Molecular Characterization Of Trichinella Serine Proteinase And Its Potential In Inducing Immune Protection

Trichinellosis is a widespread zoonotic parasitic disease worldwide.Humans and animals are mainly infected by eating meat and meat products containing Trichinella larvae.The outbreak of trichinellosis poses a huge threat to global public health.The pathogenic stage of Trichinella spiralis is mainly in the larvae period.Muscle larvae invade the intestinal mucosa by developing into intestinal infective larvae（IIL）after being ingested by the host,and develop into adult worms after 4 times of molting.The new born larvae circulate throughout the body with lymphatic vessels and blood,and the new larvae that reach the striated muscles develop into muscle larvae.Therefore,the key to the research is to prevent the Trichinella spiralis larvae invade into the intestinal mucosa.Current research indicates that Trichinella spiralis invading the intestinal mucosa may be related to its excretory secretion（ES）protein.In the early stage of this research group,a Trichinella spiralis proteinase（Ts Serp）was identified from the ES protein.This proteinase belongs to the family of serine proteases,and serine proteinase have the function of degrading host tissues during parasite infection,assisting parasites in immune escape,etc.This study cloned and expressed Ts Serp（Gen Bank:AY028974.1）.The biological characteristics,antigenicity and tissue localization of rTsSerp were identified.Through in vitro invasion experiments,ADCC,anti-rTsSerp serum antibodies and cytokine measurements,Ts Serp was revealed role of Trichinella spiralis invasion and protective immunity induced by Ts Serp.Materials and methods1.Trichinella species,experimental animals,adjuvant,cell line typesThe species of Trichinella spiralis（ISS534）used in this research originated from the domestic pigs in Nanyang,Henan,and were kept by the laboratory for generations.The experimental animals were female BALB/c mice and Kunming mice from Henan Experimental Animal Center.Adjuvants are MONTANIDETM ISA 201 VG and Freund’s adjuvant.Experimental mouse intestinal epithelial cells（IEC）were kept in this laboratory after isolation and passage.2.Bioinformatics analysis,expression level and localization of Trichinella serine proteinaseUsing NCBI,SWISS-MODEL,signal P server-5.0 and other bioinformatics online software to predict and analyze information such as Ts Serp structure domain,physical and chemical properties,signal peptide and transmembrane domain.The Ts Serp gene with the signal peptide region removed was cloned into the p ET-32a expression vector（with a TRX-tag tag）to construct a recombinant expression plasmid p ET-32a/Ts Serp,rTsSerp was obtained by prokaryotic expression,and the purified rTsSerp was injected subcutaneously Immunize mice and obtain immune serum after 4 immunizations.Western blotting was used to analyze the antigenicity of rTsSerp;RT-PCR,ELISA,and immunofluorescent test（IFT）were used to observe the transcription and expression levels of Ts Serp of Trichinella spiralis in different stages of Trichinella spiralis and positioning in worm tissue.3.The role of rTsSerp in the process of Trichinella spiralis invading IEC and intestinal mucosa in vitro100 Trichinella larvae（ML）were incubated with bile for 2 h and activated to IIL,then mixed with rTsSerp or anti-rTsSerp serum of different concentrations,added to the IEC monolayer（or injected into the isolated small intestine）,and cultured at 37℃After 2 hours,the number of larvae that invaded IEC and intestinal mucosa was observed under a microscope to evaluate the role of rTsSerp and its antiserum in the process of Trichinella invading the mucosa.4.The killing effect of anti-rTsSerp antibody-mediated cytotoxicity on new born larvae（ADCC）Mix Trichinella new born larvae with mouse peritoneal exudate cells,add to 96-well plate,add different dilutions of serum（1:10,1:50,1:100,1:200）according to different groups,each group sets 3 repeat.Incubate at 37°C and 5%CO2 for 48 h.Observe and record the survival of newborn larvae at each well at 0,12,24,and 48 h.Observe the attachment of peritoneal exudate cells to Trichinella spiralis newborn