Cloning And Expression Of Trichinella Spiralis Peptidase And Its Induced Immune Protection

Trichinella infection is mainly caused by eating raw or immature meat which is infected with Trichinella spiralis.When the host ingests meat infected with T.spiralis larvae,muscle larvae is decapsulated under the action of digestive juice in the stomach and activated by bile into intestinal infective L1 larvae?IL1?,the latter invades intestinal epithelium cells?IECs?and develops into an adult stage after 4 molts.The female adults produce new larvae?NBL?after mating,and the newborn larvae invade small blood vessels/lymph vessels in the intestinal mucosa,which circulate throughout the body with blood and invade skeletal muscles and develop into encapsulated muscle larvae,completing the life cycle of T.spiralis.The invasion of IL1 larvae into the intestinal mucosa of the host is a key step in the establishment of infection by T.spiralis,but the mechanism of invasion is not yet fully understood.Proteases in the excretion and secretion?ES?protein or surface protein of T.spiralis IL1 larvae first contact with the host intestinal mucosa,which may mediate the invasion of T.spiralis larvae into the intestinal mucosa by hydrolyzing IECs.The protease is also involved in the invasion of T.spiralis and its mechanism is currently not fully elucidated.In the early stage of our study,immunoproteomics was used to identify a T.spiralis peptidase S1A subfamily?TsP;Gen Bank:XM?003379300.1?from the ES protein of T.spiralis IL1 larvae and adults,but the biological functions of TsP in larvae invasion,worm development and survival have not been reported.The main purpose of this study was to identify the biological characteristics of TsP and its role in the invasion of Trichinella larvae into the intestinal mucosa,and to observe the humoral and cellular immune responses caused by recombinant TsP?r TsP?immunized mice and the immune protection effect against T.spiralis infection effect.Material methods1.Experimental animals,Trichinella species,strains and cell4-week-old female BALB/c mice were purchased from Henan Experimental Animal Center.The T.spiralis used in this research is T.spiralis T1 from Nanyang,Henan Province?the international standard species label is ISS534?.The strain is Escherichia coli BL21.IEC was isolated from the small intestine of mice and used for experimental research after cultured into a cell monolayer.2.The bioinformatics analysis of TsPTsP m RNA sequence and CD_S sequence was obtained from NCBI,and bioinformatics analysis software?Target P,TMHMM,SMART,Prot Param,SWISS-MODEL,Prot Scale,PSIPRED,Signal P?were used to analyze the physical and chemical properties of TsP;Bio Edit and MEGA were used to perform TsP multiple sequence alignment and evolutionary tree analysis.3.The antigenicity analysis of r TsPRecombinant TsP expression plasmid p QE-80L/Tsp was transfected into BL21 to induce protein expression.After purification,r TsP was obtained,and several mice were immunized to prepare anti-r TsP immune serum.The crude antigen and ES antigen prepared by T.spiralis larvae were collected,and r TsP antigenicity was analyzed by Western blot.4.The expression of TsP in different larval stagesThe c DNA of all T.spiralis stages were collected to perform RT-PCR,to detect TsP transcription in different stages.The collection of different larvae stages?muscle larvae,IL1,adult and NBL?was to prepare soluble antigens.The expression and localization of TsP was observed in different worm stages by Western blot and Immunofluorescence assay?IFA?.5.Analysis of the combination of r TsP and IECs by Far-Western and ELISAIECs soluble protein,nuclear protein,and cytoplasmic protein were prepared to observe the binding of r TsP and IECs protein by ELISA,then using Far-Western to further observe the binding of r TsP and IECs protein,nuclear protein and cytoplasmic protein.6.Detection the binding of r TsP to IECs and murine intestinal mucosa by IFAThe binding and cell localization of r TsP and IECs was observed by IFA and confocal microscopy.The normal mouse intestinal mucosa tissue sections were prepared to observe the specificity of r TsP binding to the intestinal mucosa by IFA.7.Larval invasion of IECs and murine intestinal mucosa in vitroAfter bile-activated IL1 larvae were incubated with monolayer IECs for 2 h,the invasion of IECs by larvae was observed.The small intestine of normal mice with a length of 2 cm was cut out,and after ligation at both ends,the IL1 larvae incubated with anti-r TsP serum or r TsP,then were injected into the intestinal cavity and incubated for 2 hours.8.The humoral/cellular immunity and immune protection induced by r TsP in BALB/c mice90 mice were divided into 3 groups?r TsP group,ISA 201 adjuvant group and PBS control,30 mice in each group?,and r TsP were vaccinated subcutaneously?20?g/mouse?at