| Trichinellosis is a parasitic infection caused by eating uncooked or raw meat?usually pork?containing Trichinella larvae.It can cause symptoms ranging from generalized fever,nausea,vomiting,abdominal pain,diarrhea,and myalgia to more severe encephalitis and myocarditis.The further development of Trichinella spiralis invading intestinal epithelial cells?IECs?is the key to its infection host.This process is not only mechanical damage,but may also be related to some hydrolytic proteases of Trichinella spiralis.A Trichinella spiralis aminopeptidase?TsAP?was found in the protein increased after co-cultivation of Trichinella spiralis infectious larvae in vitro with IECs.It is speculated that this protein may be related to the invasion of Trichinella spiralis host IECs.In this study,the TsAP gene was cloned and expressed,and recombinant TsAP?rTsAP?was obtained.The transcription and expression levels of TsAP in different stages of Trichinella spiralis were analyzed by q PCR and Western blot.Immunofluorescence?IFA?was used to analyze the location of the worm.The enzymatic activity of rTsAP was determined by hydrolysis of a specific substrate,Leucine-p-nitroanilide?L-p NA?.Western blot,ELISA and IFA techniques were used to analyze the binding effect of rTsAP and IECs protein.The effect of TsAP on Trichinella spiralis invasion,development and fertility was proved by RNA interference technology and invasion experiments.Materials and Methods1.Trichinella species,laboratory animals,cells,plasmids and strainsTrichinella spiralis?T1?were kept in Kunming mice of this laboratory;experimental animals were BABL/c mice purchased from Henan Experimental Animal Center;cells were IECs,which were isolated and passaged from normal mouse small intestine for this laboratory;both the clone and the expressing bacteria were BL21,the cloning vector was p MD19-T,and the expression vector was p QE-80L.2.Bioinformatics analysis of TsAPThe NCBI online website was used to predict the domains and enzyme active sites of TsAP?Gen Bank accession no:XP?003377703.1?.Using Bio Edit software,TsAP was sequence aligned with other Trichinella aminopeptidases,and the phylogenetic tree of TsAP was constructed using the MEGA7.0 adjacency method.3.Cloning expression and antigenicity analysis of TsAPThe TsAP gene was ligated into the cloning vector p MD19-T.After sequencing,the TsAP gene was ligated into the p QE-80L expression vector by double digestion to construct the recombinant expression plasmid p QE-80L/TsAP.After the recombinant protein rTsAP was expressed and purified through a nickel column,mice were immunized to prepare anti-rTsAP immune serum.The p QE-80L/TsAP/BL21 prior to induction,p QE-80L/TsAP/BL21 after induction,and the purified rTsAP were analyzed by Western blot to analyze the antigenicity of rTsAP.4.Expression and localization of TsAP in various stages of Trichinella spiralisTrizol was used to extract RNA from each worm stage[muscle larvae?ML?,intestinal infectious larvae?IIL?,adult worms?AW?,newborn larvae?NBL?],and reverse-transcribed into c DNA.The transcription of TsAP gene at different stages of Trichinella spiralis was analyzed by q PCR.Soluble antigens were prepared in each worm stage,and the expression of TsAP in each worm stage of Trichinella spiralis was analyzed by Western blot.Fresh parasites from different stages were collected to make paraffin sections,and immunofluorescence was performed to analyze the expression and localization of TsAP at different stages of Trichinella spiralis.5.Enzyme activity determination of rTsAPThe rTsAP has the ability to catalyze the hydrolysis of specific substrate L-p NA to produce p NA.The peak wavelength of light absorption of p NA is 405 nm.The enzyme activity of the rTsAP is calculated by measuring the change in absorbance at 405 nm.By measuring the OD405 of different temperatures,different p H values and the presence of different metal ions and inhibitors,the optimal reaction temperature,p H value of the enzyme reaction,and the effects of different metal ions and inhibitors on enzyme activity were observed.6.Binding effect of TsAP and IECs,and its effect on Trichinella spiralis invasion into IECsThe combination of TsAP and IECs was proved by Western,ELISA and IFA technology.The rTsAP and IIL were inoculated into the IEC monolayer,and a BSA and inhibitor control group was set at the same time,which proved the role of TsAP in Trichinella invasion into IECs.The anti-rTsAP immune serum and IIL were inoculated into the IEC monolayer.Trichinella spiralis infected mouse serum was used as a positive control and normal mouse serum was used