Cloning And Expression Of Trichinella Spiralis Serine Proteinase And Its Role In Invasion Of The Host Intestinal Epithelium

Background and PurposeTrichinellosis is a zoonotic disease that is widespread worldwide.It poses a serious threat to human public health and also causes serious economic problems.Trichinellosis is mainly caused by raw or semi-raw eating pork,other animal meats and meat products containing live larval cysts.A variety of serine protease（SP）has been identified in parasites.These serine proteases are related to the development and nutrition of parasites,invasion of tissues and cells,blood coagulation,and immune evasion.The SP superfamily contains His,Asp and Ser active sites at the C-terminus and plays an important role in the process of food digestion,apoptosis and differentiation,tissue remodeling,and immune defense.It may be used as a diagnostic antigen and immunity for parasitic diseases.Protected candidate molecules,at the same time,may also serve as candidate targets for therapeutic drugs.In the early stage of this research group,a Trichinella spiralis serine protease（TsSP1.1,Gen Bank:ACA28930.1）was identified in the surface protein of Trichinella spiralis muscle larvae.In this paper,TsSP1.1was cloned and expressed,and the expression of TsSP1.1 was cloned and expressed.The biological characteristics and its role in the invasion of intestinal epithelial cells（IECs）by larvae were studied.Materials and Methods1.Trichinella species,experimental animals,cells,plasmids,strainsThe Trichinella species used in this experiment is the Trichinella species（T1）preserved in the laboratory.The animals used in the experiment were Kunming mice purchased from Henan Animal Experimental Center.The experimental mouse small intestinal epithelial cells（IECs）were preserved in our laboratory after early isolation.The cloning and expression bacteria is Escherichia coli BL21,the cloning vector is p MD19-T,and the expression vector is p QE-80L.2.Biological analysis of serine protease from Trichinella spiralisUse NCBI online website to predict the structural domain and enzyme active site of TsSP1.1（Gen Bank:ACA28930.1）.The Bio Edit software was used to align TsSP1.1 with the sequences of serine proteases of 11 species including other parasites,mice and humans,and the phylogenetic tree of TsSP1.1 was constructed by the neighbor joining method of MEGA7.3.Clonal expression of TsSP1.1 and analysis of its antigenicityPrimers were designed according to the protease gene sequence,and the c DNA of Trichinella spiralis muscle larvae was used as a template for PCR amplification.The amplified TsSP1.1 gene was ligated to the cloning vector p MD19-T,and after double enzyme digestion,it was combined with the expression vector p QE-Connect with 80L to construct the recombinant expression plasmid p QE-80L/TsSP1.1.The rTsSP1.1 purified by the nickel column was immunized to mice to prepare anti-rTsSP1.1 serum.The antigenicity analysis of rTsSP1.1 was performed using western blot technology.4.Expression and localization of TsSP1.1 at various stages of Trichinella spiralisTrichinella spiralis muscle larvae（ML）,intestinal infectious larvae（IIL）,adult worm（AW）and newborn larvae（NBL）were collected to prepare soluble antigens of different stages of Trichinella spiralis.The expression of TsSP1.1 in different stages was analyzed by western blot.The indirect immunofluorescence technique（IIF）was used to observe the expression of TsSP1.1 in different stages of Trichinella spiralis and its localization.5.rTsSP1.1 enzyme activity assaySerine protease has the ability to catalyze the hydrolysis of the substrate N-benzoyl-L-arginine ethyl ester（BAEE）to produce BA.The peak wavelength of BAEE’s light absorption is the change in the absorbance at 253 nm.By measuring the OD₂₅₃value of the reaction at different temperatures and different p H values,the effect of the optimal temperature and p H value of the rTsSP1.1 enzyme reaction on the enzyme activity was observed.In addition,the renatured rTsSP1.1 protein was subjected to matrix gel electrophoresis（gelatin zymography analysis）to detect its enzyme activity.6.Binding of rTsSP1.1 to IECs and its role in T.spiralis invasion of IECsThe combination of rTsSP1.1 and IECs was proved by Western blot technology.Inoculate rTsSP1.1 and Trichinella intestinal infectious larvae（IIL）into IEC monolayers to observe the role of rTsSP1.1 in the invasion of IECs by Trichinella.The anti-rTsSP1.1 serum and IIL were inoculated into the IEC monolayer.Trichinella infection serum was used as a positive control and normal serum was used as a negative control to observe the blocking effect of specific anti-rTsSP antibody blocking on IIL from invading IECs.7.Animal studies on the efficacy of anti-rTsSP1.1 antibodies in blocking intestinal mucosal invasion by Trichinella spiralisThirty mice were divided into 3 groups（10 in each group）,anti-rTsSP1.1 antibody group,infected serum group and normal serum group.After incubating muscle larvae with the above three kinds of serum（1:100）at 37°C for 2 hours,the mice were orally infected（500/mouse）,and the number of adult worms,morphology and female reproduction were observed 5 days after infection.8.Statistical