Characterization Of Two Trichinella Spiralis Adult-specific DNase ? And Their Capacity To Induce Protective Immunity

Trichinellosis is a worldwide foodborne parasitic zoonosis caused by eating unprocessed or undercooked meat containing infective Trichinella larvae.Human trichinellosis is mainly caused by Trichinella spiralis,and pork and its products are the main source of this infection.Trichinellosis also has a great hazard on human health,meat productions and food safety.Therefore,development of vaccine is needed to interrupt the Trichinella transmission from animals to humans.In our previous studies,T.spiralis adult-specific DNase II-1?TsDNase ?-1,GenBank:AAY32316.1?/TsDNase ?-7?GenBank:AAY32322.1?were identified from AW excretory–secretory?ES?with early infected serum by Western blot and shotgun LC/MS-MS.Deoxyribonuclease II?DNase II?is a ubiquitous endonuclease that exerts its biochemical function by hydrolyzing phosphodiester bond to degrade DNA,and play an important role in evading host immune defense and pathogen invasion.As the DNA vaccine with a eukaryotic expression vector,the expressed protein is folded and modified more correctly and the structure is more similar to the native protein.With attenuated Salmonella typhimurium as a vector,oral immunization can more effectively induce a mucosal immune response,and is more conducive to killing and excreting parasites in the gut of host.In this study,rTsDNase ?-1 and rTsDNase ?-7 were cloned and purified,then immune serum was obtained.Transcription,expression and localization of TsDNase ?-1 and TsDNase ?-7 in different developmental stages of Trichinella spiralis were identified by RT-PCR,Western blot and indirect immunofluorescence assay?IIFT?;rTsDNase ?-1/7 immune serum inhibited IIL invasion into intestinal epithelial cells?IEC?monolayer and anti-rTsDNase ?-1/7specific antibody could mediated the killing of macrophages against Trichinella spiralis newborn larvae?NBL?;The oral DNA vaccines of TsDNase ?-1 and TsDNase ?-7 delivered by attenuated Salmonella were constructed respectively,and the immune responses and protection against challenge infection of mice,which immunized with rTsDNase ?-1/7 and oral DNA vaccines,were observed.The protective immunization induced by TsDNase ?-1 and TsDNase ?-7 confirmed that TsDNase ? were candidate target proteins for anti-Trichinella vaccines.Materials and Methods1.Experimental animals,Trichinella species,bacterial strains and experimental cellsThe experimental animals used in this study were 6-week-old SPF female BALB/c mice which were purchased from Henan Provincial Laboratory Animal Center.Trichinella spiralis?T1,ISS534?were kept by passage in mice every 6 months.Attenuated Salmonella typhimurium?cyaSL1344 strain was obtained from Key Lab of Animal Disease and Public Health,College of Animal Science and Technology,Henan University of Science and Technology.The mouse IEC used in the experiment were isolated by our group;the baby hamster kidney cell?BHK-21?was from the Cell Resource Center of Henan CDC.2.Sequence analysis and identification of TsDNase ?-1 and TsDNase ?-7Sequence analysis of the domain,physicochemical properties and antigenic sites of TsDNase ?-1 and TsDNase ?-7 using online software such as NCBI,ExPasy and BepiPred.Prokaryotic expression plasmids of TsDNase ?-1 and TsDNase ?-7 gene from 3d adult?AW?cDNA were constructed and the recombinant proteins were expressed and purified.The transcription,expression and localization of TsDNase ?-1/7 in Trichinella spiralis muscle larvae?ML?,intestinal infective larvae?IIL?,AW and NBL were analyzed by Real-time PCR,Western blotting and IIFT.The effects of TsDNase ?-specific antibodies during Trichinella invasion were observed by in vitro invasion of IEC monolayer and ADCC assay.3.Immune response induced by rTsDNase ?-1 and rTsDNase ?-7 immunizationThe purified rTsDNase ?-1,rTsDNase ?-7 and rTsDNase ?