as a control to observe the blocking effect of specific antibody blocking on IIL invasion into IECs.7.Effect of interfering TsAP gene expression on IIL invasion into IECsTsAP-specific si RNA?si RNA-842?was designed,and the optimal concentration and time of si RNA-842 interference were analyzed by Western blot.Measure the natural enzyme activity in the soluble protein of the muscle larvae after interference,and then activate the interference muscle larvae into intestinal infective larvae?IIL?through bile.In vitro invasion experiments were conducted to observe the blocking effect of TsAP gene expression silencing on the invasion of Trichinella larvae IECs.8.Experiment of larval attack infection after interference of TsAP gene60 BALB/c mice were divided into 3 groups?3?M si RNA-842,Control si RNA or PBS group?,and the interfered muscle larvae were orally inoculated into 10 mice,and each mouse was infected with 300 ML.At 6 days after infection,10 mice were killed in each group to collect AW counts,and the length was measured by taking pictures.Thirty females were randomly selected from each group and cultured in a 24-well plate with RPMI-1640.After 72hours,the NBL produced by the females of each group was counted,and the length was measured by taking pictures.At 60 days after infection,the remaining mice were sacrificed,the muscle larvae per gram?LPG?was calculated and the length was measured.9.Statistical AnalysisThe data obtained in this experiment were collated using Excel 2010,statistical analysis was performed using SPSS 20.0,and the corresponding statistical method was selected according to the experimental method and data type used,including one-way analysis of variance and chi-square test.Inspection level is?=0.05.Results1.Bioinformatics analysis of TsAPTrichinella aminopeptidases contain two domains of the M17 family and belong to metalloproteinases.There are 8 enzyme active sites,which are 264 Lysine?Lys?,269 Aspartic Acid?Asp?,276 Lysine?Lys?,287 Aspartic Acid?Asp?,and 348 Aspartic Acid?Asp?,350glutamic acid?Glu?,352 arginine?Arg?,376 leucine?Leu?,they are tightly arranged in the protein molecular space structure and constitute a substrate binding catalytic site.The evolutionary tree shows that the 11 species/genotypes of Trichinella are well supported and have a close relationship with intestinal parasites Trichuris trichiura.Within the genus Trichinella,two different clades were found:one was the encapsulated clade,and the other was the non-encapsulated clade.2.Cloning expression and antigenicity analysis of TsAPThe TsAP gene was ligated into the cloning vector p MD19-T.The sequencing results showed that the TsAP gene had 98%similarity with the TsAP gene sequence on Gen Bank.After double digestion,the TsAP gene was ligated into p QE-80L to construct the recombinant expression plasmid p QE-80L/TsAP.After adding 0.8 m M IPTG,the cells were collected for SDS-PAGE after induction at 16°C for 24 h.A protein band appeared at a molecular weight of 55.7 k Da.Solubility analysis showed that rTsAP was more in the supernatant.The purified rTsAP immunized mice were used to prepare anti-rTsAP immune serum.Western blot analysis of p QE-80L-TsAP/BL21 prior to induction,p QE-80L/TsAP/BL21 after induction,and the purified rTsAP showed that rTsAP could be recognized by Trichinella infected mouse serum,anti-rTsAP immune serum,and His monoclonal antibody,which proves that it has good antigenicity.3.Expression and localization of TsAP in various stages of Trichinella spiralisThe q PCR results showed that the difference of TsAP transcription levels in different stages of Trichinella spiralis was statistically significant?F=110.851,P<0.001?.The intestinal infectious larvae,adult larvae and newborn larvae were 3.03 times?P<0.001?,1.72times?P<0.01?and 0.14 times?P<0.01?of muscle larvae,respectively.Western blot results showed that anti-rTsAP serum could recognize the natural TsAP in crude antigens of Trichinella spiralis at different stages,and the expression levels of TsAP in different stages were different?F=55.706,P<0.001?.The expression of TsAP protein in intestinal infectious larvae and adult larvae was 1.60 times?P<0.001?and 1.71 times?P<0.001?of muscle larvae,respectively,and that of newborn larvae was 1.14 times?P>0.05?.The results of immunofluorescence tests on larval sections show that TsAP is mainly located in the cortex of Trichinella spiralis and embryos of female adults.4.Enzyme activity determinationThe enzyme activity of rTsAP gradually increased with the increase of rTsAP protein concentration,and it stabilized at a concentration of 0.032?g/?L.The