Clonal Expression Of Trichinella Spiralis Trypsin And Its Induced Protective Immunity

Trichinellosis is a food-borne zoonotic parasitic disease that is distributed worldwide,mainly due to raw or semi-lived meat infected with encapsulated infective larvae.The intestinal epithelium is the first natural barrier forTrichinella spiralis to invade the host.The direct contact of T.spiralis with the intestinal epithelium is the key to the establishment of infection.Trypsin is a major subfamily member of serine protease superfamily in helminths.Serine proteases may play an important biological function during the infection of T.spiralis,and may be involved in the degradation of various host tissue components,larval invasion and migration of T.spiralis.In recent years,through proteomic and immunohistochemical studies,some serine proteases have been found in excretory/secret?ES?and surface antigens at different developmental stages of T.spiralis.After co-culture of T.spiralis intestine infective larvae?IIL?larvae and intestinal epithelial cells?IECs?monolayer,the IIL larvae invaded the IEC monolayer and produced some serine proteases to act on the IECs monolayer,thereby promoting its invasion into the IECs monolayer.T.spiralis serine proteases may promote larval invasion of the intestinal mucosa.However,immunization of mice with certain single recombinant serine proteases induced only partial protective immunity against infection by T.spiralis infection.Therefore,there is a need to identify other serine proteases of T.spiralis and evaluate the immune protection they cause.Trypsin belongs to the family of serine proteases PA and has functions of digestion,anticoagulation,fibrinolysis,development,fertilization,apoptosis and immunity.In this study,recombinant TsT?rTsT?was expressed and purified by molecular cloning of T.spiralis trypsin?TsT,Genbank:XP?003374437.1?.The expression of TsT by RT-PCR and indirect immunofluorescence assay?IFA?It was analyzed with the localization of worms;anti-rTsT mouse serum was used for in vitro invasion tests and in vivo animal experiments.The rTsT protein was used to immunize mice subcutaneously and the type of immune response induced by the protein and the immune protection effect on the mice were detected,indicating that the protein can be used as a candidate target for vaccines against trichinella infection.Materials and Methods1.laboratory animals and Trichinella speciesExperimental mice are Kunming mice and BALB/c mice;T.spiralis is a geographical strain of Henan pig origin?ISS534?;expression plasmid used in the experiment is p QE-80L;expression strain is BL21;cells used in the experiment are mouse small intestinal epithelial cells.2.TsT bioinformatics analysisThe biological analysis website was used to analyze the physical and chemical properties of TsT?accession No:XP?003374437.1?.Predict the molecular weight,p I and hydrophobicity of TsT on the Ex Pasy website;predict the TsT signal peptide on the Signal P-5.0 website;predict the transmembrane structure of TsT on the TMHMM Server v.2.0 website;perform TsT subcellular localization analysis on the Target P-2.0 Server website;Predict the secondary and tertiary structure of TsT on the SWISS-MODEL web page;predict the B cell epitope of TsT on the Bcepred website;use Bio Edit software to compare the sequence of T.spiralis TsT with other species or genotypes of T.spiralis and other parasites;Use MEGA 7's adjoining method to perform 1000 repeated calculations to construct TsT phylogenetic tree.3.Cloning and expression of TsT and analysis of its antigenicityThe TsT gene was ligated into the cloning vector p MD19-T,identified by PCR and enzyme digestion,and then sent to sequencing;after selecting the strain with the correct sequencing results for double digestion,the TsT target fragment was ligated to the expression vector p QE-80L to construct recombinant expression plasmid p QE-80L/TsT of TsT.The rTsT protein was expressed in E.coli and purified using Ni-NTA-Sefinose resin,and the mice were immunized with rTsT to prepare mouse anti-rTsT immune serum,and the soluble crude antigen and excretory antigen of T.spiralis muscle larvae were collected for SDS-PAGE and Western blot to analyze the antigenicity of rTsT protein.4.Expression and immunofluorescence localization of TsT in different stages of T.spiralisCollect worm bodies at different stages,and analyze the transcription level of TsT gene in different stages of T.spiralis?ML,IIL,3 d adult worms?AW?,and newborn larvae?NBL??by RT-PCR.IFA was used to observe the expression of TsT in T.spiralis at different developmental