Biological Properties Of Trichinella Spiralis Aminopeptidase P And Its Potential In Inducing Immune Protection

Background and purposeTrichinellosis is a zoonotic disease caused by ingesting raw or semi-raw meat containing Trichinella larvae cysts.The disease can cause gastrointestinal symptoms such as nausea,vomiting,abdominal pain,diarrhea,myalgia,general fever and eyelid edema,and even more serious encephalitis and myocarditis.Trichinella invades intestinal epithelial cells（IECs）and further development is the key to infecting the host.Trichinella spiralis aminopeptidase P（TsAPP;GenBank:EFV57850）was identified by mass spectrometry in the early stage of this research group from the new protein produced by the in vitro co-cultivation of Trichinella spiralis intestinal infectious larvae（IIL）and IECs.Therefore,it is suggested that the protein may be related to the IECs invaded by Trichinella spiralis.The TsAPP gene was cloned and expressed,and a recombinant TsAPP（rTsAPP）was obtained.Using RT-PCR and Western blot to analyze the transcription and expression of TsAPP in different stages of T.spiralis,and the immunofluorescence antibody test（IFA）was used to analyze the location of TsAPP in the parasite.The enzyme activity of rTsAPP was determined by the hydrolysis method of the specific substrate—L-alanyl-L-proline（H-Ala-Pro-OH）.Western blot,ELISA and IFT were used to confirm the binding of rTsAPP to hemoglobin（Hb）.Preliminary studies of our group have shown that the Trichinella cathepsin X（TsCX,GenBank:XM₀03372890.1）recombinant protein can promote the invasion of Trichinella spiralis larvae into mouse IECs,and may be a potential target molecule for the vaccine against the intestinal infectious larvae.The rTsAPP and recombinant Trichinella cathepsin X（rTsCX）were subcutaneously immunized to detect the humoral and cellular immune responses of the immunized mice and the immune protection after larval challenge.Materials and Methods1.Trichinella species,laboratory animals and strainsT.spiralis was used in this study,which was preserved in KM mice in our laboratory;the experimental animals were BABL/c mice purchased from Henan experimental animal center;the clone and expression strain was BL21..2.Biological information of TsAPPThe sequences of TsAPP and aminopeptidase P from other species of Trichinella spiralis were aligned by BioEdit software,and the phylogenetic tree of TsAPP was constructed by the adjacency method of mega7.0.The domains and active sites of TsAPP were predicted on NCBI and smart websites.3.Cloning,expression and antigenicity analysis of TsAPPThe TsAPP gene was ligated to pMD19-T.After induction,rTsAPP was expressed,and the purified rTsAPP was used to immunize mice to prepare anti-rTsAPP serum.The antigenicity of rTsAPP was analyzed by Western blot.4.Expression and localization of TsAPPExtract RNA from various stages of Trichinella spiralis,and reverse transcribed into cDNA,and analyzed by RT-PCR TsAPP gene transcription level in different stages.The soluble antigens of each stage were prepared,and the expression of TsAPP in each stage was analyzed by Western blot.Collect fresh complete worms of different stages to make paraffin sections,and use IFA to observe the expression and location of TsAPP in different stages of Trichinella spiralis.5.rTsAPP enzyme activity determinationAminopeptidase P can hydrolyzes H-Ala-Pro-OH（L-alanyl-L-proline）to produce Pro.Proline can react with ninhydrin under acidic conditions The direct product produced is red with a characteristic absorption peak of 520 nm.By measuring the change in absorbance at 520 nm,the enzyme activity of aminopeptidase P can be calculated.This article observes the optimal temperature and pH value of rTsAPP enzyme reaction.6.Interaction between rTsAPP and hemoglobin（Hb）Western blot,ELISA and IFA were used to detect the specific binding of rTsAPP to mouse and porcine hemoglobin（Hb）,and anti-recombinant Trichinella aminopeptidase（rTsAP）was used as a control.7.ADCCThe newborn larvae were mixed with the peritoneal exudate cells of mice and then added into the