-1/7 mixed proteins,adjuvant and PBS were used to immunize BALB/c mice respectively.At 0,10,20 and 30 days after first immunization,mouse serum were collected from tail vein blood,then IgG,IgG1 and IgG2a levels induced were detected by indirect ELISA.4.Construction and identification of TsDNase ?-1 and TsDNase ?-7 DNA vaccineThe eukaryotic recombinant plasmids pcDNA3.1-TsDNase ?-1 and pcDNA3.1-TsDNase ?-7 were constructed and transferred into BHK-21 cells using Lipo2000.The in vitro transcription and expression of the plasmids were identified by RT-PCR and IIFT.The recombinant plasmids were transformed into attenuated Salmonella by electroporation.One week after the mice were orally immunized,spleens and mesenteric lymph nodes?MLN?were collected.Then the transcription and expression of the plasmids in immunized mice were identified by RT-PCR and IIFT.5.Immune response induced by TsDNase ?-1 and TsDNase ?-7 DNA vaccine immunizationSerum,intestinal lavage fluid,spleen cells and MLN cells were collected at 0,10,20 and30 days after first immunization.The levels of antibody IgG,IgG1,IgG2a in serum,and total IgA and specific IgA levels in intestinal fluid were measured by ELISA.The spleen cells and MLN cells were stimulated with rTsDNase ?-1 and rTsDNase ?-7 for 72h,and the supernatant was collected.The cytokines?IFN-?,IL-4,IL-10?productions were detected by sandwich ELISA.In addition,TsDNase ?-1/7 on the surface of worms at different development stages were detected by IIFT by using DNA vaccine immunization serum and intestinal lavage fluid as the primary antibody.6.Immune protection of rTsDNase ? and DNA vaccineAfter mice were immunized with subcutaneous injection of rTsDNase ? and oral DNA vaccine,they were challenged with 300 Trichinella spiralis muscle larvae at 10 days after the3^rd immunization.The mice were sacrificed 5 days after the infection,and the adult worms in the intestine were collected and the reduction of adult worm was calculated;while ML were collected 42 days after infection and calculated the reduction of muscle larvae burden to observe the immune protection.7.Statistical analysisThe data was analyzed using SPSS version 19.0 software.The analysis of intragroup or intergroup difference was performed using One-way ANOVA,Student's t-test or chi-square test.All data of OD values,cytokine production,and worm recovery was shown as the mean±standard deviation?SD?.P<0.05 was justified as statistically significant.Results1.Sequence analysis and identification of TsDNase ?-1 and TsDNase ?-7The sequencing results showed that the complete cDNA sequences of TsDNase ?-1 and TsDNase ?-7 were 1221 and 1161 bp,the predicted ORF encoded 347 and 348 amino acids,molecular weights were 38.06 and 38.22 kDa,and pI were 7.58 and 7.56 respectively.Western blot analysis showed that the natural TsDNase ?-1 or TsDNase ?-7 in IIL,AW,NBL crude extracts and AW ES proteins could be recognized by the anti-rTsDNase ?-1 and rTsDNase ?-7 serum.Real-time PCR results TsDNase ?-1 and TsDNase ?-7 were transcribed during all the developmental stages of Trichinella spiralis;IIFT results showed TsDNase ?-1 and TsDNase ?-7 were expressed on the surface of AW and NBL,and mainly located in the cuticle and stichosome of the worm tissue sections.The in vitro invasion of IIL with rTsDNase ?-1 and rTsDNase ?-7 immunized serum was 40.13%and 32.65%,which was lower than pre-immune serum?4.01%,?₁²=105.012,?₂²=70.118,P<0.01?.The results of ADCC assay shows mortality of NBL in rTsDNase ?-1?66.84%?and rTsDNase ?-7?63.39%?immune sera were significantly higher than that of pre-immune serum?15.30%,t₁=26.420,t₂=22.599,P<0.01?,and the cytotoxicity had relationship with the dilution of anti-rTsDNase ?-1 and anti-rTsDNase ?-7 antibody?F₁=69.569,F₂=68.231,P<0.01?.2.Immune response induced by rTsDNase ?-1 and rTsDNase ?-7 immunizationHigh titer antibodies were induced by the immunization of rTsDNase ?-1,rTsDNase ?-7and mixed protein,and IgG1 levels in serum rTsDNase ?-1?t20d=13.318,t30d=56.608,P<0.01?,rTsDNase ?