| Trichinellosis caused by Trichinella spiralis is a globally distributed serious parasitic zoonosis and acquired by eating raw or undercooked meat contaminated with the infective larvae of the genus Trichinella.In recent years,Trichinellosis outbreaks have occurred in neighboring countries such as South Korea,Thailand,Laos,and Vietnam.So trichinellosis has become an emerging and re-emerging zoonotic disease with health,social,and economic impacts in developing countries.At present,the diagnosis of trichinellosis mainly focuses on Trichinella antigens in the muscle larval stage.The International Trichinellosis Commission(ICT)recommends that the ES antigen of Trichinella spiralis muscle larvae be used as the gold standard for the diagnosis of trichinellosis.However,when the Trichinella spiralis muscle larvae ES antigen is used to detect the serum of Trichinella-infected mice or pigs,anti-Trichinella antibodies are not detected until 3 to 4 weeks after infection;and patients infected with Trichinella are 28 days after infection.the positive rate of antibodies against Trichinella can reach 100%.Therefore,there is a"window phase"of 2 to 3 weeks after Trichinella infection.In the life history of Trichinella,IIL and adults are the invasive phase of Trichinella spiralis to the host intestinal mucosa.The ES protein is in direct contact with the intestinal mucosa and interacts with it.The immune system that can contact the host first causes the host’s immune response;therefore,The excretory and secretory antigens of intestinal infectious larvae and adults contain the major components that stimulate the host to produce an immune response.In the early stage after Trichinella infection,antibodies against these antigens were also the first to appear in anti-Trichinella antibodies.Since the Trichinella developed into a cystic larvae(muscle larvae)at 4weeks after infection in host and encapsulated in the collagen sac,the muscle larvae are later exposed to the host’s immune system,so the host produces anti-muscle larva antibodies are later.Hence,there is an urgent need to screen and indentify new antigens for early diagnosis of trichinellosis from early worms infected with Trichinella.This study used serological diagnostic methods to detect the early diagnostic value of Trichinella spiralis adult ES antigen for trichinellosis.The Trichinella spiralis putative serine protease(TsSP)was screened and to clone and identify its function.Lastly,we were detected the early diagnosis of rTsSP against Trichinella spiralis infection and its potential application value of vaccine target.Materials and methods1.Trichinella spp,experimental animals,serum,vectors,strains and cell linesTrichinella spp were subcultured in our laboratory.Animals used of the experiment were purchased from the experimental animal center.Serum:Serum of different species of Trichinella spiralis,Toxoplasma gondii,Schistosoma japonicum and A.cantonensis infected mice,and other parasitic infected patients.Cell line:IEC,C2C12,frozen in liquid nitrogen in this laboratory.Vector and strain:cloning vector pGEM-T,expression vector pQE-80L,host strain BL21,frozen for laboratory.2.Early diagnostic value of Trichinella spiralis adult worm ES antigenThe sensitivity and specificity of AW ES-ELISA for the diagnosis of trichinellosis were examined.At the same time,30 female BALB/c mice were prepared and infected with different doses of Trichinella(100,300,500 larvae per mouse).AW ES-ELISA was used to detect anti-Trichinella antibody levels at different doses of infection and the different days infection.The sensitivity of AW ES-ELISA to early and mild infection of Trichinella was examined.Finally,AW ES ELISA was used to detect the serum of patients with early and late Trichinella spiralis and other parasites,and to further evaluate the sensitivity and specificity of AW ES for the early diagnosis of trichinellosis.3.Trichinella spiralis putative serine protease(TsSP)bioinformatics analysis,cloning,expression and identificationAfter analyzing the early infected serum identification protein of adult ES,a potential early diagnostic antigen(TsSP)was selected from the identified proteins.The online domain of NCBI,ExPasy,SignalP3.0 serve and TMHMM Server v.2.0 