well.The immune sera of each group（1:50,1:100,1:200,1:400,1:800）with different dilutions,and each group was set with 3 wells.The survival of each well was observed and recorded at 0,24,48 and 72 hours.The living is mobile,and the dead is rigid and inactive.The percentage of dead larvae in total larvae observed in each experiment was used to determine the cytotoxicity of different groups of serum.8.Immune responses by mice immunized with rTsAPP and rTsCXDivide 250 BALB/c mice and immunize them with rTsAPP,rTsCX,rTsAPP+rTsCX,ISA 201 adjuvant and PBS by subcutaneous injection.A total of 3 immunizations,with an interval between each immunization After 2 weeks,the sera of different groups of mice were collected after each immunization,and the levels of specific IgG,IgGl,IgG2a and IgA in the serum of each group of mice were detected by the crude muscle larvae antigen with ELISA.Before immunization,2 weeks after the last immunization,and 2 weeks after the larval challenge,ELISA was used to detect the total sIgA and specific sIgA in the intestinal fluid.9.Immune produced by the combined immunization of rTsAPP and rTsCX in miceTwo weeks after the last immunization,300 ML were used to infect all the immunized mice by oral challenge.7 days and 42 days after infection,10 animals in each group were dissected.The adult and muscle larval load of Trichinella spiralis were observed.The reduction rate of rTsAPP,rTsCX,rTsAPP+rTsCX immunized mice was calculated,and the rate of rTsAPP,rTsCX,rTsAPP+rTsCX was evaluated.And the pathological changes of small intestine tissues and muscles of mice in different immunization groups 7 days after infection and 42 days after infection were observed.10.Statistical analysisAccording to the experimental methods and data,the types of corresponding statistical methods are selected,including t-test,one-way ANOVA and chi square test.The level was a=0.05.Results1.Bioinformatics analysis of TsAPPTsAPP is 1887 bp,628 amino acids,71.4 kDa,and pI 5.84.It has no signal peptide and belongs to metalloprotease.TsAPP contains proline aminopeptidase,M24 family and two creatinase family domains.There are 6 enzyme active sites,respectively 403 histidine（His）,416 aspartic acid（Asp）,427 aspartic acid（Asp）,494 histidine（His）,509 glutamic acid（Glu）,539 glutamic acid（Glu）.Using BioEdit software,TsAPP was compared with the aminopeptidase sequences of 12 other parasites,mice and humans,and it was found that it had high homology with other Trichinella species.analysis of aminopeptidase P of 20 species with MEGA software showed that Trichinella spiralis and cystic type Trichinella spiralis have a close evolutionary status.2.Antigenicity analysis of TsAPPSolubility analysis showed that rTsAPP was more in the precipitate.Western blot results show that rTsAPP can be recognized by infection serum and His antibody,which proves that rTsAPP has good antigenicity.3.Expression and localization of TsAPPRT-PCR and Western TsAPP mRNA and protein were expressed in the four different stages of Trichinella spiralis.IFA results show that TsAPP is mainly located in the cortex,stichosome and embryos.4.Enzyme activityThe optimal reaction temperature of rTsAPP is 50℃,and the optimal pH value is 9.5.Mn2+,Co2+,Fe2+all enhance the enzymatic activity of rTsAPP,and the order of effect is Mn2+>Co2+>Fe2+.The hydrolysis of H-Ala-Pro-OH by rTsAPP conforms to Miman’s kinetics.The kinetic parameters Vmax is 220 μmol min.lmg-land Km is 31.82 mM.5.Binding of rTsAPP to hemoglobin HbFar-western results show that anti-rTsAPP and rTsAP serum can recognize 5 bands of porcine Hb（63.4,55.7,29.9,23.6,15.7 kDa）,and infection serum can recognize 2 bands of porcine Hb（29.9,15.7 kDa）,rTsAPP can specifically bind to porcine Hb protein,suggesting that TsAPP may be related to the nutrition uptake of Trichinella spiralis.ELISA results show that rTsAPP can bind to Hb,and the OD value is correlated with Hb protein concentrationIt correlated with the incubation concentration IFA results later showed that rTsAPP can specifically bind to mouse Hb.6.NBL destruction by ADCCThe results of