Prokaryotic Expression And Activity Analysis Of GcGAS And Preliminary Study On The Antiviral Activity Of Goat 2′3′-cGAMP

The second messenger molecule 2 ’3’-c GAMP is a cyclic dinucleotide synthesized by cyclic GMP-AMP synthase(c GAS)catalyzed with ATP and GTP as substrates.stimulator of interferon genes(STING)can be used as a natural immune agonist for stimulator of interferon genes(STING).It induces the expression and secretion of cytokines such as interferons(IFNs),cytokines(inflammatory factors)and chemokines(chemokines),activates the natural immune response of the host,and thus plays a role in antiviral,anti-tumor and immune regulation.Therefore,2 ’3’-c GAMP has great potential to be developed into antiviral drugs,antitumor drugs,immune adjuvants and immune enhancers,and has broad application prospects for the prevention and control of major diseases in humans and animals.In this study,a soluble prokaryotic expression vector of goat c GAS(gcGAS)gene was constructed,expressed and purified,and the bioactive gcGAS protein was obtained.The second messenger molecule 2 ’3’-c GAMP was synthesized by enzymatic reaction in vitro.Secondly,the antiviral effect of 2 ’3’-c GAMP on Pseudorabies virus(PRV)was investigated from the cellular level.In order to lay a foundation for the development of safe and efficient antiviral drugs,immune adjuvants and immune enhancers.Specific research results are as follows:1.Soluble prokaryotic expression of gcGAS recombinant protein in goats.The full-length gcGAS gene was amplified by RT-PCR using Shanbeiwhite velvet goat c DNA as the base template.The amino acid structure of gcGAS and its enzyme active region were analyzed,and then the enzyme active region was cloned into p ET-28a-SUMO vector,and the optimized expression conditions,purification conditions and biological activity analysis were carried out.The results showed that the recombinant expression plasmid of gcGAS was successfully constructed to induce expression in Ecoli Rosetta(DE3),and the target protein with high purity and concentration was obtained after the optimized conditions.2.gcGAS activity analysis and 2 ’3’-c GAMP biosynthesis of goats.The purified gcGAS can catalytically synthesize 2 ’3’-c GAMP with high purity and correct structure in vitro,and induce the secretion of cytokines IFN-β and TNF-α at the cellular level,indicating that the recombinant gcGAS has bioenzyme activity.3.Preliminary study on the role of 2 ’3’-c GAMP in the process of resistance to virus infection.Firstly,cell culture and proliferation were performed.Different doses of 2 ’3’-c GAMP were selected to stimulate cells,and the concentration changes of TNF-α and IFN-βwere detected at different time points.The optimal immune dose of 2 ’3’-c GAMP was determined according to the time,concentration or dose and concentration curve.The virulence of PRV virus was determined,and the infection complex number with MOI=1 was selected to infect the cells.2 ’3’-c GAMP prevention and treatment were performed at different time points before and after virus infection,and different prevention and treatment schemes were established.The results showed that the 2 ’3’-c GAMP prevention and treatment group could significantly reduce the virus titer,and play a role in in vitro virus inhibition,compared with the treatment group only receiving virus infection without 2 ’3’-c GAMP prevention and treatment.In summary,the prokaryotic expression vector of gcGAS was successfully constructed in this experiment,the soluble expression of the recombinant protein was achieved by exploring and optimizing the induction conditions,and the target protein gcGAS with high purity and high concentration was successfully obtained.2 ’3’-c GAMP was catalyzed by enzyme reaction using gcGAS as enzyme molecules in vitro.Then,Raw267.4 and PK-15 cells were stimulated with the synthesized 2 ’3’-c GAMP,and the results showed that the stimulation of 2 ’3’-c GAMP successfully induced the secretion of cytokines IFN-β and TNF-α.The above results proved that the laboratory preparation method of 2 ’3’-c GAMP was successfully established in this experiment,and the synthetic cost of 2 ’3’-c GAMP was saved.Finally,by establishing the evaluation model of 2 ’3’-c GAMP against pseudorabies virus in vitro,the effect of 2 ’3’-c GAMP against pseudorabies virus was preliminarily studied and discussed.