was used to predict the domain,physicochemical properties,signal peptide and transmembrane domain of TsSP;Then TsSP were cloned and expressed in E.coli,The purified rTsSP was subcutaneously inoculated into the mice to obtain rTsSP immune serum.The immunogenicity and antigenicity of rTsSP were detected by Western-blot.The transcription and transcription of TsSP gene in different developmental stages of Trichinella were detected by RT-PCR and q-PCR.The level of TsSP protein was detected by indirect immunofluorescence(IFA)on the surface and inside of different developmental stages;paraffin-embedded and frozen tissue sections were observed in different stages,and TsSP was observed the specific localization in different stages by IFA;preparation of soluble proteins in different stages of worm,detection of TsSP expression and expression levels in different stages by ELISA and Western blot.4.Specificity of binding of TsSP to host IEC and its functionThe specific binding of rTsSP to mouse intestinal epithelial cells(IEC)was analyzed by Far-Western,ELISA and IFT.Different dilutions of anti-rTsSP serum,infectious serum and normal serum were mixed with intestinal infectious larvae(IIL),respectively,and then inoculated into IEC monolayer for 2 h in vitro for larval in vitro invasion experiments.The inhibitory effect of different serum on IIL invasion into IEC was observed under microscope..The killing effect of anti-rTsSP serum on the worm was detected by antibody-dependent cell-mediated cytotoxicity(ADCC).The larval activity and macrophage adhesion were observed microscopically,and the larval mortality was calculated,and ML after the action of ADCC were were infected with the mice to calculat the worm reduction rate.5.Early diagnosis of rTsSP for trichinellosisDifferent doses of Trichinella were infected with 3 doses of severely(500 larvae/mouse),moderate(300 larvae/mouse)and mild(100 larvae/mouse),The blood was collected on alternate days.rTsSP-ELISA was used to detect the recognition of rTsSP on the early infection of Trichinella spiralis and the sensitivity and specificity of rTsSP Trichinella infected mice.Simultaneous detection of Trichinella spiralis infected pig serum,Trichinella infection in early and late patient serum and other parasitic patient serum,further evaluation of the potential application value of rTsSP for early diagnosis of trichinellosis.6.Immune protection of rTsSPIn order to evaluate the immunoprotective effect of rTsSP,each mouse was immunized with 20μg rTsSP subcutaneously,immunized once every 10 days,and immunized 3 times.The anti-rTsSP antibody level and the induced humoral immune response were detected after immunization,Then to calculat reduction of the worm and muscle larvae respectively;using the cholera toxin subunit(CTB)as an adjuvant,the mice were inoculated with rTsSP by intranasal route.Immunization once every 10 days,a total of 3 times,respectively,to detect the total and special sIgA level in the intestine after intranasal immunization;the number of sIgA cells and goblet cells secreted in the intestine;and the changes of cytokine levels induced by spleen cells and mesenteric lymph nodes The worm reduction rate of adult and muscle larvae collected for different days after infection.7.Statistical analysisAll the results and data were statistically analyzed by SPSS 17.0 analysis software and graphed by Graphpad Prism.Statistical analysis was performed by one-way ANOVA,repeated data analysis of variance,t-test and chi-square test.The test level wasα=0.05.Results1.Early diagnosis and immunoproteomics analysis of adult worm ES antigenAdult worm ES-ELISA method was used to detect the sera of Trichinella spiralis infected mice with different doses of different days.The results showed that AW ES,AW crude and ML ES were detected sera of infected with 100 Trichinella spiralis,Anti-Trichinella antibodies were detected at 8,12 and 12 after infection.and anti-Trichinella antibodies was detected at 10,8,and 10 days in the high-dose group(500 larve mouse).Using these three antigens to detect the serum of early and late patients with Trichinella spiralis showed that sensitivity and specificity of AW ES were 100%and 97.48%.2.Bioinformatics analysis of TsSPThe predicted