【摘要】 目的 构建金黄仓鼠载脂蛋白A5(Apoa5)基因表达载体以及在不同组织中的表达差异分析，为深入研究Apoa5基因在金黄仓鼠不同组织中功能奠定基础。方法 提取金黄仓鼠肝脏组织总RNA，采用RT-PCR方法对金黄仓鼠Apoa5基因的cDNA进行克隆，构建Apoa5基因表达载体并将其转染293T细胞，分别采用RT-PCR和Western blot方法检测mRNA及蛋白质表达水平。采用荧光定量PCR检测并分析Apoa5 mRNA在8个组织中的表达量。结果 经PCR检测鉴定,Apoa5基因表达载体构建成功,经测序分析金黄仓鼠Apoa5基因cDNA全长1 243 bp,编码366个氨基酸，与人、大鼠、小鼠等物种比对，核苷酸和氨基酸序列均具有较高的同源性，金黄仓鼠与人、大鼠、小鼠的氨基酸同源性分别为75%、84%、84%。转染293T细胞48 h后，RT-PCR和Western blot结果表明，Apoa5基因在mRNA和蛋白质水平都有表达。荧光定量PCR结果显示，Apoa5 mRNA在肝脏表达量最高，在脑和睾丸中中度表达，在心、脾脏、肺、肾、卵巢组织中低度表达。结论 成功构建Apoa5基因表达载体并分析其在不同组织中的表达差异，为制备Apoa5转基因金黄仓鼠动物模型以及研究Apoa5基因在金黄仓鼠脂类代谢中的功能奠定基础。更多还原

【Abstract】 Objective To construct apolipoprotein a5(Apoa5) expressing plamid and describe the gene expression profile of Apoa5 in golden hamster. Methods Total RNA was extracted from liver tissue of golden hamster, cDNA of Apoa5 was cloned by RT-PCR, and then subcloned into plasmid. The plasmid of pEF6/V5-His-Apoa5 was transfected into 293T cells, the expression was examined by RT-PCR and Western blot. The expression of Apoa5 in 8 organs, including brain, heart, lung, liver,spleen, kidney, testis, and ovary of golden hamster were also detected by real time PCR. Results Apoa5expression vector was constructed. Apoa5 mRNA and APOA5 protein was successfully detected in293T cells 48 h after transfection. Sequencing results showed that the cDNA of Apoa5 contained 1 243 bp,it encodes 366 aa. The Apoa5 gene in golden hamster has high homology with human, mouse and rat.The Apoa5 gene in golden hamster shares 75%, 84%, 84% amino acids with human, rat, and mouse.Real time PCR results showed that gene expression of Apoa5 was high in liver, brain, testis, but low in heart, lung, and kidney. Conclusion The Apoa5 expression vector was constructed successfully, and Apoa5 expression profile in golden hamster had been described. It may be as the foundation for establishing Apoa5 transgenic golden hamster and researching the function of Apoa5.更多还原