optimal reaction inhibitor 1,10-Phenanthroline has an inhibitory effect on rTsAP enzyme activity,other protease inhibitors have no effect on enzyme activity.The hydrolysis effect of rTsAP on Leu-p NA obeyed simple Michaelise-Menten kinetics,with kinetic parameters Vmax of 28.99?M·min-1 and Km of 14.04 m M.5.Combination of rTsAP and IECsFar-Western results showed that the IECs protein was recognized by anti-rTsAP antibody12 bands?49.8,45.6,42.3,38.6,36.8,35.5,32.9,30.7,28.7,27.3,25.3,17.3 k Da?,and was recognized by infected Trichinella mice serum with 4 bands?42.3,38.8,35.8,28.7 k Da?but could not be recognized by normal mouse serum.It was proved that rTsAP can specifically bind to IECs protein and may be related to the invasion of Trichinella spiralis into the small intestine IECs of mice.ELISA results showed that rTsAP could bind to IECs protein,and the OD value was related to the IECs protein coating concentration?r=0.878,P<0.01?,which increased with the increase of IECs protein concentration?F=127.553,P<0.001?;OD value was also related to the incubation concentration of rTsAP?r=0.868,P<0.05?,which increased with the increase of rTsAP concentration?F=938.272,P<0.001?.The results of IFA and confocal microscopy showed that the binding sites of rTsAP and IECs were mainly in the cytoplasm.6.Effect of rTsAP and anti-rTsAP immune serum on Trichinella invasion of IECsThe difference in the invasion rate between rTsAP concentration?12,16,20,24?g/m L?and the control group was statistically significant?P<0.01?.The promotion rates for Trichinella invasion to IECs were 23.61,28.98,31.77 and 32.48%.It showed that rTsAP had a promotion effect on the invasion of Trichinella spiralis,and the promotion rate was related to the concentration of rTsAP?r=0.916,P<0.001?,and increased with the increase of the concentration?F=215.761,P<0.001?.The differences in the invasive rate of rTsAP immune serum at dilution?1:100,1:200,1:400,1:800?and the normal serum group were statistically significant?P<0.01?,and the inhibitory rates of Trichinella invasion to IECs were respectively were 35.66,27.62,21.02,and 18.61%.Demonstrated that anti-rTsAP immune serum has an inhibitory effect on Trichinella spiralis invasion IECs,and its inhibition rate is related to the dose of anti-rTsAP immune serum?r=0.924,P<0.001?,which decreases with the increase of dilution of anti-rTsAP immune serum?F=175.096,P<0.001?.7.Silencing TsAP gene blocking IIL invasion of IECsWestern blot results showed that TsAP protein expression was inhibited by 44.64,57.98,and 40.71%at si RNA-842 concentrations of 2?M,3?M,and 4?M?P<0.01?;TsAP protein expression was inhibited by 23.59,43.17,and 50.90%at 1 d,3 d,and 5 d after interference?P<0.01?.The results showed that the interference effect was the best when the concentration of si RNA-842 was 3?M and the culture time was 5 days,and si RNA-842 was specific forTsAP interference.After si RNA-842 interference,the inhibition rate of TsAP enzyme activity was49.72%?F=1246.154,P<0.001?compared with the PBS group.The inhibition rate of silencing TsAP gene on larval invasion of IECs was 32.35%?F=921.579,P<0.001?.8.Effects of TsAP gene interference on Trichinella spiralis invasion,development and reproductionsiRNA-842 treated group compared to the PBS group,6 d adult worm reduction rate was39.58%?F=50.033,P<0.001?,female fertility?neonate larvae yield?decreased significantly?F=171.278,P<0.001?,muscle larvae worm reduction rate was 56.03%?F=180.217,P<0.001?.Compared to the PBS group,si RNA-842 treated group was significantly shorter length of the adult female?F=43.018,P<0.001?,male adults also significantly shorter length?F=94.371,P<0.001?,shortening the length of neonate larvae?F=78.893,P<0.001?,muscle larvae reduced length?F=129.492,P<0.001?.Control si RNA was no significant difference between the PBS group?P>0.05?.In conclusion1.Trichinella spiralis aminopeptidase?TsAP?is expressed in all stages of Trichinella spiralis development,and is mainly distributed in the cuticles and female adult embryos.2.The rTsAP has the highest enzyme activity at a temperature of 50°C and a p H value of 8.0,inhibitor 1,10-Phenanthroline has an enzymatic activity inhibition on rTsAP.3.The rTsAP can specifically bind to IECs and promote the invasion of Trichinella larvae into IECs;anti-rTsAP immune serum or silenced TsAP gene can inhibit the invasion of Trichinella larvae into IECs.4.TsAP plays a vital role in the invasion,development,and reproduction of Trichinella spiralis,and can be used as a candidate target for anti-Trichinella vaccine. |
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