results showed that TsSP gene(GI:164521948)has a full length of 1372 bp and encodes 429 amino acid residues with a molecular mass of 47.55 kDa,an isoelectric point of 8.73,an instability index of 42.25,and a fat index of 71.52.The average hydrophilicity is-0.360,which is considered to be a hydrophilic protein.The N-terminus of the protein has a distinct hydrophobic region and no transmembrane region.TsSP is predicted to be a secreted protein with a signal peptide,and the cleavage site is between amino acid residues 18-19,indicating that the mature peptide begins at amino acid 19 and has a signal peptide SP sequence,which is thought to be secreted outside the cell..This protein is a highly conserved structural domain Tryp-SPC between positions 37-277.I-TASSER predicts that TsSP belongs to the serine protease family.3.Cloning,expression and identification of TsSPThe PCR primers and the internal primers with specific restriction sites were designed,and the TsSP gene of 1236 bp was amplified by nested RT-PCR.The TsSP gene was ligated into the cloning vector pGEM-T,and TsSP was digested.The gene was ligated to the expression vector pQE-80L to construct a recombinant expression plasmid,and transfected into E.coli BL21 competent cells.The recombinant plasmid was successfully constructed by double enzyme digestion.For the recombinant plasmid that were successfully ligated,IPTG was used to induce expression,and the cells were collected for SDS-PAGE.It was found that an obvious band appeared at 45.2 kDa,but the uninduced bacteria did not see this band,indicating that the TsSP recombinant protein expression was successful.Western blot analysis showed that rTsSP protein could be recognized by Trichinella spiralis-infected mouse serum and anti-rTsSP serum,indicating that the recombinant protein has good antigenicity and immunogenicity.The results of RT-PCR and qPCR showed that the TsSP gene was transcribed and had no difference in transcription levels in different developmental stages(ML,IIL,3dAW,6dAW and NBL)of Trichinella spiralis.The results of Western blot and ELISA showed that TsSP was expressed in different stages of Trichinella spiralis,and the expression levels of IIL and NBL were relatively high.The results of IFA showed that TsSP protein was expressed in the above five stages,and it was mainly located in the cuticles,stichosomes,embryos of the parasite.4.Specificity of binding of TsSP to host IEC and identified its function4.1 Specificity of binding of rTsSP to intestinal epithelial cells The specific binding of rTsSP to mouse intestinal epithelial cells(IEC)was analyzed by Far-Western,ELISA and IFT.Anti-rTsSP immune serum and enzyme-labeled secondary antibody were added.The results showed that the anti-rTsSP immune serum recognized 24 protein bands with molecular weights ranging from 14.4kDa to 86.5kDa,whereas rTsSP does not bind to the C2C12 protein,indicating that rTsSP can bind specifically to the IEC protein.ELISA assay also found that rTsSP binds to IEC protein in a dose-dependent manner.IFT found that rTsSP specifically binds to IEC and intestinal mucosa;confocal microscopy results indicate that the binding site of rTsSP to IEC is cell membrane and cytoplasm.4.2 Inhibition or blocking effect of anti-rTsSP antibody on larval invasion of IEC In vitro invasion experiment showed that the sera at the same serum dilution(1:50),the larval invasion rates of anti-rTsSP serum,infected serum and normal sera were 31.48%,15.84%and 84.16%(P<0.01),respectively.The invasion of larvae was dose-dependent and decreased with increasing serum dilution(P<0.01),indicating that anti-rTsSP antibodies have significant inhibitory effects on larva invasion into IEC.4.3 Anti-rTsSP antibody-dependent ADCC killing effect on Trichinella spiralis larvae ADCC results showed that anti-rTsSP serum promoted the adherence of PM to NBL and ML.When 1:100 dilutions of anti-rTsSP serum was supplemented and incubated for 72 h at 37°C,the ADCC caused a significant death of NBL and ML(52 and 44.67%cytotoxicity,respectively),in comparison with the larvae treated by pre-immune serum(P<0.01).The cytotoxicitywas the dose-dependent of anti-rTsSP antibody(P<0.01),which had a decreasing trend following serum dilution elevation.The