Cloning And Expression Of Cathepsin B Of Trichinella Spiralis And Its Role In Larval Invasion Of Mouse Intestinal Epithelial Cells

Trichinosis is a food-borne zoonotic parasitic disease,mainly caused by raw or semi-raw meat containing the encapsulated larvae.Trichinella can cause a variety of clinical manifestations such as fever,facial edema,and muscle pain.Serious infections can die from complications.Larvae are the main pathogenic stage of Trichinella,and it is especially important to look for proteases that play a key role in the invasion of intestinal mucosa.Cathepsin B is a papain-like cysteine protease.Previous studies have found that cathepsin B can degrade the host adhesive protein,fibronectin,type I and type IV collagen,which is related to the invasion of parasites and tissue migration,so we speculate that the protein may be involved in the invasion of Trichinella spiralis into the host.In this study,the caterpillar cathepsin B?TsCB?was cloned and expressed,and its bioinformatics properties were analyzed.Purified recombinant TsCB?rTsCB?was used to prepare immune serum,and then TsCB was subjected to immunofluorescence localization and RT-PCR analysis.And to study its binding to mouse intestinal epithelial cells?IECs?and its function in invasion of IEC,to explore the role of TsCB in Trichinella invasive host IEC.Materials and methods1.Trichinella species,experimental animals,cells and strainsThe Trichinella species used in this experiment were Henan Nanyang pig-derived geographical strains.Experimental animals including Kunming mice and BABL/c mice were purchased from Henan Experimental Animal Center.Cells were isolated from the experimental mice intestinal epithelial cells,rTsCB The expression strain was BL21.2.Bioinformatics analysis of TsCBPrepasy website was used to predict the basic physicochemical properties of TsCB protein;SignalP 4.1 Server was used to Predict whether TsCB has signal peptide;TsCB protein transmembrane structure was predicted on TMHMM website;SWISS-MODEL webpage was used to predict secondary and tertiary structure of TsCB.Information related to TsCB?TsP-03306?was found in NCBI to predict its enzyme activity sites.Using BioEdit software,Trichinella spiralis cathepsin B was sequenced with cathepsin B from human,mouse and other parasites and phylogenetic tree analysis was performed.3.Cloning expression and antigenicity analysis of TsCBThe TsCB gene was ligated into the expression vector PQE-80L,and the recombinant protein rTsCB was expressed in prokaryotic cells.The mouse anti-rTsCB immune sera were prepared by immunizing mice with affinity purification on a nickel column,and ML crude antigen and ES antigen were collected for SDS-PAGE and Western blot experiments were performed to analyze the antigenicity of rTsCB.4.Expression and fluorescent immunolocalization of TsCB in Trichinella spiralisRT-PCR was used to analyze the transcriptional levels of TsCB gene in different stages of muscle larvae?ML?,intestinal infectious larvae?IIL?,adult worm?AW?,and newborn larvae?NBL?,and collect worms from different stages.The fresh worms were made into paraffin sections,and IFA experiments were performed to detect the expression and localization of TsCB in different developmental stages of Trichinella spiralis.5.The combination of TsCB and IECs,and its role in Trichinella invasion into IECsELISA,Western blot,Far-Western and fluorescence confocal microscopy were used to detect the binding of TsCB and IECs after incubation,and the interaction between TsCB and IECs was further verified by the separation of IECs cytoplasm and nucleus.Then,the in vitro larval invasion experiment was carried out.The IIL larvae were added to the DMEM semi-solid medium and covered on the surface of the IECs monolayer.The rTsCB and rTsCB immune serum were added simultaneously in the medium.After incubation for 2 hours,the larvae invaded the IECs.6.Interference with the expression of TsCB gene to block the invasion of IIL into IECsDesign a small interfering RNA?siRNA 596?,silence the expression of TsCB in muscle larvae,determine the optimal concentration and duration of siRNA 596 interference,and perform in vitro invasion experiments on ML after interference,to observe the blocking effect of silencing TsCB gene expression on larval invasion of IECs.7.Immune response and immune protective effect induced by rTsCB immunization in miceThirty BALB/c mice?female?were randomly divided into