Functional Analysis Of Trichinella Spiralis Serine Protease 1.2 And Serine Protease Inhibitor By SiRNA Mediated RNA Interference

Trichinella is a major food-borne parasitic nematode,trichinellosis is mainly resulted from the ingestion of Trichinella larvae present in raw or semi-raw meat products.Trichinella adults are parasitic in the small intestine of the host,but the mechanism by which larvae invade the intestinal mucosa is not fully understood.Protease can catalyze the hydrolysis of protein polypeptide chains,and the multi-function proteases secreted in the parasites play an important role in the invasion and pathogenesis of parasites.Trichinella serine protease TsSP1.2 gene and serine inhibitor TsSPI were obtained from excimer-secretory?ES?.In the early stage of the laboratory,it was found that TsSP1.2 gene?GenBank Accession No.:EU302800?and TsSPI inhibitory factor?GenBank accession no.XP₀03377380.1?may be involved in the invasion of intestinal epithelial cells?IEC?by Trichinella spiralis infectious larvae.RNA interference?RNAi?refers to the phenomenon of highly-specific degradation of homologous mRNA induced by double-stranded RNA?dsRNA?,which is highly conserved during biological evolution.The aim of this study was to determine the role of TsSP1.2 and TsSPI in the invasion and growth of Trichinella spiralis larvae by RNAi.The specific small interfering RNA?siRNA?was introduced into the muscle larvae?ML?by electroporation,and the interference effect was verified by qPCR and Western blot.At the same time,the two genes interact with each other as an unrelated gene for RNAi-specific studies.In addition,after selecting the optimal siRNA and introducing the larvae of Trichinella spiralis,the larval survival rate was observed in vitro and the invasion was performed after the bile was activated into the intestinal larvae?IL1?to observe whether RNAi silencing TsSPI can inhibit larval-intestinal epithelial cell?IEC?invasion and development.The RNAi-treated muscle larvae were orally inoculated into mice,and the inhibitory effects of silencing TsSP1.2 and TsSPI on larval development,survival and fecundity were observed.Materials and Methods1.Trichinella,experimental animals and cellsThe Trichinella used in this experiment were all from Trichinella?T1?preserved in Kunming mice of this laboratory.BALB/c mice used in the experiment were male 6-week old and purchased from the Experimental Animal Center of Henan Province.The cell was a mouse IEC isolated.2.Activation and protein expression of the cells,purification and preparation of antiserumThe previously frozen TsSP1.2 and TsSPI recombinant bacteria in our laboratory were activated.Induction of expression of rTsSP1.2 and rTsSPI,sonication and purification of proteins,immunization of mice,and acquisition of anti-rTsSP1.2 and anti-rTsSPI immune serum.3.Design and synthesis of siRNAAccording to the gene number of TsSP1.2 and TsSPI,the siRNA was designed on online design software siDirect version 2.0.Three siRNAs were screened for each gene,and the siRNAs named TsSP1.2 were siRNA-534,siRNA-818,and siRNA-303 according to the site where the mRNA was targeted.The siRNA of TsSPI was siRNA-445,siRNA-653,siRNA-882.In addition,a Control siRNA was randomly scrambled according to the target sequence of the siRNAs,and a FAM fluorescent label was added to the 5'end of the Control siRNA base to experimentally observe whether or not the siRNA was introduced into the Trichinella spiralis.4.Introduction of siRNA into Trichinella by electroporationUsing the method of transient electric shock,2??different siRNA were introduced into Trichinella spiralis,transferred to RPM1640 for culture in vitro,and the fluorescence signal was observed after 18h.5.Changes in gene transcription levels and protein expression levels after RNAiThe transcript levels and protein expression levels of TsSP1.2 and TsSPI were observed by qPCR and Western blot.After 2,4,6 and 8 days after transfection of siRNA into muscle larvae.At the same time,the interference effects of different doses of siRNA?1,2,3???were observed.6.RNA interference is gene specificTsSP1.2 and TsSPI were used for gene silencing with 2??siRNA,and the two groups were used as a control group to verify RNAi gene specificity.7.Survival rate of RNA after RNAi and its effect on IEC invasionAfter electroporation treatment of Trichinella spiralis muscle larvae,cultured in vitro for7 days,the morphological changes of muscle larvae were observed,and the survival rate was calculated.1,1.5,2,2.5,3?M siRNA-534,siRNA-653 