黑色素浓集激素受体2基因siRNA重组腺病毒载体构建及鉴定

Construction and identification of human MCHR2 gene siRNA recombinant adenovirus vector

目的:构建黑色素浓集激素受体2(MCHR2)siRNA重组腺病毒载体,并在稳定表达MCHR2的中国仓鼠卵巢(CHO)细胞中鉴定其干扰作用.方法:人工合成靶向MCHR2的siRNA干扰序列,用分子克隆的方法克隆到穿梭载体pSES-HUS上,得到pSES-HUS-MCHR2siRNA,并与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,得到pAdeasy-SES-HUS-MCHR2siRNA,在HEK293细胞中包装成重组腺病毒,感染稳定表达MCHR2的CHO细胞株,RT-PCR及Western Blot检测腺病毒对细胞MCHR2表达的影响.结果:成功构建MCHR2siRNA重组腺病毒载体.MCHR2siRNA重组腺病毒显著抑制CHO细胞中MCHR2的表达.结论:构建的Adeasy-MCHR2 siRNA腺病毒能有效地抑制CHO细胞中MCHR2表达,为研究MCHR2的功能及其与肥胖的关系提供了有效的工具.

AIM： To construct the recombinant adenovirus vector for siRNA targeting melanin-concentrating hormone receptor 2 （MCHR2） gene and to identify its inhibitive effect on the expression of MCHR2 gene in CHO cells stably expressing MCHR2 gene. METHODS： The siRNA sequence targeting MCHR2 gene was synthesized and cloned into the shuttle plasmid pSES-HUS to form the vector pSES-HUS-MCHR2siRNA. It was homogenously recombinated with adenovirus backbone plasmid pAdeasy-1 in E coli BJ5183. Then the recombinant adenovirus vector was transfected into HEK293 cells and adenovirus was packaged and ampliied. The CHO cells stablly expressing MCHR2 gene were infected by the adenovirus. The target gene was detected by RT-PCR and Western Blot. RESULTS： The recombinant adenoviral vector containing siRNA targeting MCHR2 gene was successfully constructed. The expression of MCHR2 in the CHO cells was significandy inhibited by MCHR2siRNA recombinant adenovirus. CONCLUSION： The recombinant adenovirus Adeasy-MCHR2- siRNA significantly inhibits the expression of MCHR2 in the CHO cells, which can provide a valid tool to study the function of MCHR2 and the relationship with obesity.