关键词: 猪cGAS, 非酶连接, 丙酸诱导, 原核表达

Abstract: 【Objective】cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase), as a new type of nucleic transferase was found in mammalian cells recently. It can identify cytoplasmic DNA and catalyse ATP and GTP to generate the second messenger cGAMP, cGAMP binds to STING by dependent way, leading to activate transcription factor IRF3. Then the inherent immunity was starting. This experiment was conducted to construct a prokaryotic expression plasmid, pBbB3a-His6-NusA- cGAS, containing porcine cGAS gene. The gene expression was induced in E. coli, and the cGAS protein was obtained. Then a foundation of the research on the synthesis of cGAMP in vitro and its function in innate immune progress was made.【Method】 The open reading frame (ORF) of cGAS was amplified from the cDNA of porcine spleen, and cloned it into propionate inducible plasmid pBbB3a-His6-NusA-LIC by ligation-independent cloning (LIC) technology. Identification of individual clone was performed by bacteria liquid PCR followed by DNA sequencing. Plasmids were extracted from the confirmed bacteria and transformed into E.coli BL21 (DE3). When the bacteria grew to logarithmic phase, sodium propionate was used to induce the expression of His6-NusA- cGAS fusion protein, 20 mmol·L^-1 sodium propionate at 20℃, 180 r/min were induced by 2 h, 4 h, 6 h, 8 h, 10 h and 0 h, then the optimal induction time was determined. Sodium propionate at 0, 5, 10, 15, 20, 25, 30, 35, 40 and 45 mmol·L^-1, respectively, and at 20℃, 180 r/min induced for 6 h to determine the best induction concentration of sodium propionate. Under the conditions of 20, 30 and 37℃, 20 mmol·L^-1 sodium propionate was used at 180 r/min to cultivate for 6 h to determine the best temperature induction. The His6-NusA-cGAS fusion protein was induced by sodium propionate and identified by SDS-PAGE and Western Blot. 【Result】 (1) Porcine cGAS gene’ORF, which is 1 494 bp in length, was successfully cloned; (2) The propionate inducible plasmid pBbB3a-His6-NusA-cGAS was constructed; (3) At 37℃, 20 mmol·L^-1 sodium propionate to induce for 6 h, the His6-NusA-cGAS fusion protein’s expression amount was the highest. (4) His6-NusA-cGAS fusion protein was efficiently expressed in soluble form with a molecular weight of about 111.87 kD.【Conclusion】 These results indicate that cGAS fusion protein was successfully expressed in E.coli BL21 (DE3) and this will provide technology and methods for cGAS’s fusion protein expression in vitro.

Key words: porcine cGAS, ligation-independent cloning, propionate induction, prokaryotic expression