larvae and the survival of new born larvae,and determine the killing effect of anti-rTsSerp serum-mediated cytotoxicity on new born larvae.5.Humoral and cellular immune responses induced by rTsSerp immunization in miceDivide 200 mice into 4 groups（50 mice in each group）and immunize them with subcutaneous injections of rTsSerp,TRX-tag,ISA 201 adjuvant and PBS,respectively,for a total of 4 immunizations,each immunization interval is 14 days,After immunization,the sera of different groups of mice were detected by rTsSerp-ELISA for rTsSerp-specific Ig G,Ig G1,Ig G2a,Ig M and Ig E levels in the sera of each group.At the same time,the spleen and mesenteric lymph nodes of mice were collected 2 weeks after the last immunization,and the spleen cells and mesenteric lymph node cells were collected by grinding,washing,separation and other methods to detect Th1（IFN-γ,IL-2）and Th2（IL-4）Cytokine levels.6.Immunoprotective effect of rTsSerp immunized miceTwo weeks after the last immunization,all immunized mice were orally challenged with 300 Trichinella larvae.Ten mice were sacrificed 6 days and 56 days after infection respectively.The number of adults of Trichinella spiralis and the load of muscle larvae were observed.The deworming rate of mice in the rTsSerp immunized group was calculated to evaluate the immune protection effect of rTsSerp.In addition,the histopathological changes of masseter muscle 56 days after infection in different immunization groups were observed.7.Statistical analysisThe analysis software SPSS 26.0 was used to process the experimental data.The data was expressed in the form of mean±standard deviation.Chi-square test and analysis of variance were mainly used.The comparison between the two groups used t test.The test level wasα<0.05.Result1.Bioinformatics analysis,expression level and localization of Trichinella serine proteinaseThe analysis results of the bioinformatics software show that the Ts Serp gene is 1445 bp in length,encodes 421 amino acids,and has a p I of 6.33.Ts Serp contains a trypsin-like serine protease domain,and amino acids 1-27 are the signal peptide region of the gene.The amplified Ts Serp gene was cloned into p ET-32a expression vector to construct recombinant expression plasmid p ET-32a/Ts Serp,and transfected into E.coli BL21（DE3）to induce expression,and the induced fusion protein was purified.SDS-PAGE and Western blot results showed that the molecular weight of the fusion protein expressed by the recombinant bacteria was about 65.7 k Da.The molecular weight of the rTsSerp protein is equal to the sum of the c DNA clone（44.7 k Da）+the TRX-tag encoded protein（21 k Da）of the vector,and rTsSerp can be identified by the primary antibody with his tag and the mouse serum infected with Trichinella spiralis.RT-PCR and Western blotting results showed that Ts Serp m RNA and protein were expressed in 4 different developmental stages of Trichinella spiralis,and Ts Serp protein expression levels in muscle larvae and intestinal infectious larval stages were significantly higher than those in adult and neonatal larvae（χ²=62.405,P<0.001）;the results of indirect immunofluorescence tests showed that Ts Serp was mainly localized in the epidermis,rod-shaped body and female embryos,suggesting that Ts Serp is a larval invasion.protein.2.The role of in vitro simulated rTsSerp in the invasion of intestinal mucosa in miceAfter incubating rTsSerp protein and TRX-tag tag protein with 100 IILs,add IECs monolayer,observe the invasion and count after 2 hours.The results showed that the effect of rTsSerp on larval invasion of IECs was rTsSerp dose-dependent（r=0.996）,and the change in the concentration of the TRX-tag protein did not affect the invasion rate.When the concentration of Ts Serp and TRX-tag protein was 15μg/m L,the Ts Serp group and TRX The invasion rate of larvae in the-tag group was 86.96%and 61.18%,respectively（P<0.01）,indicating that rTsSerp can promote the invasion of larvae into IEC.Different dilutions of anti-rTsSerp serum,infection serum and normal serum were mixed with IIL,respectively,inoculated into IEC monolayer and incubated in vitro for 2 h.The inhibitory effect of different serum on IIL invasion into IEC