mice inoculated orally with ML treated by ADCC assay with anti-rTsSP serum exhibited a 89.40%reduction of intestinal AW at 3 dpi and a 36.50%reduction of ML at 42 dpi,when compared with ML treated by pre-immune serum.This results suggest that specific anti-rTsSP antibodies could obviously kill the ML and decrease its infectivity by an ADCC fashion5.Diagnostic value of rTsSPThe results showed that anti-rTsSP antibodies were detected in rTsSP-ELISA at 8,7 and 7days after infection in severe,moderately and mildly infected mice.And the positive rate of antibody detection reached 100%at 16,10 and 10 days after infection;the sensitivity and specificity of rTsSP-ELISA for detecting Trichinella-infected mice were 100%.The sensitivity of rTsSP-ELISA for the diagnosis of early and late Trichinella patients was 95.24%(20/21)and100%(32/32),respectively.The sensitivity for the diagnosis of early Trichinella patients was significantly higher than that of ML ES(75%).The results showed that the specificity of rTsSP-ELISA for the diagnosis of trichinellosis(99.49%)(197/198)was significantly higher than that of ML ES-ELISA 91.41%(181/198)(P<0.05).6.Immune protection of rTsSP6.1 Subcutaneous inoculation of rTsSP induced immune response The results showed that serum rTsSP-specific antibody IgG levels were significantly increased after the first and second immunizations,and the anti-rTsSP antibody IgG titer was 1:10~5 at 10 days after the last immunization;IgG1 levels were 10,20,30 and 40 days after immunization.Significantly higher than IgG2a(P<0.01),indicating that rTsSP immunized mice induced a Th2-type humoral immune response.Compared with the PBS group,the worm reduction rate of the adult rats in the rTsSP immunization group and the adjuvant control group was 52.70%and 10.30%(P<0.05)at 5 days after infection;42 days after infection,the muscle larvae of the rTsSP immunization group and the adjuvant control group.The worm reduction rates were 52.1%and 3.66%,respectively(P<0.05).The results showed that subcutaneous immunization of rTsSP induced significant immune protection.6.2 Intranasal immunization with rTsSP induced immune response The results showed that rTsSP nasal immunized mice can cause significant levels of total sIgA and TsSP-specific sIgA in the intestine;serum TsSP-specific antibody IgG/IgM/IgA levels also increased significantly;More goblet cells/acid mucin and IgA secreting cells were detected in the duodenum of immunized mice.The levels of IFN-γ,IL-4 and IL-10 were significantly increased in the rTsSP immunized group compared with the control group.The infection was challenged with 300 larvae 10 days after the last immunization.Compared with the PBS control group,the adult worm reduction rates at 3,5,7 and 9 days after infection were 49.95%,61.30%,68.30%and 71.10%,respectively(P<0.001).The worm reduction rates of muscle larvae in the 42d-infected and adjuvant groups were 62.1%and 13.9%,respectively(P<0.001).The results showed that rTsSP induced a significant intestinal sIgA response and a systemic Th1/Th2 mixed immune response after nasal immunization in mice,and had a significant protective effect against Trichinella challenge infection.Conclusions1:Trichinella spiralis AW ES ELISA antigen for detection of trichinosis has high sensitivity and specificity,provide a new source of antigen diagnosis for early diagnosis of trichinosis;2:Cloned and expressed TsSP protein and TsSP is transcribed and expressed in the various stages of Trichinella,mainly located in stichosome,cuticle and embryo of the worm;3:rTsSP protein can specifically bind to intestinal epithelial cells,and anti-rTsSP antibody can inhibit the invasion of Trichinella spiralis into IECs in vitro,and anti-rTsSP antibody can mediate the killing effect of macrophages on NBL and ML.TsSP may be the major invasive proteina of Trichinella to IEC;4:rTsSP-ELISA for detection of trichinosis has high sensitivity and specificity,and can be used as an early diagnostic antigen for potential trichinellosis;5:rTsSP protein was induced by nasal/subcutaneous immunization of mice,and induced a mucosal and systemic Th1/Th2 mixed immune response,and a significant protective effect against Trichinella challenge infection. |
PDF Full Text Request