Cloning and expression of Toxoplasma gondii silent information egulator 2(TgSir2) and preparation of its polyclonal antibody
ZHANG Fang-fei1, CAO Jia-xin2, WANG Chen-hong3, MAO Yi-jie1, HUA Qian-qian4, LIU Shu-xian5, TAN Feng5, HU Xin6
1. Renji College, Wenzhou Medical University, Wenzhou 325035, China; 2. School of Ophthalmology & Optometry, Wenzhou Medical University, Wenzhou 325035, China; 3. The Second Clinical Medical College, Wenzhou Medical University, Wenzhou 325035, China; 4. Clinical Laboratory, Dongyang People's Hospital, Jinhua 322100, China; 5. School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou 325035, China; 6. School of Life Science and School of Medical Laboratory Science, Wenzhou Medical University, Wenzhou 325035, China
Abstract：To obtain the specific polyclonal antibody against Toxoplasma gondii silent information regulator 2 (TgSir2). Searching the homologous sequence in Toxoplasma genome used the amino acid sequence of Saccharomyces cerevisiae silent information regulator 2 (ScSir2) as template and predicting the signal peptide. TgSir2 cDNA was inserted into prokaryotic expression vector pET28b. After identification, the constructed plasmid pET28b-TgSir2 was transformed into E. coli BL21 cells and induced by isopropyl-β-D-thinoglucoside (IPTG) for expression of the protein. The recombinant protein was purified via Ni-NTA affinity chromatography. Western blotting assay was conducted with anti-His tag monoclonal antibody as the primary antibody. Rabbits were immunized with purified recombinant protein. The specific anti-TgSir2 polyclonal antibody was obtained and Western blotting was performed to detect the reaction by the antibody. The TgSir2 in Toxoplasma genome was found through homologous comparison, signal peptide analysis indicated that 1-15 amino acids were the sequence of its signal peptide. pET28b-TgSir2 plasmid was identified by PCR amplification, restriction enzyme digestion and sequencing. SDS-PAGE analysis showed that the recombinant TgSir2 protein was expressed highly induced by IPTG. Western blotting showed that both TgSir2 protein from prokaryotic expression and tachyzoite lysates can be recognized by the specific anti-TgSir2 polyclonal antibody. The obtained polyclonal antibody against TgSir2 can specifically react with the endogenous TgSir2 protein.
张芳菲, 曹佳欣, 王晨红, 毛懿杰, 华倩倩, 刘淑贤, 谭峰, 胡昕. 弓形虫沉默信号调节子2(TgSir2)的克隆表达与多克隆抗体制备[J]. 中国人兽共患病学报, 2019, 35(2): 120-125.
ZHANG Fang-fei, CAO Jia-xin, WANG Chen-hong, MAO Yi-jie, HUA Qian-qian, LIU Shu-xian, TAN Feng, HU Xin. Cloning and expression of Toxoplasma gondii silent information egulator 2(TgSir2) and preparation of its polyclonal antibody. Chinese Journal of Zoonoses, 2019, 35(2): 120-125.
2019, Vol. 35 
