Cloning,Expression And Functional Characterization Of The TsSPI Gene Of Trichinella Spiralis

Trichinellosis is a zoonotic parasitic disease caused by the host eating live bursa larvae.After the live larvae enter the host,they are activated by the host's bile and gastric juices to become infected larvae of the intestine.Within 30-48h of the infection,the insects grow into adult worms and grow into adults.After mating,males generally die immediately and are excreted with the feces of the host.The females continue to develop,and 5 to 6 days of infection begin to produce newborn larvae.The newborn larvae will circulate through the lymphatic vessels and blood vessels of the host to various parts of the body.However,only newborns that reach the striated muscle can continue to develop into muscle larvae.The attachment and invasion of Trichinella spiralis to the host intestinal epithelial cells is the key to the infection of the host,but its invasion mechanism is not yet clear.We identified the Trichinella spiralis serine protease inhibitor?TsSPI?in the secretory protein of Trichinella spiralis in the early stage,and it is speculated that the protein may be involved in the invasion of Trichinella spiralis.Serine protease inhibitors inhibit the activity of serine proteases and are a group of structurally conserved protein superfamilies.Members of this family are involved in key biological processes such as the vertebrate coagulation cascade,complement activation,inflammation,apoptosis,cell development and fibrinolysis.In this experiment,molecular cloning expression of the T.spiralis serine protease inhibitor?TsSPI,ID:XM₀03377332.1;protein ID:XP₀03377380.1?was used to purify the recombinant TsSPI?rTsSPI?and qPCR analysis of TsSPI and immunofluorescence antibody localization.Analysis;analysis of the inhibitory function of rTsSPI;in vitro invasion and ADCC experiments with anti-rTsSPI serum.The aim was to reveal the potential function of TsSPI in the invasion of Trichinella spiralis.Materials and Methods1.Trichinella,experimental mice,plasmids,strains,and cellsT1 Trichinella spiralis?ISS534?Henan Geographical strain,experimental mouse BALB/c and Kunming mice,expression plasmid pQE-80L,expression strain BL21,cell-based IECs isolated.2.TsSPI bioinformatics analysisPhysical and chemical properties prediction and hydrophobicity analysis of TsSPI?GenBank accession no.XP₀03377380.1?was performed on Ex Pasy website;transmembrane structure was predicted on TMHMM website;signal peptide prediction was performed on SignalP 4.1 Server;subcellular localization analysis was performed on TargetP1.1 Server.The SWISS-MODEL web version predicts the secondary structure of TsSPI;PyMOL software predicts the tertiary structure.Using the BioEdit Sequence Alignment Editor software,the sequences of the T.spiralis TsSPI were compared with the sequences of 22 other serine protease inhibitors from other parasites,mice and humans;the serine protease inhibitors of the 22 species were tested using MEGA's proximity method.The factors were aligned and mapped 1000 times to construct a phylogenetic tree.3.Cloning and Expression Analysis of Enzymatic Activity of TsSPIThe rTsSPI was expressed in E.coli and affinity purified using a nickel column;the antigenicity of rTsSPI was analyzed by SDS-PAGE and Western blot.The BAEE inhibition at the A253nm was used to calculate the inhibition of trypsin.4.Expression,immunolocalization of TsSPI and functional detection during the invasion of Trichinella spiralisReal-time PCR was used to analyze the transcription of TsSPI gene in different stages of worms.IFA was used to detect the expression and localization of TsSPI in different stages of development.The rTsSPI and normal mouse intestine were analyzed by Far-Western blot with IFA and fluorescence confocal microscopy.The effect of anti-rTsSPI mouse serum on the invasion of Trichinella spiralis larvae into intestinal epithelial cells?IECs?was assessed by in vitro invasion assay.The ADCC assay was used to evaluate the killing effect of anti-rTsSPI mouse serum on NBL.5.Immune response and protective effect induced by rTsSPI immunized mice60 female BALB/c mice were divided into 3 groups?immunized group,adjuvanted group,and PBS group?.Each mouse was immunized subcutaneously with rTsSPI?20?g/body,immunized 4 times,at intervals of 2 weeks?,and immunized.Before and after each immunization with mouse serum,the serum antibody IgG,IgG1,and IgG2a levels were detected by ELISA.Seven days after the last immunization,each mouse was orally infected with 300 Trichinella spiralis MLs.Six days after the infection,10 caterpillars were killed in each group.Adults were collected from the intestine and the worm reduction rate was calculated.At the 35th day after infection,muscle larvae were collected from 10 mice in each group and the worm reduction rate was calculated to evaluate the immune protective effect of rTsSPI in mice.6.Statistical AnalysisThe data were analyzed using PASW Statistics 18 software.The data were presented as mean±standard deviation.The two groups were compared using the t-test.The other one-way analysis of variance was used.The test level was?