analysisUse Excel 2010 to sort out the data obtained in this experiment,use SPSS 21.0 for statistical analysis,and select the corresponding statistical methods according to the experimental methods and data types used,including t-test,one-way analysis of variance,and chi-square test.The inspection level isα=0.05.Result1.Bioinformatics analysis of the Trichinella spiralis serine protease TsSP1.1The TsSP1.1 gene has a full length of 1424 bp,a molecular weight of 51 k Da,encodes453 amino acids,has a p I of 6.25,has a signal peptide,is secreted into the periplasm,has no transmembrane region,and has an obvious hydrophobic region at the N-terminus,which belongs to serine protease.Using the NCBI online website to predict the TsSP1.1 domain and enzyme active site,the results show that TsSP1.1 contains a serine protease family domain with 3 enzyme active sites（Asp,Ser and His）.Using Bio Edit software,TsSP1.1 was compared with the serine protease sequences of 11 other parasites,mice and humans,and it was found that it had high homology with otherTrichinella species.The MEGA7 software was used to compare the serine proteases of 17 species and the phylogenetic analysis showed that Trichinella spiralis and other cystic type Trichinella spiralis are located on the same branch.2.Clonal expression of TsSP1.1 and analysis of its antigenicitySDS-PAGE analysis revealed that rTsSP1.1 was present in the precipitate,indicating that the rTsSP1.1 was an insoluble protein.The results of SDS-PAGE after affinity purification by His-tag Ni-column showed that there was a single protein band at a molecular weight of 51k Da,indicating that rTsSP1.1 has a better purification effect.Western blot showed that rTsSP1.1 can be recognized by infection serum and anti-rTsSP1.1 serum,but not by normal serum,which proves that rTsSP1.1 has good antigenicity.3.Expression and localization of TsSP1.1 at various stages of Trichinella spiralisWestern blot results showed that the anti-rTsSP1.1 serum could recognize the natural TsSP1.1 in the crude antigen of Trichinella spiralis ML,IIL,AW and NBL stages,which proved that TsSP1.1 gene was expressed in ML,IIL,AW,and NBL stages.Fresh paraffin sections were collected and tested for IIF.It was found that bright green fluorescence appeared on the body surface and internal organs of ML,IIL,and AW.The results showed that the TsSP1.1 gene was in the three stages of ML,IIL,and AW.It is expressed,and TsSP1.1 is mainly located in the cortex,stichosome and embryos of female adult worms.4.rTsSP1.1 enzyme activity assayThe enzymatic activity of rTsSP1.1 was measured with N-benzoyl-L-arginine ethyl ester（BAEE）as the substrate.The result showed that rTsSP1.1 has the enzymatic activity of natural serine protease,and the optimum temperature is 45℃,the optimum The p H is 8.5.When the p H is 8.5,rTsSP1.1 can hydrolyze gelatin.5.Binding of rTsSP1.1 to IECs and its role in the invasion of IECs by T.spiralis larvaeFar–western blot results show that IECs protein can be recognized by rTsSP1.1 serum and infection serum after co-incubation with rTsSP1.1,while C2C12 can not be recognized by anti-rTsSP serum and infection serum after co-incubation with rTsSP1.1.The results of the in vitro invasion test showed that when the rTsSP1.1 protein concentration reached 5μg/m L,rTsSP1.1 had a significant effect on larval invasion of IECs（χ²=2.335,P﹤0.05）,and rTsSP1.1 had a significant effect on larval invasion of IECs.The promotion effect is dose-dependent with rTsSP1.1 protein（r=0.939）,and it increases with the increase of the dose of rTsSP1.1（F=23.465,P<0.0001）.When the serum dilution is 1:100,the larval invasion rate of anti-rTsSP1.1 serum,infection serum,and normal serum group are 41.54%,31.63%and 77.95%,respectively（P<0.01）.The inhibitory effect of rTsSP1.1 antibody（1:100-1:800）on larval invasion is dose-dependent（correlation coefficient r=0.960）,which decreases with the increase of serum dilution（P<0.01）,indicating anti-rTsSP1.1 Antibodies can prevent larvae from invading IECs.6.Animal experiments to verify the effect of anti-rTsSP1.1 antibody on larvae invasion into intestinal mucosa and worm development5 days after challenge infection,the anti-rTsSP1.1 serum and the infection serum groups had 26.08%and 46.06%of adult worms reduction rates,respectively（P<0.01）,but the difference between male and female worms in each group was not statistically significant（P>0.05）,suggesting that the anti-rTsSP1.1 antibody blocked the TsSP1.1 part of the worm body to prevent the larvae from invading the intestinal mucosa.Conclusion1.Constructed TsSP1.1 recombinant expression plasmid p QE-80L/TsSP1.1 and expressed and purified rTsSP1.1.2.TsSP1.1 is expressed in different developmental stages of Trichinella spiralis,and is mainly located in the cortex,stichosome and embryos of female adult worms.3.rTsSP1.1 has the enzymatic activity of natural serine protease,which can specifically bind with IECs,and has obvious promotion effect on the invasion of Trichinella spiralis larvae into IECs and intestinal mucosa.4.TsSP1.1 is a protein related to Trichinella invading the host intestinal mucosa,and can be used as a candidate target antigen for the anti-Trichinella vaccine.