Study On TsGLS Gene Silencing On The Acid-Resistance Of Trichinella Spiralis And Immunoprotection Of Its Recombinant Protein

Trichinosis is one of the most important food-borne zoonotic diseases and is distributed throughout the world.Trichinosis not only causes significant economic losses,but also endangers human health.This experiment used RNA interference technology to explore the importance of the glutamine-dependent acid resistance mechanism of Trichinella spiralis.Three specific Ts GLS-siRNAs were introduced into Trichinella spiralis muscle larva by immersion method,and a Ts GLS-siRNA with the best silencing effect was screened.The larvae transfected with siRNA were placed in a culture solution of p H2.5,p H4,p H6.6,and p H9,and the effects of Ts GLS gene silencing on the acid resistance of muscle larvae were detected from the survival rate,gene expression level,enzyme activity of the bacterium,p H change of the culture medium,and Gln deletion.The muscle larvae transfected with siRNA were orally inoculated into mice,and the effect of Ts GLS gene silencing on the acid resistance of muscle larvae,the larval rate of muscle larvae and the acid resistance of the first generation was observed.By gene level and protein level analysis,the expression of Ts GLS in muscle larvae of siRNA-881 group was 72.82% and 55.44%lower than that of PBS group,and siRNA-881 with the best interference effect was successfully screened from three Ts GLS-siRNAs.In the detection of the effect of Ts GLS gene silencing on the acid resistance of muscle larvae in vitro.The survival rate showed that the survival rate was significantly lower than that in other acidic environments at p H2.5 and 2 h,and the worm reduction rate of siRNA-881 group was only 48.33% of the PBS group.The results of enzyme activity assay showed that glutaminase activity was the highest at p H2.5 and 0.5 h,which was 1.67U/mg,p H4 was second,and there was no activity at p H9.The p H change of the culture solution showed that the p H of the culture solution was measured by a p H meter,and the p H change was most remarkable when the initial p H of the culture solution was p H4.After incubation for 1 h in a p H4 environment,the p H increase was 1.7 lower than that of the PBS group after siRNA-881 interference,and the gap was smaller with time.By detecting the effect of glutamine deficiency on acid resistance,the results showed that the addition of 10 μM glutamine in vitro increased Ts GLS gene expression,and siRNA-881 silenced Ts GLS better than BPTES.In the detection of the effect of Ts GLS gene silencing on the acid resistance of muscle larvae,the worm reduction rate of the 7 d adult and the larval rate of the 35 d muscle larvae were 61.64% and 66.71%,respectively.Theresults of detecting the thickness of the nanny cells showed that the thickness of the siRNA-881group(15.90±1.301)was reduced by 39.78% compared with the PBS group.The siRNA treatment of the larval Ts GLS gene silencing heritability analysis,compared with the parental,the gene expression of the child generation decreased by 32.70% compared with the parent.This proves that Trichinella spiralis has a glutamine-dependent acid resistance system,and RNA interference technology has also significantly reduced the acid resistance of muscle larvae.In order to evaluate the immunoprotective effects of Ts GLS and Ts GAD recombinant proteins induced by Trichinella spiralis,the recombinant proteins of Ts GLS,Ts GAD and Ts GLS+Ts GAD were mixed with adjuvants to immunize BALB/c mice.Detection of changes in specific antibody levels in serum,and detection of cytokine levels produced by recombinant proteins in vitro stimulated by mouse spleen lymphocytes.After boosting the mice,the worm reduction rate of the7-day adult and 35-day muscle larvae was measured and compared by the challenge test,and the cytokine levels produced on the 7th,14 th,21st,and 35 th day after the challenge of the immunized mice.Serum-specific antibody assays showed that mice immunized with Ts GLS and Ts GAD recombinant proteins induced a mixed Th1/Th2 immune response in the body.The results of Ig G,Ig G1,and Ig G2 a in the serum of each immunized group showed that the levels of Ig G,Ig G1,and Ig G2 a antibodies increased,and the antibody level of Ig G1(Th2)increased most significantly.The results of proliferation experiments showed that the number of spleen lymphocytes increased significantly after stimulation with Ts GLS and Ts GAD recombinant protein,and the most significant stimulation with Ts GAD recombinant protein.The results of cytokine secretion in Elisa assay showed that IFN-γ,IL-12,IL-4 and IL-10 levels were significantly increased.Elisa detection of serum cytokines showed that levels of IL-4 and IL-10 reached the highest level on day 14 after challenge,and IFN-γ and IL-12 levels reached the highest on day 7 after challenge.The results of the challenge test showed that the adults and muscle larvae in Ts GLS,Ts GAD and Ts GLS+Ts GAD mice were 35.55%,49.24%,37.34% and 47.05%,60.01%,and 46.55%.The above results indicated that both Ts GLS and Ts GAD recombinant proteins could induce immunoprotective effects in mice,and Ts GAD recombinant protein had the best immune effect.Two experiments have shown that Trichinella spiralis muscle larvae have a glutamine-dependent acid resistance system,and RNAi technology has also significantly reduced the acid resistance of muscle larvae,providing a new idea for the prevention of Trichinosis.Glutaminase can be used as a candidate for Trichinosis vaccine.