ADCC test revealed that after culture at 37℃ for 72 h,anti-rTsAPP antibodies mediated the PECs adhesion to the NBL and damage of the NBL.When 1:50-1:400 dilutions of immune serum（anti-rTsAPP serum,anti-rTsCX serum and anti-rTsAPP+rTsCX serum）were added,the ADCC resulted in an evident death of the NBL（33.9,28.8,15.1 and 11.9%cytotoxicity;34.9,33.0,19.5%and 11.3%cytotoxicity;40.4,35.1,25.0 and 14.9%cytotoxicity）.Compared with adjuvant serum and PBS serum,the mortality of newborn larvae in three immune groups at different serum concentrations was statistically significant（F1:50=116.806,P=0.0001;F1:100=31.936,P<0.005;F1:200=94.964,P<0.005;F1:400=63.131,P<0.005）The cytotoxicity was dose-dependently related with immune serum（anti-rTsAPP serum,anti-rTsCX serum and anti-rTsAPP+rTsCX serum）antibodies（rrTsAPP=0.970,P<0.01;rrTsCX=0.976,P<0.01;rrTsAPP+rTsCX=0.994,P<0.01）.the cytotoxicity showed an elevating trend with the prolongation of culture time（F24h=65.837,P=0.0001;F48 h=91.978,P<0.005;F72h,119.039,P=0.0001）.7.Humoral and cellular immune responses caused by the combined immunization of rTsAPP and rTsCXThe mice were immunized with rTsAPP,rTsCX,and the combination of the two.After 3 immunizations,the levels of different types of antibodies in the serum were measured.The results showed that as the number of immunizations increased,the antibody levels of the mice in each group gradually increased,and after immunization The IgG levels of the three immunization groups at 2,4,6 weeks higher than PBS control groups（F=585.162,P=0.0001）.In addition,the levels of IgGl and IgG2a in the immunized group were also significantly increased,but the IgG1 level was significantly higher than that of IgG2a（t6w=7.350,t8w=7.663;P=0.0001）,indicating that rTsAPP immunization induced an immune response dominated by Th 2 type.The results of cytokine detection showed that the levels of IFN-γ and IL-4 after immunization in the three immunization groups were significantly higher than those before immunization（F=20.032,P=0.0001;F=8.686,P=0.001）,and also significantly higher In the adjuvant group and the PBS group（F=62.031,P=0.001;F=37.918,P=0.001）.The levels of IFN-γ and IL-4 continued to increase 2 weeks after challenge infection.The results showed that mice immunized with rTsAPP,rTsCX or the combination of both induced a systemic and local intestinal mucosal Thl/Th2 mixed immune response.8.Immune protective effect caused by the combined immunization of rTsAPP and rTsCX in miceCompared with the PBS group,mice immunized with rTsAPP,rTsCX and the two proteins were challenged 7 d,and the intestinal worm reduction rates were 44.69,49.22 and 63.99%（F=79.278,P=0.0001）.The rate of adult worms reduction in the combined immunization group was significantly higher than that of the single protein immunization group（F=18.881,P=0.0001）.The length of females shorter than that in the PBS group（F=7.439,P=0.0001）.The results of HE staining of small intestine pathological sections of immunized mice infected with Trichinella showed that the number of goblet cells in the intestinal epithelium of the three immunized groups was significantly less than that of the adjuvant and PBS groups（F=71.843,P=0.0001）,and the width of intestinal villi was significantly smaller than that of the two control groups（F=36.608,P=0.0001）,showed that rTsAPP,rTsCX and the combination of the two proteins all alleviated the intestinal inflammatory response after Trichinella infection.Conclusion1.The recombinant Trichinella spiralis aminopeptidase P（rTsAPP）was expressed and purified.2.TsAPP is expressed in all stages of Trichinella development,and mainly located in the cortex and embryos.3.rTsAPP has the enzymatic activity of natural aminopeptidase P.When the optimal reaction temperature is 50℃ and pH is 9.5,Mn2+ and Co2+can enhance the enzymatic activity of rTsAPP,and EDTA can inhibit the enzymatic activity of rTsAPP.4.rTsAPP can specifically bind to porcine and mouse hemoglobin.5.The combined immunization of rTsAPP and rTsCX induced a mixed Th1/Th2 immune response and a significant protective effect.