three groups:rTsCB immunization group,adjuvant group and PBS group.Each mouse was immunized with 20?g rTsCB protein subcutaneously at intervals of 2 weeks for a total of 4 times.After the last immunization,each mouse was infected with 300 ML,for 6 days,then the mice were killed,the adults in the intestinal tract and the cultured newborn larvae were collected,the worm reduction rate and the yield of the newborn larvae were calculated,and the body length of the mice was measured.The morphological changes were observed.8.Statistical processingAll data in this experiment were processed and analyzed by SPSS17.0 statistical software.The data were average±standard deviation.The statistical methods were selected according to the type of experiment and data,including chi-square test,one-way ANOVA and t-test.The test level was?<0.05.Results1.Bioinformatics analysis of TsCBThe TsCB?accession No:NW₀03526941.1?gene bioinformatics software and online website analysis predicted that the TsCB gene sequence is 1071 bp in length,encoding 356amino acids,with a molecular weight of approximately 40.23 kDa and an isoelectric point of7.86.The TsCB gene has a signal peptide?1-29aa?,a distinct hydrophobic region at the N-terminus,a transmembrane region?12-35aa?,and a structural domain of peptidase_C1A,located at 102-351aa,located at Extracellular membrane.The TsCB secondary structure contains 7?-helices and 13?-sheets.The tertiary structure predicts that there are 4 enzyme active sites:histidine?His?,glutamine?Gln?,and cysteine.?Cys?,asparagine?Asn?,they are closely arranged in the molecular structure of the protein.2.Cloning expression and antigenic analysis of TsCBThe TsCB gene was ligated into the cloning vector PMD19-T.After double digestion,the TsCB was ligated into the expression vector pQE-80L,and the recombinant expression plasmid pQE-80L/TsCB was constructed and transformed into competent cells.The positive clones were sequenced and identified.The single colony with successful connection was added with 2 mM IPTG.After 6 hours of induction at 37°C,the cells were collected for SDS-PAGE.It was found that pQE-80L/TsCB showed a protein band at 39.7 kDa.Soluble analysis showed that rTsCB existed as inclusion bodies.Murine anti-rTsCB serum was prepared from rTsCB-immunized mice.Western blot analysis showed that the purified rTsCB protein could be recognized by anti-rTsCB serum,but could not be recognized by the Trichinella-infected mice serum,and had no cross-reactivity with normal mouse serum;anti-rTsCB serum could recognize Trichinella spiralis natural TsCB in the crude antigen in different stages?ML,IIL,AW,NBL?,but not the TsCB in the ES antigen,indicating that TsCB is a component of the Trichinella spiralis protein.3.Expression and fluorescent immunolocalization of TsCB in Trichinella spiralisThe results of RT-PCR showed that the TsCB gene was transcribed in different stages of Trichinella spiralis?ML,IIL,AW,NBL?.The immunofluorescence of TsCB showed that TsCB was mainly stichosome,cuticles and female adult embryos.4.Indirect ELISA detects the combination of rTsCB and IECsAfter incubation with 10?g/mL rTsCB with different concentrations of IECs,the ELISA results showed that the optimal coating concentration of IECs was 3.2?g/mL.Then,the plate was incubated at different concentrations and incubated with different concentrations of rTsCB protein.The results showed that rTsCB fully bound to IEC protein when rTsCB concentration reached 1.75?g/mL.The OD value increased with the concentration of IEC package?F=298.187,P<0.01?,and the correlation was significant?r=0.743,P<0.05?.In addition,the OD value also increased with the concentration of rTsCB.Large and increased?F=458.891,P<0.01?,there was also a significant correlation?r=0.953,P<0.01?,indicating that the recombinant protein can bind to IECs.5.Western blot analysis of specific binding of rTsCB to IECsAfter rTsCB was incubated with live IECs for 2h,Western blot showed that the anti-rTsCB immune sera recognized 7 bands of IECs,while the infected serum recognized 5bands,and normal serum could not be identified.However,anti-rTsCB serum did not recognize the IECs protein after incubation with BSA.Western blot analysis of IECs cytosolic protein and nuclear protein after incubation with rTsCB showed that anti-rTsCB serum recognized 5 cytoplasmic protein bands and 3 nuclear protein bands,indicating that the binding sites of TsCB and IECs are IEC cytoplasm