every 2 weeks with 4 times.Two weeks after each immunization,the levels of anti-TsP antibody Ig G and Ig G1/Ig G2a in sera of immunized mice were detected by r TsP-ELISA.ELISA was used to detect the levels of total s Ig A and specific s Ig A in the intestine of mice after 8 weeks immunization,and to detect the changes of cytokines IFN-?and IL-4 in spleen and mesenteric lymph node cells of mice.Two weeks after the last immunization,300 mice were challenged with T.spiralis infection,and after 6d and 30d infection,the mice were sacrificed after anesthesia.The intestinal adults and muscle larvae were collected.The larval reduction rate of muscle larvae was observed,and the changes of worm body morphology and infiltration of inflammatory cells in the intestine and masseter muscle of mice were observed to evaluate the immune protection effect of r TsP immunized mice against T.spiralis infection.9.Statistical processingThe experimental data was processed with SPSS and Graphpad software.Statistical analysis was performed using the T test,one-way ANOVA and chi-square test,and the test level was?=0.05.Results1.Analysis of related properties of TsP bioinformaticsAnalysis of TsP?Gen Bank:XM?003379300.1?used online software such as NCBI,Ex Pasy,Signal P 3.0 serve and TMHMM Server v.2.0.The results showed that the TsP sequence was 780 bp,encoding a total of 259 amino acids,and the relative molecular weight was 28.7 k Da.The p I is 8.32.TsP has no signal peptide,a clear hydrophobic region at the N-terminus,and no transmembrane region.TsP protein subcells are localized as other types.It has good antigenicity and has a trypsin-like serine protease domain.TsP tertiary structure prediction revealed that TsP has 4 alpha helices and 16 beta sheets.Histidine?His?,serine?Ser?and aspartic acid?Asp?constitute a catalytic triad of TsP enzyme activity.2.The antigenicity analysis of r TsPThe recombinant plasmid p QE-80L/TsP was introduced into BL21 and induced by 0.8m M IPTG at 30?for 6 h.The expressed product was purified by nickel column to obtain r TsP.The prepared anti-TsP immune serum titer was 1:10⁵.Western blot results showed that r TsP could be recognized by T.spiralis-infected mouse serum and anti-r TsP immune serum,but it could not be recognized by normal mouse serum,indicating that r TsP has good antigenicity.3.The expression of TsP in each worm stageWestern blot showed that TsP was expressed in soluble proteins in all stages of T.spiralis?anti-r TsP serum can recognize the natural TsP protein bands in muscle larvae,IL1,adult and NBL crude antigens?.IFA results showed that TsP was expressed in different stages of T.spiralis?muscle larvae,IL1,3-day and 6-day adults,and NBL?,and was mainly located in the epidermis of T.spiralis and female embryos.4.Analysis of specific binding of r TsP to IECs by Far-Western and ELISAThe results of Far-Western of IECs protein showed that after incubation of IECs protein with r TsP,9 bands?23.4-46.4 k Da?could be recognized by r TsP immune serum,and after incubation of IECs protein with IL1 ES protein,r TsP immune serum was identified with two bands?51.6 k Da and 60.5 k Da?;however,after incubation of IECs protein with BSA protein,it cannot be recognized by r TsP immune serum.In order to further identify the interaction between r TsP and IECs components,IECs were incubated with r TsP firstly and then the nucleus and cytoplasmic proteins were separated.It was found that IECs protein can be recognized by r TsP immune serum as 5 bands?29.0-72.8 k Da?,IECs nuclear protein can be recognized by r TsP immune serum as 2 bands?43.8 k Da and 29.0 k Da?,IECs qualitative protein can be recognized by r TsP immune serum as 1 protein band?29.0 k Da?.And r TsP cannot bind to C2C12 protein.It indicates that the binding site of r TsP and IECs is in the cytoplasm and nucleus of IECs.After coating with different concentrations of IECs protein and incubating with 5?g/m L r TsP for ELISA,it was found that when the IECs protein coating concentration is less than0.20?g/m L,the higher IECs protein concentration hada stronger binding between r TsP and IECs protein,showing adose dependence relation for IECs and r TsP;ELISA results after 2?g/m L of IECs protein coating and incubation with different concentrations of r TsP protein showed that the higher the r TsP protein concentration when the r TsP incubation concentration was less than 0.06?g/m L,the relationship between r TsP and IECs protein had a stronger binding effect,the results showed that r TsP can bind to IECs protein,and this binding had a dose-dependent relationship between r TsP and IECs protein.5.r TsP and IECs combination and locoalization by IFA and confocal detectionAfter co-incubating r TsP with IECs,IFA and confocal microscopy revealed that r TsP could specifically bind to IECs,and the donor site was in the nucleus