Yang Shuangjuan, Xu Chengquan, Wu Jiangwei, Yang Gongshe
Laboratory of Animal Fat Deposition and Muscle Development, Northwest A & F University, Yangling 712100, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Apr;26(4):462-9.
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 1.2x10(9) 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.
为构建表达细胞因子信号转导抑制因子3（SOCS3）的重组腺病毒载体并获得感染性腺病毒颗粒，从质粒pcDNA3-SOCS3中扩增SOCS3基因，并亚克隆至腺病毒穿梭质粒pAdTrack-CMV。序列确认后，重组穿梭质粒pAdTrack-CMV-SOCS3经Pme I酶切线性化，然后转化至BJ5183感受态细胞，通过pAdTrack-CMV-SOCS3与腺病毒骨架质粒pAdEasy-1在BJ5183中的同源重组获得重组质粒pAd-SOCS3。pAd-SOCS3经Pac I酶切线性化，通过脂质体转染至HEK293细胞。重组腺病毒在HEK293细胞中包装并扩增。纯化后，通过组织培养感染剂量50（TCID50）测定病毒滴度。用重组腺病毒感染猪原代脂肪细胞，通过荧光显微镜观察绿色荧光蛋白（GFP）的表达，并通过RT-PCR和Western印迹鉴定SOCS3基因。限制性内切酶和PCR分析表明重组腺病毒载体构建正确，病毒滴度达到1.2×10⁹ PFU/mL。RT-PCR和Western印迹结果显示，感染重组腺病毒的猪原代脂肪细胞中SOCS3 mRNA和蛋白表达显著增加。总之，本研究成功构建了含SOCS3基因的重组腺病毒，有助于进一步研究SOCS3的功能。
