定了基础。

Cloning and Eukaryotic Expression of Human ING4 Gene

Objective To clone the gene of human construct pEGFP-ING4 eukaryotic vector. Methods Total and eDNA of ING4 was then amplified by RT-PCR. The ING4 derived from human placenta and RNA was extracted from human placenta resultant PCR product was inserted into pEGFP-C2 vector. The positive recombinant pEGFP-ING4 clone was analyzed by restriction endonuclease digestion and DNA sequencing. The pEGFP-ING4 plasmid was transfected into human MCF-7 cells, and expression of ING4 was detected by fluorescent microscopy and immunohistochemical staining. Results RT-PCR product was shown as a 750 bp specific fragment. Restriction enzyme digestion and DNA sequencing revealed that ING4 clone was successfully constructed. The green fluorescence was visible in transfected human MCF-7 cells under fluorescent microscopy. Immunohistochemieal staining detection showed that ING4 was expressed in transfected ceils, displaying as brown granules. Conclusion The gene encoding human ING4 derived from human placenta was obtained and cloned into pEGFP-ING4 plasmid. Expression of the ING4 protein was detected in MCF-7 cells following transfection. The established pEGFP-ING4 construct may be used to further study the function of INCA and its tumor suppressive mechanisms.