SOCS3基因重组腺病毒的构建及其在猪脂肪细胞中的表达

Construction of SOCS3 recombinant adenovirus and its expression in porcine primary adipocytes

摘要:

本研究旨在构建细胞因子信号转导抑制因子3 (Suppressor of cytokine signaling 3，SOCS3) 的重组腺病毒表达载体，获得有感染性的病毒颗粒。以pcDNA3-SOCS3质粒为模板扩增SOCS3基因，将其亚克隆至腺病毒穿梭载体pAdTrack-CMV，经测序验证后，重组的穿梭质粒用Pme I酶切线性化后转化到BJ5183感受态细菌中与其内的骨架载体pAdEasy-1进行同源重组，获得的重组质粒pAd-SOCS3，经Pac I线性化后转染至HEK293细胞中进行包装和扩增，纯化后用TCID50法测定病毒滴度。以重组的病毒感染原代培养的猪脂肪细胞后，荧光显微镜下观察报告基因GFP的表达，RT-PCR和Western blotting检测细胞内SOCS3 mRNA和蛋白的表达。重组腺病毒载体pAd-SOCS3经酶切及PCR鉴定正确，病毒滴度为1.2×109 PFU/mL；感染原代培养的猪脂肪细胞后，荧光显微镜观察可见报告基因GFP的表达；RT-PCR和Western blotting检测到细胞中SOCS3 mRNA和蛋白的表达显著提高。本研究成功构建了SOCS3基因的重组腺病毒，感染原代培养的猪脂肪细胞可稳定表达SOCS3蛋白，为深入研究SOCS3的功能奠定了基础。

Abstract:

In order to construct recombinant adenovirus vector expressing Suppressor of cytokine signaling 3 (SOCS3) and obtain infectious adenoviral particles, SOCS3 gene was amplified from plasmid pcDNA3-SOCS3 and subcloned into the adenovirus shuttle plasmid pAdTrack-CMV. After sequence confirmation, the recombinant shuttle plasmid pAdTrack-CMV-SOCS3 was linearized by Pme I, and then transformed into BJ5183 competent cell, the recombinant plasmid pAd-SOCS3 was obtained by homologous recombination between pAdTrack-CMV-SOCS3 and the adenoviral backbone plasmid pAdEasy-1 in BJ5183. The pAd-SOCS3 was linearized by Pac I and transfected into HEK293 cells via liposome. The recombinant adenovirus was packaged and amplified in HEK293 cells. After purifying, virus titer was determined by tissue culture infectious dose 50 (TCID50). Using the recombinant adenoviruses to infect porcine primary adipocytes, the expression of green fluorescent protein (GFP) was observed by fluorescent microscopy, and SOCS3 gene was identified by RT-PCR and Western blotting. Restriction enzyme and PCR analysis demonstrated that the recombinant adenovirus vector was constructed correctly, and the virus titer reached 1.2×109 PFU/mL. The result of RT-PCR and Western blotting showed that SOCS3 mRNA and protein expression was remarkably increased in porcine primary adipocytes infected with recombinant adenovirus. In conclusion, this study successfully constructed the recombinant adenovirus containing SOCS3 gene, and can be helpful for further research on the function of SOCS3.