stages and its tissue localization in T.spiralis.5.The combination of TsT and IECs and their function detection during the invasion of T.spiralisThe binding of TsT to normal mouse intestinal epithelial cells?IECs?was detected by Far-ELISA and Far-Western.Through the in vitro invasion test of larvae,we observed the promotion or inhibition effect of rTsT and anti-rTsT mouse serum on T.spiralis invasion of small intestinal epithelial cells.The killing effect of anti-rTsT serum on NBL was evaluated by ADCC test.6.Detection of immune response induced by rTsT and evaluation of immune protection effect90 female BALB/c mice were divided into 3 groups?30/group?and were injected subcutaneously with rTsT,ISA 201 adjuvant or PBS,respectively.The mice were immunized with rTsT?20?g/mouse?,immunized once every 2 weeks,for a total of 4 immunizations.Collect sera at 0,2,4,6,and 8 weeks after immunization,and use ELISA to detect the levels of mouse serum specific antibodies Ig G,Ig G1,Ig G2a,and total s Ig A and rTsT specific s Ig A in intestinal lavage fluid after immunization,spleen cells and mesenteric lymph node cells of immunized mice were collected,stimulated with rTsT protein and incubated at 37°C for 72h.The level of cytokines?IFN-?and IL-4?produced by splenocytes and mesenteric lymph node cells before and after immunization with rTsT was detected by double antibody sandwich ELISA.Two weeks after the last immunization,each mouse was challenged with 300 muscle larvae.At 5 and 30 days after infection,the intestinal adults and muscle larvae of the immunized mice were collected,counted,and the worm reduction rate was calculated.Evaluate the immune protection effect of rTsT.7.Statistical processingUse SPSS 21.0 statistical analysis software to perform statistical analysis on the data.Mainly adopt t test,one-way analysis of variance and chi-square test,the value is expressed as mean±standard deviation,test significance level:?=0.05.Result1.TsT bioinformatics analysisThe full-length sequence of T.spiralis trypsin TsT gene is 891 bp,encoding 296 amino acid residues,with a molecular weight of 33 k Da and a p I of 9.42.The protein has a signal peptide,no transmembrane region,and a clear hydrophobic region at the N-terminus.Subcellular localization indicates that it is a secreted protein with good antigenicity and a complete trypsin-like serine protease domain.Secondary structure prediction shows that TsT has 3?helices,16?sheets and 20 random coils;the prediction result of tertiary structure shows that TsT has 3 active sites,namely histidine?His?,serine?Ser?and aspartic ammonia Acid?Asp?.2.Cloning and expression of TsT and analysis of its antigenicityThe results of SDS-PAGE showed that the induced recombinant bacteria had a protein band at 30.9 k Da.After solubility analysis,the protein was mainly in the precipitate and existed as inclusion bodies.Western blot analysis showed that rTsT protein can be recognized by anti-his monoclonal antibody and mouse infected sera,but not by mouse normal sera,indicating that the recombinant protein is T.spiralis trypsin TsT and has good antigenicity.3.Expression and immunofluorescence localization of TsT in different stages of T.spiralisThe results of RT-PCR analysis showed that the TsT gene was transcribed in different stages of T.spiralis?ML,IIL,AW and NBL?.IFA showed that TsT was mainly expressed in the cortex of 5d and 6d adult stages.IFA results of worm sections showed that TsT was located.In the cut surface of the worm body and the female adult embryo.4.Far-western and ELISA detect the combination of rTsT and IECsFar-Western results show that IECs protein can be recognized by anti-rTsT immune serum and mouse infected serum after incubation with rTsT,but it cannot be recognized by mouse normal serum;rTsT can bind to 10 bands of mouse IECs protein,which proves rTsT can interact with IECs,but rTsT cannot bind to C2C12 protein,indicating that rTsT has specific binding to IECs protein.ELISA test results also show that rTsT can bind to IEC protein,and this combination has a dose-dependent relationship between rTsT and IECs protein.The optical density?OD?value of ELISA increases with the increase of IECs protein concentration?F=356.446,P<0.001?,and It is dependent on the amount of IECs protein?r=0.874,P<0.001?;when IECs protein is incubated with different concentrations of rTsT,the OD value increases with the increase of rTsT protein concentration?F=498.378,P<0.001?,OD value was correlated with rTsT protein concentration?r=0.981,P<0.001?.5.Promoting or inhibiting effect of rTsT and its antiserum on T.spiralis invasion of IECsrTsT was added to semi-solid medium containing IECs and co-cultured with T.spiralis IIL larvae for 2 h.The results showed that the larval invasion rate of the rTsT group and Bull Serum Albumin?BSA?group were 89.94%and 76.17%,respectively??