and Control siRNA were introduced into Trichinella spiralis muscle larvae by electroporation,and PBS was used as a control group.After the worms were added to the medium containing RPM1640 for 18 hours,100IL1 and 500?L of semi-solid medium were mixed uniformly,and inoculated into a 24-well plate which had been covered with a single layer of IEs,and placed at 37°C,5%CO₂.After incubation for 2 hours in the incubator,the in vitro invasion of IEC by muscle larvae was observed,and the inhibition rate of RNAi on IEC invasion was calculated.8.Effect of RNAi on the Infectivity,development and fertility of TrichinellaEighty BALB/c mice were equally divided into 4 groups?20 animals per group?:TsSP1.2 group,TsSPI group,Control siRNA group and blank control group.Each group was orally infected at a dose of 300 ML/mouse.After 6 days of infection,mice in each group were sacrificed 10 times to recover adult intestinal tracts.The length of male and female worms was measured and the worm reduction rate of adult?AW?was calculated.100 adult worms were cultured in vitro,and newborn larvae?NBL?were collected 72 hours later.Observe the length of the NBL worm and calculate the fecundity of the female.The remaining mice were sacrificed 35 days after infection,muscle larvae were collected,the length of the worms was measured,and the larval rate of muscle larvae was calculated.Results1.Introduction of siRNA:Under the fluorescence microscope,it was found that there was a clear fluorescence in the larvae after RNAi treatment and the whole body of the worm was bright,while the untreated larvae had no fluorescence signal.2.Effect of RNAi on expression levels of TsSP1.2 and TsSPI genes in muscle larvae2.1 The effect of RNAi on expression of TsSP1.2 gene:qPCR results showed that the siRNA-534,siRNA-818,siRNA-303 treatment group muscle transcription level decreased57.05,57.37 and 27.18%relative to PBS group?P<0.05?,respectively,and protein expression levels decreased 69.65,68.97and 37.09%,respectively?P<0.05?;1,2,3??siRNA-534 was introduced into the worms,the transcription of TsSP1.2 was decreased 44.29,53.81 and79.16%?P<0.05?.Protein levels were decreased 35.83,44.05 and 63.28%?P<0.05?.Muscle larvae by 2?M siRNA-534 interference induced the gene expression levels were decreased by56.44 and 35.46%in the cultured days?P<0.05?,and the protein levels were decreased by84.48 and 67.35%,respectively?P<0.05?.The TsSP1.2 transcription level after transfection with control siRNA had no distinct decrease in transcription and protein expression levels?P>0.05?.2.2 Effect of RNAi on expression of TsSPI gene in T.spiralis muscle larvae:qPCR analysis showed that the siRNA-445,siRNA-653,siRNA-882 treatment groups had lower TsSPI gene transcription levels than the PBS control group in 20.50,42.09 and 12.13%?P<0.05?.Protein expression levels decreased 63.52,80.26 and 10.61%?P<0.05?;1,2 and 3??siRNA-653 were introduced into the worm 1d,TsSPI The gene transcription levels were decreased by 23.57,36.90 and 60.78%?P<0.05?,and the protein expression levels were decreased by 26.47,75.13 and 89.18%?P<0.05?;2?M siRNA-653 interfered with muscle larvae at 4d and 6d,the gene expression decreased significantly by 75.75 and 69.79%?P<0.05?,and the protein expression decreased by 69.23 and 50.63%?P<0.05?.There was no significant difference in TsSPI gene transcription and protein expression between Control siRNA group and PBS group?P>0.05?.3.Gene specific analysis of RNA interferenceThe expression of TsSP1.2 and TsSPI protein was observed by RNAi 1d after introduction of siRNA-TsSP1.2 and siRNA-TsSPI into larvae.The results showed that the protein expression level of siRNA-TsSP1.2 treatment group was decreased by 42.63%compared with PBS group,and the expression level of siRNA-TsSPI protein was not statistically significant?P>0.05?;siRNA-TsSPI protein expression level Compared with the PBS group,the decrease was 30.21%,and the difference of TsSP1.2 protein expression level was not statistically significant?P>0.05?.4.Survival rate of insects after RNAiThe larval mortality of siRNA-534,siRNA-653,Control siRNA group and PBS group was 5.16,4.80,4.49 and 3.16%after one day electroporation?P>0.05?.At 7 days after silencing,the mortality of the four groups was 30.34,34.08,33.13 and 32.73%,respectively?P>0.05?.The results showed that RNAi had no significant inhibitory effect on the viability of larvae.5.Inhibition of RNAi on IEC invasion5.1 Inhibition of TsSP1.2 gene silencing on Trichinella invasion of IEC:The inferencing of TsSP1.2 with siRNA-534 reduced larva intrusion of IEC.The intrusion rate of the IL1transfected with 2,2.5 and 3?M siRNA-534 was 42.82,38.66 and 32.88%,respectively.