and nucleus.6.Far-Western and IFA verify rTsCB interaction with IECsFar-Western results showed that IECs protein can be recognized by anti-rTsCB serum and Trichinella infected mice serum,but not by normal mouse serum;anti-rTsCB serum can recognize 8 bands in IEC whole cell proteins,in cytoplasmic proteins Seven bands and six bands in the nuclear protein,again indicating that rTsCB can bind to IECs,confocal microscopy found that rTsCB can specifically bind to IECs,more intuitively indicating that rTsCB can bind to IEC,binding site in IECs cytoplasm and nucleus.7.The promotion and inhibition of rTsCB and anti-rTsCB serum on in vitro invasion of IECs by Trichinella spiralisIn vitro invasion showed that rTsCB significantly promoted the invasion of IIL larvae into IECs in a dose-dependent manner?r=0.985,P<0.01?,and enhanced the invasive effect with the increase of anti-rTsCB protein concentration?F=341.596,P<0.01?,but BSA did not promote larval invasion.After IIL is added to the IECs monolayer,the larvae can invade the cell monolayer.Compared with the PBS group,anti-rTsCB serum?1:100-1:600?significantly inhibited larval invasion of IECs?P<0.01?.The anti-rTsCB antibody was dose-dependent?r=0.974,P<0.01?,and decreased with the increase of serum dilution?F=90.963,P<0.01?.In addition,mouse pre-immune serum and adjuvant-immunized mouse serum did not significantly inhibit larval invasion.8.Blocking effect of silencing TsCB gene on IIL invasion of IECsDifferent concentrations of siRNA 596 were transfected into Trichinella spiralis muscle larvae by electroporation.Western blot analysis showed that the expression of TsCB was inhibited by 33.18%and 70.74,respectively.The results showed that when the concentration of siRNA 596 was 1?M,1.5?M,2?M and 2.5?M,the expression of TsCB was inhibited by19.61%,41.35%,75.47%and72.38%,respectively.The expression level of TsCB protein was inhibited by 89.49%after 2?M of siRNA 596 was introduced into the worm for 2 days.The results of in vitro invasion experiments showed that the siRNA 596 interference group had a statistically significant difference in larval invasion rate compared with PBS group??2=42.632,P<0.01?,and the IEC inhibition rate of silent TsCB gene on larva invasion was49.96%.9.Analysis of immune response types and immune protective effects induced by rTsCBAfter immunization with rTsCB,the levels of IgG antibody in serum increased significantly,and the levels of IgG1 and IgG2a also increased significantly after the second immunization,but the level of IgG1 increased more significantly(t_4W=4.350,t_6W=4.247,t_8W=2.902,P<0.01).Therefore,Th2 type immune response was mainly induced in mice immunized with rTsCB.5 days after infection,the worm reduction rate of mice immunized with rTsCB was 52.81%,which was significantly different from that of PBS group?F=63.80,P<0.01?.There was no significant difference in the number of insects between adjuvant group and PBS?F=8.400,P>0.05?.The length of female and male adults was measured respectively.the results showed that the length of female adults in rTsCB immunized group was significantly shorter than that in PBS group and adjuvant group?F=19.390,P<0.01?,but there was no significant difference in the length of male adults among the three groups?F=1.849,P>0.05?.The in vitro NBL yield for 72 h of adult females from rTsCB-immunized mice was significantly lower than that of control mice?F=11.153,P<0.01?.Moreover,the length of NBL produced by female adults from immunized mice was clearly shorter than that from PBS control mice?F=24.788,P<0.01?.In conclusion1.The recombinant prokaryotic expression plasmid pQE-80L/TsCB of Trichinella spiralis cathepsin B?TsCB?was successfully constructed and induced to express rTsCB;2.TsCB is expressed in all developmental stages of Trichinella,mainly located in the rods of the worm body,stratum corneum and female embryos;3.rTsCB can specifically bind to mouse intestinal epithelial cells and promote the invasion of intestinal epithelial cells by Trichinella spiralis.Anti-rTsCB serum can significantly inhibit the invasion of intestinal epithelial cells by larvae.Silencing TsCB gene also inhibited the rotation.The silent TsCB gene also significantly inhibited the invasion of the small intestinal epithelial cells by the Trichinella spiralis;rTsCB has certain immune protective effect on mice.TsCB is a protein involved in the invasion of host intestinal epithelial cells by Trichinella.