²=6.771,P<0.001?,rTsT has a significant promotion effect on IIL larval invasion to IECs,which has a dose-dependent relationship with rTsT?r=0.987,P<0.001?.With the increase of rTsT protein concentration,the promotion effect is significantly enhanced?F=12.749,P<0.001?.However,BSA did not promote IIL invasion of IECs.Anti-rTsT serum was added to semi-solid medium containing IECs and co-cultured with T.spiralis IIL for 2h.The results showed that the larval invasion rate of anti-rTsT serum group,mouse infected serum group and mouse normal serum group was 49.52%,42.19%and 79.14%??²=37.376,P<0.001?.It shows that anti-rTsT immune serum can significantly inhibit the invasion of T.spiralis into mouse intestinal epithelial cells.The inhibitory effect of anti-rTsT serum on larval invasion of IECs is dose-dependent of anti-rTsT antibody?r=0.980,P<0.001?,and shows a downward trend with the increase of serum dilution?F=809.173,P<0.001?.6.rTsT protein-induced immune responseAfter the second and third subcutaneous immunization,the Ig G antibody level of the rTsT protein group increased significantly,but the difference in antibody level between the ISA 201 adjuvant group and the PBS group was not statistically significant?P>0.05?;after the rTsT protein immunization at 4,6 and 8 week,Ig G1 antibody levels were significantly higher than Ig G2a(t_4w=7.327,t_6w=5.961,t_8w=20.751,P<0.001),indicating that rTsT immunized mice induced Th2 type humoral immune responses.In addition,after the last immunization,the total Ig A and TsT-specific s Ig A in the rTsT group were significantly higher than those before immunization.The difference in specific s Ig A levels in the intestinal fluids of the three groups was statistically significant?F=16.042,P<0.001?.The rTsT protein immunization group was significantly higher than the ISA 201 group and the PBS group?t₁=3.776,t₂=4.452,P<0.001?.The spleen and mesenteric lymph node cells of rTsT immunized mice were stimulated by rTsT protein,and the levels of cytokines?IFN-?and IL-4?produced were significantly increased(spleen-t_IL-4=6.093,P<0.01;t_IFN-?=4.275,P<0.01;MLN-t_IL-4=7.81,P<0.001;t_IFN-?=7.911,P<0.001).At 8 weeks after immunization,the levels of cytokines?IFN-?and IL4?produced by spleen and mesenteric lymph node cells of rTsT immunized mice were significantly higher than those in the adjuvant group and PBS 0.001;F_IFN-?=54.738,P<0.001).7.Killing effect of anti-rTsT antibody-mediated ADCC on newborn larvae of T.spiralisTo add different sera?anti-rTsT mouse serum,infection mouse serum and normal mouse serum?and newborn larvae to the culture plate containing mouse peritoneal macrophages,and the ADCC role mediated by anti-rTsT antibody at different times after the ADCC action was observed.The killing effect of caterpillar newborn larvae.The results showed that when the serum dilution was 1:100,the cytotoxicity of anti-rTsT mouse serum to NBL was 21.97%,which was significantly higher than 8.97%of the normal mouse serum group??²=6.733,P<0.01?.This cytotoxicity is dependent on anti-rTsT antibodies,and the mortality of larvae decreases with the increase of antibody dilution?F=101.254,r=-0.880,P<0.001?.With the extension of incubation time,the mortality rate of NBL gradually rise?F=88.941,r=0.993,P<0.001?.8.Immunoprotective effect of rTsT immunization on micerTsT immunized mice were challenged with T.spiralis 5 days and 30 days after infection.The intestinal worm reduction was 33.17%?F=79.988,P<0.001?,and the muscle larvae reduction rate was 37.8%?F=8.683,P<0.001?.Conclusion1.The recombinant expression plasmid p QE-80L/TsT of T.spiralis trypsin?TsT?was constructed,and rTsT was successfully expressed and purified.2.TsT is transcribed in all stages of T.spiralis,but it is mainly expressed in the adult stage of 5-6 days,and is located in the cortex of the worm body and the female embryo.3.TsT can specifically bind to mouse intestinal epithelial cells and promote the invasion of T.spiralis into small intestinal epithelial cells.Anti-rTsT antibodies can inhibit the invasion of larvae,and can mediate the killing effect of macrophages on newborn larvae.4.Subcutaneous immunization of mice with rTsT induces a systemic Th1/Th2 mixed immune response,which can produce partial immune protection against Trichinella infection,and TsT can be used as a candidate molecular target for anti-Trichinella vaccine.