(?²_2?M=11.095,?²_2.5?M=14.892,?²_3?M=23.878,P<0.001).The suppressive effect was dose-dependent of siRNA 534?r=0.976?,showing an increasing trend with the elevation of siRNA 534 dose?F=61.430,P<0.01?.However,no remarkable suppression on larva intrusion was detected when control siRNA was used5.2 Inhibition of TsSPI gene silencing on Trichinella invasion of IEC:When the IL1 larvae were added onto the IEC monolayer and cultured for 2 h,the IL1 intrude into the IEC and migrated in monolayer.The silencing of TsSPI by siRNA-653 suppressed the larval penetration of the IEC.The penetration rate of worms treated with2,2.5 and 3?M siRNA-653was 43.68,36.07 and 30.54%,respectively(?²_2?M=12.789,?²_2.5?M=17.185,?²_3?M=28.474,P<0.001).The inhibitory effect was siRNA dose-dependent?r=0.981?and exhibited an elevating trend with the increase of the siRNA concentration?F=78.440,P<0.001?.However,no apparent inhibition on the larval invasion was observed by using control siRNA.6.Effects of RNAi on the infectivity,development and fecundity of T.spiralis6.1 TsSP1.2-siRNA group:RNAi composition of insect load?48.31±7.29?was significantly lower than Control siRNA group?114.15±12.68?and PBS group?112.77±9.99?6 days after siRNA-534 treated(F_adults=162.493,P<0.001);the yield of newborn larvae after 72 h of female adult culture were 53.33±8.26,78.17±6.33 and 76.17±6.78,respectively(F_NBL=39.547,P<0.001);35 days after infection,from 3 groups of mice The number of muscle larvae recovered was 1076.40±348.31,3341.79±643.72 and 3682.34±1090.84(F_AW=35.530,P<0.05).The results showed that the worm reduction rates of adult and muscle larvae at 6d and 35d after inoculation with TsSP1.2-siRNA were 57.16%and 71.46%,respectively.The length measurement of the worm body showed that the length of the female adult in the siRNA group?1496.48±96.19??m was lower than that of the siRNA control group?1807.70±105.88??m and the PBS group?1999.05±36.42??m;the length of the male adults in the siRNA group and the siRNA control group were respectively?867.92±28.11??m and?949.89±20.01??m were also significantly smaller than the PBS group?996.29±70.27??m(F_femal=50.965,F_male=6.205,P<0.05).The lengths of the newborn larvae of the three groups were?73.72±9.69??m,?116.50±2.59??m,and?119.28±4.64??m?FNBL=39.547,P<0.0001?.The length of muscle larvae in the siRNA group?843.44±39.60??m was also significantly shorter than that in the control group?1133.12±55.54??m and in the PBS group?1146.38±20.70??m?F=57.836,P<0.001?.6.2 TsSPI interference group:The same experimental composition of worm load 40.92±8.29was less than 114.15±12.68 of Control siRNA group and 112.77±9.99 of PBS group(F_adults=191.887,P=0<0.001),worm reduction rate 63.71%?P<0.05?;the yield of newborn larvae after 72 h was 40.67±6.32,78.17±6.33 and 76.17±6.78,respectively?F=112.406,P<0.001?.The recovered muscle larvae were 1012.29±131.41,3341.79±643.72 and 3682.34±1090.84?FML=57.014,P=0.00<0.05?.In the interference group,the worm reduction rate of ML reached 72.38%?P<0.05?.The lengths of the same three groups of male and female adults were significantly different.The female adult in the experimental group was 1517.98±90.74,which was lower than the control group 1816.36±96.52 and 2023.11±15.73?m,and the male adults were715.32±57.60,949.89±20.01 and PBS group 996.29±70.27?m,(F_femal=40.683,F_male=40.008,P<0.05).The length of the newborn larvae was 69.85±4.22?m,116.50±2.59 and119.28±4.64?FNBL=57.836,P=0.00?.The muscle larva length experimental group817.44±43.25?m was also shorter than the control group of 1133.12±55.5 4?m and the PBS group 1146.38±20.70?m,there was a significant difference?F=39.547,P<0.001?.Conclusions1.Electroporation successfully introduced siRNA into T.spiralis muscle larvae.2.siRNA can effectively reduce the transcription level of TsSP1.2 and TsSPI genes by electroporation,and also significantly reduce their protein expression.It was also demonstrated that RNAi is gene specific.3.Using RNAi technology to silence the TsSP1.2 and TsSPI gene,effectively inhibited the invasion of intestinal epithelial cells by muscle larvae.At the same time,RNA silencing reduced the fecundity of females,and resulted in the significant changes of morphology of all stage worms,further confirming that TsSP1.2 and TsSPI play a vital role in the life cycle of T.spiralis,and they might be the promising molecular target for anti-Trichinella vaccine.