was observed under microscope.The results showed that the larval invasion rate of anti-rTsSerp serum,infected serum and normal serum group was 36.47%,27.13%and 68.96%（P<0.01）when the same serum was diluted（1:100）,and the anti-rTsSerp antibody against larvae.The inhibitory effect of invasion was antibody-dose dependent（r=0.986）,which decreased with increasing serum dilution（P<0.01）,indicating that anti-rTsSerp antibodies prevented the invasion of IEC by larvae.In addition,after the rTsSerp,TRX-tag,anti-rTsSerp serum,anti-TRX-tag serum or PBS treated muscle larvae were injected into the isolated small intestine and incubated for 2h,the invasion rate of the larvae into the intestinal mucosa was 71.33%,45.67%,55.34%,57.6%and 56.00%.It showed that rTsSerp significantly promoted larval invasion（χ²=6.000,P<0.0001）,while anti-rTsSerp serum significantly inhibited larval invasion of intestinal mucosa（χ²=4.900,P<0.0001）.3.The killing effect of anti-rTsSerp antibody-mediated cytotoxicity on new born larvae（ADCC）When the serum concentrations of anti-rTsSerp were 1:10,1:50,1:100,and 1:200,the mortality of ADCC-mediated new born larvae was 24.35%,24.47%,22.02%,and 17.68%,which was significantly higher than that of no immunization.The mortality rate of neonatal 0.01).And the killing effect on larvae increases with the increase of serum concentration,which has a dose-dependent antibody（r=0.903,F=119.518,P﹤0.01）,this kind of killing effect also increases with the extension of culture time（r=0.921,F=18.356,P﹤0.01）.4.Humoral and cellular immune responses induced by rTsSerp immunization in miceAfter BALB/c mice were immunized with rTsSerp 4 times,the level of serum specific antibodies（Ig G,Ig G1/Ig G2a,Ig E and Ig M）gradually increased with the increase of the number of immunizations,while the antibody levels in the adjuvant group and PBS group no significant changes.In addition,Ig G1 and Ig G2a antibody levels also increased significantly,but Ig G1 levels were significantly higher than Ig G2a(t_4w=14.246 t_6w=22.291 t_8w=14.244).It indicated that rTsSerp mainly induced Th 2 type immune response.Compared with the adjuvant group and PBS group,the levels of Ig E and Ig M in the rTsSerp group also increased significantly（F=404.328,P<0.01）.Cytokine test results showed that IFN-γ,IL-2 and IL-4levels were significantly higher than those of TRX-tag,adjuvant and PBS control groups 8weeks after rTsSerp immunization and 2 weeks after challenge infection（P<0.0001）.The results showed that rTsSerp subcutaneously immunized mice induced a Th1/Th2 mixed Th1/Th2 immune response between the whole body and local intestinal mucosa.5.Immunoprotective effect of rTsSerp immunized miceCompared with the PBS group,the worm reduction rates of intestinal adults and muscle larvae were 47.36%（F=120.677,P<0.01）and 54.85%（F=131.244,P<0.01）).Pathological section HE staining results showed that the label group,adjuvant group and PBS group had more obvious pathological changes around the larval cysts,that is,there were a large number of inflammatory cells such as eosinophils and lymphocytes infiltration around the larval cysts.The rTsSerp immunization group significantly reduced the infiltration of inflammatory cells around the larval cysts（r=0.993,P<0.01）,suggesting that rTsSerp immunization can reduce muscle inflammation afterTrichinella infection.Conclusion1.The recombinant expression plasmid p ET-32a/Ts Serp expressed rTsSerp which was purified.Ts Serp is transcribed and expressed in all developmental stages of Trichinella spiralis,but its expression level is higher in muscle larvae and intestinal infectious larvae than other period.In the period,it is mainly located in the cuticle and stichosome of the Trichinella.2.rTsSerp can promote Trichinella spiralis to invade IEC and intestinal mucosa.Anti-rTsSerp antibody mediates the killing effect of ADCC on new born larvae.3.rTsSerp immunization induced mice can induce significant humoral and cellular immune responses and significant immune protection effects.4.Ts Serp is a major protein of Trichinella spiralis that invades the intestinal mucosa of the host,and can be used as a candidate target antigen for anti-Trichinella vaccine.