-7(t_20d=17,507,t_30d=22.466,P<0.01)and mixed proteins(t_20d=7.821,t30d=24.942,P<0.01)immunized mice were significantly higher than IgG2a levels at 20 and30 days after first immunization,indicating that these immunizations induced Th1/Th2 mixed type immune response with Th2 dominant.3.Construction and identification of TsDNase ?-1 and TsDNase ?-7 DNA vaccineThe results of restriction enzyme digestion and sequencing confirmed that?cyaSL1344/pcDNA3.1-TsDNase ?-1 and?cyaSL1344/pcDNA3.1-TsDNase ?-7 DNA vaccines were successfully constructed.After transfection into BHK-21 cells in vitro,Real-time PCR and IIFT assay showed that TsDNase ?-1 and TsDNase ?-7 genes were transcripted and proteins were expressed in the transfected cells.RT-PCR and IIFT experiments were also performed on the spleen and MLN of mice immunized with DNA vaccine at one week after first immunization.The results showed that the TsDNase ?-1 and TsDNase ?-7 were successfully transcribed and expressed in the spleen and MLN.4.Immune response induced by TsDNase ?-1 and TsDNase ?-7 DNA vaccine immunizationAfter the second and third immunizations of?cyaSL1344/pcDNA3.1-TsDNase ?-1 and?cyaSL1344/pcDNA3.1-TsDNase ?-7,the serum IgG levels of the mice were significantly increased,and the IgG1 levels were significantly higher than IgG2a levels?P<0.01?;total IgA and specific IgA levels in intestinal lavage fluid also increased significantly.At 10,20 and 30days after first immunization,the spleen cells and MLN cells of the mice were stimulated by recombinant proteins,the IFN-?,IL-4 and IL-10 production were significantly higher than the empty plasmid and PBS group?P<0.01?.It indicated that TsDNase ?-1 and TsDNase ?-7DNA vaccines induced a Th1/Th2 immune response and mucosal immune responses.5.Immune protection of recombinant protein and DNA vaccineThe reduction of adult worm from rTsDNase ?-1 immunized group was 40.36%,which was higher than that of rTsDNase ?-7 immunized group?34.86%,t=2.480,P<0.05?,while the reduction of larval burden was 50.43%and 42.33%,respectively,which was no statistically difference?t=1.008,P>0.05?between two immunized groups.The reduction of adult worms from TsDNase ?-1 DNA vaccine group was 53.85%,which was higher than that of TsDNase ?-7 DNA vaccine group?46.15%,t=3.253,P<0.05?,while the reduction of larval burden was 59.26%and 53.41%,respectively,which was no statistically difference?t=1.685,P>0.05?between two immunized groups.The results indicate that both recombinant proteins and DNA vaccines can induce partial immune protection on mice.The reduction of adult worms in mice immunized with TsDNase ?-1 or TsDNase ?-7 DNA vaccine was significantly higher than that of the recombinant protein immunized group?t1=2.670,t2=2.812,P<0.05?;The reduction of larval burden in mice immunized with DNA vaccine was not statistically significant compared with the recombinant protein immunized group?t1=1.641,t2=1.429,P>0.05?.Conclusion1.Recombinant plasmids pQE-80L-TsDNase ?-1/7 had been constructed and rTsDNase ?-1/7 proteins were expressed successfully.TsDNase ?-1/7 are transcribed in different development stages of Trichinella,and expressed in all stages except ML,mainly located on the cuticle,stichosome and embryo.2.The?cyaSL1344/pcDNA3.1-TsDNase ?-1/7 DNA vaccines were successfully constructed. Both DNA vaccine-immunized mice induced Th1/Th2 mixed immune responses with Th2 dominant and specific mucosal immune responses.3.Oral immunized with TsDNase ?-1 and TsDNase ?-7 DNA vaccines,and subcutaneous injection of recombinant proteins can produce partial immune protection against Trichinella spiralis.There is no statistically difference in the reduction of muscle larvae burden among different immunized groups,and the reduction adult worm of TsDNase ?-1 DNA vaccine is higher than any other immunized group.4.TsDNase ? were Trichinella invasion related proteins and the potential target molecules for anti-Trichinella vaccine.