Prokaryotic Expression, Purification and Immunological Characterization of Micronemal Protein 16 of Toxoplama gondii

• Published in: Zhongguo jishengchongxue yu jishengchongping zazhi Vol. 34; no. 3; p. 198
• Main Authors: Li, Jin, Cui, Yong, Yin, Kun, Liu, Gong-zhen, Xiao, Ting, Xu, Chao, Wei, Qing-kuan, Huang, Bing-cheng, Sun, Hui
• Format: Journal Article
• Language: Chinese
• Published: China 01.06.2016
• ISSN: 1000-7423

To prokaryotically express three gene fragments of micronemal protein 16 （TgMIC16） of Toxoplasma gondii, and analyze the immunoreactivity of the three recombinant protein products. Primers were designed for three fragments of TgMIC16 gene which encode proteins within the functional domain. Reverse-transcription PCR was used to generate cDNA from RNA, and the three fragments were amplified on the cDNA by PCR using the designed primers. The PCR products were double-digested, inserted into the pET-32a（+） plasmid, and transformed into Escherichia coli TOP10 cells. Plasmids extracted from positive clones were confirmed by BamHⅠ/HindⅢ double digestion and sequencing, and further transformed into E. coli Rosetta cells. Protein expression was induced by IPTG, and confirmed by SDS-PAGE. The expressed recombinant proteins were purified with Ni-NTA affinity chromatography and their immunoreactivity analyzed with Western blotting. The amplified three fragments were 1 806, 1 290 and 855 bp in size. Double digestion and se