<0.05.Result1.The bioinformatics analysis of the serine protease inhibitor TsSPI of Trichinella spiralis and its cloning,expression and identificationThe domain,physicochemical properties,signal peptides and transmembrane domains of Trichinella spiralis TsSPI were analyzed using NCBI,Ex Pasy and other online websites.The results showed that the gene sequence has a full-length 1050 bP,encoding 349 amino acid residues,a protein molecular mass of 39.59 kDa,and an isoelectric point of 5.78;TsSPI belongs to the family of serine protease inhibitors.The TsSPI gene with a size of 1122 bp was amplified by nested PCR.The TsSPI gene was cloned into the expression vector pQE-80L by one-step cloning.The recombinant plasmid pQE-80L/TsSPI was constructed and induced by IPTG and analyzed by SDS-PAGE.A clear band appeared at 42.5 kDa,and the protein was expressed both in the supernatant and in the pellet,demonstrating that the expressed rTsSPI is a soluble protein.Western blot analysis showed that rTsSPI protein could be recognized by mice infected with Trichinella spiralis serum,but could not be recognized by normal mouse serum,indicating that the recombinant protein has good antigenicity.The anti-rTsSPI serum can recognize the native TsSPI of the worm proteins and ES proteins of Trichinella spiralis at different developmental stages,indicating that TsSPI is expressed in various stages of Trichinella spiralis.2.Transcription and Expression of TsSPI in Different PeriodsReal-time PCR analysis showed that the transcriptional level of TsSPI was 1.79 times that of muscle larvae at 3d adult stage?F=15.380,P<0.05?,and 2.27 times that of muscle larvae stage at the newborn larval stage?F=51.129,P<0.05?,The transcript level in the larval stage of intestinal infection was-7.77-fold of the muscle larval stage?F=76.004,P<0.01?.The results of IFA indicated that TsSPI expressed in various stages?muscle larvae,intestinal infectious larvae,3d and 6d adult and newborn larvae?,and was mainly located in the epidermis,rods and reproductive organs of the parasite.3.rTsSPI inhibits the activity of trypsinThe results of biochemical experiments on rTsSPI showed that rTsSPI could effectively inhibit the activity of porcine trypsin and the inhibition rate was 59.33%.The recombinant protein had a certain inhibitory effect on trypsin activity between 25°C and 90°C,and the inhibition rate was highest at a temperature of 25°C.4.Far-Western and IFA detect the combination of rTsSPI and IECsThe results of Far-Western showed that rTsSPI could bind to 29 bands of IEC protein in mice,demonstrating that rTsSPI can interact with IECs,but the specific mechanism of action and the process of action are still unclear.The use of anti-rTsSPI mouse serum for IFA detection found that rTsSPI can bind to normal mouse intestinal epithelial cells but not to liver tissue of normal mice,indicating that the binding of rTsSPI to mouse intestinal epithelial cells is specific,further This shows that rTsSPI can interact with mouse intestinal intestinal epithelial cells.5.Effects of recombinant protein?rTsSPI?and its antiserum on in vitro invasion of Trichinella spiralisSera were added to semi-solid medium containing IECs and co-cultured with T.spiralis larvae.The results showed that the invasive rates of the larvae of the anti-rTsSPI sera group,the serogroups of the mice infected with Trichinella spiralis,and the normal mice were 38.7%,16.85%,and 80.08%,respectively.Statistical analysis showed that there was a statistically significant difference in the invasion rate between the anti-rTsSPI serogroup,the serogroup infected with Trichinella spiralis and the normal mouse sera??²=134.354,P<0.01?.The inhibitory effect of anti-rTsSPI serum on larval invasive IECs was significantly higher than that of normal serum??²=119.551,P<0.01?,and this inhibitory effect was antibody-dependent?F=160.236,P<0.01?,indicating serum rTsSPI serum.It can significantly inhibit the invasion of mouse intestinal epithelial cells by Trichinella spiralis.6.Anti-rTsSPI serum mediated ADCCIn the culture plate containing mouse peritoneal macrophages,different serum and newborn larvae were added,and the killing effect of ADCC on newborn larvae of Trichinella spiralis was observed.The mortalities of the mice infected with Trichinella spiralis,rTsSPI immune sera,normal sera,and PBS were 87.67%,27.67%,10.33%,and 6.33%,respectively?F=400.798,P<0.01?.The larval mortality rate of rTsSPI-immune sera was significantly higher than that of normal sera??²=10.526,P<0.05?.There was a significant positive correlation between cytotoxicity and culture time?r=0.968,P<0.01?.The mortality of larvae showed an increasing trend with the prolongation of culture time?F=117.584,P<0.01?,and the cytotoxicity was dependent on the dose of anti-rTsSPI antibody.Sex?r=-0.984,P<0.01?,larval mortality showed a decreasing trend with increasing serum dilution?F=96.813,P<0.01?.7.rTsSPI immunization in miceCompared with PBS group,the worm reduction rates of adults and muscle larvae were62.2%and 57.25%,respectively(F_adult=62.34,F_larva=27.2,P<0.01);and adjuvants were observed at 6 d and 35 d after infection with rTsSPI-immunized mice.The adult worm reduction rates of 22.27%and muscle larvae?2.04%?were significantly lower than those of the immunized group(F_adult=35.77,P<0.01;F_larvae=28.70,P<0.01).In conclusion1.This experiment successfully cloned and expressed TsSPI,a serine protease inhibitor of Trichinella spiralis,and the protein has good antigenicity.2.TsSPI exists in various stages of growth of Trichinella spiralis,and is mainly located in the epidermis,rods and genital pedicels.3.rTsSPI can effectively inhibit pancreatic enzyme activity.rTsSPI can specifically bind to intestinal epithelial cells;anti-rTsSPI mouse serum can inhibit larval invasion into intestinal epithelial cells and mediate ADCC killing neonate larvae.rTsSPI has a certain degree of immunoprotection in mice.