Prokaryotic Expression Of Trichinella Spiralis TRE Gene And Its Researchs Of Establishment Of Indirect ELISA Method And Immune Protection

Trichinella spiralis（T.spiralis）is a highly harmful foodborne zoonotic parasite that mainly parasitizes mammals such as pig,mouse,dog and cat,and sometimes infects human.The outbreak of trichinosis has brought huge economic losses to the livestock industry in China.However,in the actual detection of this disease,there are always many factors that interfere with the detection effect.For example,extremely rich samples,heavy number of samples,inaccurate detection and time-consuming and laborious quarantine methods,which will greatly increase the rate of missed detection.So far,there have been many vaccines against trichinosis,but the immune effect is far from ideal.Therefore,rapid diagnosis and vaccine development of trichinosis have important significance and research value.In this experiment,molecular biological techniques were used to prokaryotically express the recombinant of T.spiralis trehalase protein（r Ts TRE）.Based on the published Ts TRE gene sequence（XM＿003380744.1）on Gene Bank,a pair of specific primers were designed to amplify the Ts TRE gene fragment from the T.spiralis c DNA by RT-PCR technology;connect the target gene with the p MD-18T cloning plasmid and transfer it into Escherichia coli（E.coli）DH5-αreceptive cells,clone plasmids were extracted and identified using Sac I and Hind III enzymes via double restriction enzyme digestion,the plasmid with correct enzyme digestion was sent to Jilin Kumei Sequencing Company for sequencing,and was named p MD18T-Ts TRE clone plasmid.After connecting the target gene with the p Cold I expression plasmid,it was transferred into E.coli BL21 receptive cells,and the expression plasmid was extracted.Similarly,Sac I and Hind III were used to identify the connection between the target gene and the expression plasmid,and the correctly connected plasmid is the p Cold I-Ts TRE expression plasmid.Use an ITPG inducer to induce the expression of the recombinant protein.At the same time,set the ITPG induction concentration were 0.2 m M,0.4 m M,0.6 m M,0.8 m M,and 1.0 m M for 6 h,10 h,14 h,18 h,20 h,and 24 h,respectively.Determine the solubility of the protein and screen the optimal ITPG induction concentration and time for expressing the protein.At the same time,the rabbit r Ts TRE polyclonal antibody was prepared,and Indirect immunofluorescence technique（IF）was used to locate the Ts TRE protein.The results of prokaryotic expression showed that the Ts TRE gene was approximately 1548 bp,the relative molecular weight of Ts TRE protein is about 60 k Da;the homology of this gene is as high as 99.48%,and the expression of this protein was highest at ITPG concentration of 1.0 m M for 24 h.The IF results show that Ts TRE mainly exists in the rod-shaped cells and reproductive primordium of muscle larva.In this study,r Ts TRE protein was used as a coating antigen to establish an indirect ELISA antibody detection method for trichinosis.As well as,the evaluation content includes:antigen coating concentration and coating conditions,dilution and incubation conditions of the serum to be tested,type and time of blocking solution,dilution and incubation conditions of enzyme-labeled secondary antibody,TMB reaction time,critical value calculation,repeatability test,sensitivity test,specificity test,detection rate test and the contrast of grinding fluid of different tissues and organs and so on.The results showed that the determined antigen coating concentration was 100 ng/hole,the antigen coating condition is 4℃overnight,the dilution of the serum to be tested is 1:16000,the incubation condition of the serum to be tested was closed at 37℃for 2 h,the sealing liquid is selected as 5%BSA,and the sealing condition of the sealing liquid is 37℃for 2 h,the dilution of the enzyme-labeled second antibody was 1:10000,the incubation condition of the enzyme-labeled second antibody was 37℃for 2 h,and the TMB reaction time was 10 min.Moreover,the critical value was 0.384,the coefficients of variation within and between batches were 5.504%-7.630%and 4.664%-9.929%,respectively,both less than 10%,the minimum detection dilution is 1:1280,there was no cross reaction with antibodies against Clonorchis sinensis,Schistosoma japonicum,Ascaris suum,Toxoplasma gondii and Toxocara suum,with strong specificity.Finally,the clinical negative detection rate was 100%,the detection rate was proportional to the number and day of infections T.spiralis,and cardiac abrasive was detected first,followed by liver.In order to further evaluate the immunoprotective effect of r Ts TRE protein,46 BALB/c mice were randomly divided into two groups,the first group was PBS control group,and the second group was Ts TRE experimental group.At once,mice were immunized with PBS buffer and r Ts TRE protein three times,separately.Simultaneously,three mice were taken on the 7 th day after three immunizations,the splenic lymphocytes of their mice were extracted,and proliferative ability,phagocytic ability,and the ratio of CD4⁺and CD8⁺markers on the surface of T lymphocytes were evaluated.The remaining 20 mice were infected with different quantities of T.spiralis muscular larva（ML）,which 10 mice were infected with 50 ML and the remaining 10 mice were infected with 200 ML in each group.From the beginning of immunity to the end of the experiment,serum was collected once a week for the detection of antibodies（Ig G,Ig G1,and Ig G2a）,Th1（INF-γand IL-2）and Th2（IL-4 and IL-10）cytokine levels.Synchronizationand,the worm reduction rates of adult worm（AW）and ML were calculated on the post infection 7 th day（7th-pid）and 42 th day（42 th-pid）,respectively.The results showed that the proliferative and phagocytic abilities of spleen lymphocytes in the Ts TRE experimental group increased significantly（p<0.05）,the number of CD4⁺expressing T lymphocytes increased,while the number of CD8⁺expressing T lymphocytes decreased,with significant differences（p<0.05）.Compared to the PBS group,the antibody levels of Ig G,Ig G₁,and Ig G_2awere significantly increased（p<0.01）,and they decreased briefly after 7 th-pid.The level of Ig G₁type antibodies was higher than that of Ig G_2atype antibodies and the levels of Th1/Th2 cytokines were significantly increased（p<0.01）.Concurrently,the level of Th1 cytokines reached the highest level 7 th-pid,while the level of Th2cytokines reached the highest level 14 th-pid.The worm reduction rates of AW and ML of mice in the Ts TRE（50）group were 46.32%and 51.19%,and the worm reduction rates of AW and ML of mice in the Ts TRE（200）group were 40.49%and 42.37%,respectively.To sum up,the three part experiment showed that the r Ts TRE protein expressed in this experiment had good characteristics of both reactivity and immunogenicity.Tandem,indirect ELISA antibody detection method established using this protein as an immunogenic substance is good and can be used for clinical detection of trichinosis.Mice immunized with the r Ts TRE protein can induce cellular/humoral immune response,and cellular immunity is a Th1/Th2 type mixed immunity dominated by Th2.These reference data can provide theoretical basis for subsequent diagnosis and prevention of trichinosis.