and cytoplasm of IECs,which was consistent with the previous ELISA results.After incubating r TsP with normal mouse small intestine tissue sections,IFA results found that r TsP was able to bind to normal mouse small intestinal mucosa epithelium,but did not bind to normal mouse liver and lung tissue,further confirming that r TsP and IECs and the binding of murine intestinal mucosal epithelial cells was specific.6.Invasion experiments in vitroAfter incubating IL1 larvae with r TsP?or anti-r TsP serum,infected serum,normal serum?and IECs monolayer cells for 2 h,compared with the PBS group,the larval invasion rate of the r TsP group and BSA group was 83.44%and 61.34%??2=12.004,P<0.005?,r TsP has a significant promotion effect on the invasion of IL1 larvae into IECs,and this promotion of invasion has r TsP concentration dose-dependent?r=0.985,P<0.005?.But BSA has no effect on IL1 invading IECs.At the same time,it was found that the larval invasion rate of anti-r TsP serum group,infected serum group and normal serum group was 38.73%,25.85%and67.94%??2=42.337,P<0.005?.The results showed that anti-r TsP serum had part blocking effect of the infection of T.spiralis in IECs.The partial blocking effect of r TsP serum on larval invasion of IECs is dose-dependent of anti-r TsP antibody?r=0.906,P<0.05?.The anti-r TsP serum,infected serum,normal serum and IL1 larvae incubated with r TsP were injected into the 2 cm intestinal cavity after ligation and incubated for 2 h.Compared with PBS group,anti-r TsP serum group,infected serum group and normal serum group were observed.The larval invasion rate was 46.17%,34.67%and 65.17%??2=15.757,P<0.001?.The results showed that anti-r TsP serum partially blocked the invasion of T.spiralis into the small intestinal mucosa.The partial blocking effect of r TsP serum on the invasion of larvae into the intestinal mucosa is dose-dependent?r=0.881,P<0.05?.The larval invasion rates of the r TsP group and the BSA group were 88.17%and 66.17%??2=8.640,P<0.005?.r TsP had a significant promotion effect on the invasion of IL1 larvae into the small intestinal mucosa?r=0.973,P<0.005?.But BSA did not promote the invasion of larvae into the intestinal mucosa.7.The immune protection of r TsPAfter 4 immunizations with r TsP,the levels of specific Ig G and Ig G1/Ig G2a in serum were significantly higher than those before immunization(F_{Ig G}=932.349,F_{Ig G1}=288.240,F_{Ig G2a}=252.490,P<0.001),but ISA 201 adjuvant There was no statistically significant difference in antibody levels between the group and the PBS group?P>0.05?;Ig G1 antibody levels were significantly higher than Ig G2a at 4,6,and 8 weeks after immunization(t_4w=10.451,t_6w=9.853,t_8w=10.841,P<0.05),indicating that r TsP immunization of mice induces a Th2-type humoral immune response.Eight weeks after the last immunization,the total s Ig A and TsP-specific s Ig A in the r TsP group were significantly higher than those before immunization.The difference in specific s Ig A levels in the intestinal fluid of the three groups of mice was statistically significant?F=64.678,P<0.005?.The r TsP immunization group was significantly higher than the ISA 201 adjuvant group and the PBS group?t₁=8.407,t₂=11.18,P<0.001?.At 8 weeks after immunization,r TsP immunized mice had significantly higher levels of cytokines?IFN-?and IL4?than ISA 201 adjuvant group and PBS group(Spleen-t_IL-4=50.933,P<0.001;Spleen-t_IFN-?=53.829,P<0.001;MLN-t_IL4=66.771,P<0.001;MLN-t_IFN-?=66.097,P<0.001).After 6 days and 30 days of infection,the worm reduction rates of intestinal adults and muscle larvae in immunized mice were 38.60%and41.93%(F_AW=159.895,F_ML=132.928,P<0.001).The results showed that r TsP induced subcutaneous immunization in mice had shown a Th1/Th2 mixed immune response of systemic and intestinal mucosa and obvious immune protection.In addition,it was also found that r TsP immunization can enhance the infiltration of inflammatory cells in the intestinal mucosa of mice?may be related to intestinal deworming?,but can reduce the inflammatory response in infected mouse muscles.Conclusion1.The recombinant expression plasmid p QE-80L/TsP was constructed and the r TsP was expressed and purified;2.TsP is expressed in different stages of development of T.spiralis,and mainly located in the cuticle of the worm body and the embryo in the female uterus;3.r TsP can specifically bind to mouse intestinal epithelial cells and intestinal mucosa and help T.spiralis to invade intestinal epithelial cells;4.r TsP immunization can produce a systemic Th1/Th2 mixed immune response and local intestinal mucosal response.It has an obvious immunoprotective effect against T.spiralis infection.TsP is a protein related to the invasion of T.spiralis into the host intestinal mucosa,and it can be used as a potential target antigen for the anti-Trichinella vaccine.