| Background:Ticks are second only to mosquitoes as vectors of human and animal pathogens.The tick infection rate of grazing herbivorous livestock can reach 100%,it can easily cause tick-borne infection(e.g.piroplasmosis),These have seriously hinder the healthy development of regional economy and aquaculture.The ticks Hyalomma asiaticum(H.as)and Hyalomma anatolicum(H.an)are the widespread tick species,having major medical and veterinary significance in Eurasia and North Africa.Beside their direct pathogenic effects(skin lesions,inflammation),H.as and H.an are vectors of important diseases of livestock and in some instances of zoonoses.In search of ways to address the increasing incidence of global acaricide resistance,tick control through vaccination is regarded as a sustainable alternative approach.This has been the focus of academic attention.Anti-tick vaccine candidate antigen Cathepsin L-like cysteine protease(CPL),is a potent hemoglobinase,and plays important roles in the digestion of blood acquired from a host.Furthermore,interferon-gamma(IFN-?),is an immunomodulatory factor that plays an important role in the regulation of adaptive immunity against infection.Objective:we first used IFN-?as an adjuvant of anti-tick vaccine,with recombinant H.as CPL(r Has CPL)immune promotes the protective immunity against H.as and H.an infestation.The main research Methods and Results are as follows:(1)Studies of biological characterization on H.as and H.anIn this study,we collected hard ticks from Qitai(n=317)and Turpan(n=291).We identified H.as and H.an by morphological and molecular biology methods,respectively.We systematically observed and studied the biological characteristics of H.as and H.an at various developmental stages of their life cycles.We found that phylogenetic analysis indicated that the COI sequences of H.as and H.an in Xinjiang belong to the same cluster(homology>99.00%)with a sequence from Kazakhstan and Gansu.Morphological comparison found that there was no significant difference between H.as and H.an from eggs,larvae and nymphs,the key points of identification between adult ticks were paraproct/hypopygium and spiracular plates;The period comparison found that the appear time of“cicatricle”(later development into anus)in H.as(day19)later than H.an(day 14)in the process of egg development,the oviposition and egg hatching cycles of H.as(27.5 days and 31.5 days)longer than H.an(12.0 days and 20.5 days),and the period of engorge nymph molting and adults blooding cycles of H.as(19.0 days and 8.5 days)less than H.an(30.0 days and 13.0days).the life cycles of H.as and H.an were 131.5 and 112.5 days,respectively.(2)H.as Has CPL was cloned,expressed,and identifitionThe recombinant plasmid Has CPL/p ET-28a was successfully constructed using prokaryotic expression system,and the Has CPL was successfully expressed in E.coli,where the purified recombinant Has CPL with His-tag(approximately 18.07 k Da,0.9-1.3 mg/m L),Sera of the animals immunized with r Has CPL recognized Has CPL antigens detected by Western blot,this result indicate that Has CPL has good reactogenicity;the recombinant plasmid Has CPL/p EGFP-C3 was successfully constructed using eukaryotic expression system,the Has CPL was successfully expressed in BHK-21 cells,IFAT results indicated that the sera of immune group have anti-Has CPL antigen-specific Ig G antibodies that could detect the Has CPL-fluorescence signal in eukaryotic cells,this results indicate that Has CPL has good immunogenicity.(3)BALB/c mouse IFN-?was cloned,expressed,and identifitionThe recombinant plasmid(Mus-IFN-?/p ET-28a)of BALB/c mouse IFN-?gene was constructed by prokaryotic expression system,and the Mus-IFN-?was successfully expressed in E.coli,where the purified recombinant Mus-IFN-?with His-tag(approximately 18.51 k Da,0.5-0.8 mg/m L).The anti-His-tag antibody was used to identify r Mus-IFN-?by western blot demonstrating that anti-His-tag antibody could recognize the r Mus-IFN-?,and verified by silico analysis and IFN-?-ELISA kit test.These proteins were provided the materials for subsequent experiments.(4)The immune effect analysis of the recombinant Has CPL for against H.as tickSix rabbits were divided into two groups.The first group of three rabbits were immunized with r Has CPL and the control group of three rabbits were injected with PBS alone.All rabbits were immunized 3 times at an interval of 2 weeks.H.as adult and larva infestation were conducted after immunization.Immune efficacy was evaluated according to the levels of antibodies and cytokines in serum of rabbits,the number of engorged ticks,mass,and larval molting rate from Immune/Control rabbits respectively.We found that the r Has CPL inducing humoral(titer:102 400-409 600)and T-cell responses(elevated cytokine levels:IL-4?IL-10 and IFN-?)in the immunized group;the anti-Has CPL sera collected from the rabbits of immune group that could detect native CPL in tick proteins(MG;SG;Ovar);for the infesting ticks,the female ticks that fed on r Has CPL(429±21.44 mg)-immunized rabbits had a significantly lower engorgement weight compared to those ticks that fed on control group(789.86±38.28 mg)(P<0.05);the larva that fed on r Has CPL-immunized rabbits had a significantly lower engorged and molting rate(44.40%and 39.01%)compared to those ticks that fed on control group(70.13%and 54.99%)(P<0.05),respectively.The protection rate of r Has CPL against females and larva H.as infestation were 54.05%and 55.09%,respectively.(5)Prediction and validation of cross-protective antigen Has CPL and its the immune effect analysis for against H.an tickThe silico analysis of two CPLs(Has CPL and Han CPL)were investigated,including protein sequence similarity,predicted B-cell epitopes,antigenicity,hydrophobicity,isoelectric point,and temperature factors.The two proteins were found to have very similar structure and high homology(90.48%).The native CPL protein in the extracts of ticks'different developmental stages were verified for cross reactivity with sera against Has CPL by Dot blot,ELISA,and IHC.Finally,a cross protection experiment was conducted according to the immune method in(4),we found that the number of engorge ticks and egg weight in the immune groups were significantly lower than control groups,and the inhibition rates were 21.43%(P<0.05)and 38.84%(P<0.001),respectively.A 54.76%protection of H.an indicate that Has CPL protein may induce reactive antibody responses capable of cross-protecting against infestations by H.an.(6)Recombinant IFN-?promotes immune response to Has CPL and its the efficacy of anti-tickAll BALB/c mice were divided into three groups.The first group of mice were immunized with r Has CPL and r Mus-IFN-?(CPL+IFN-?),and the two other group of mice were injected with r Has CPL(CPL)or PBS alone.The immune pathway is divided into two methods:subcutaneous injection and intraperitoneal injection.BALB/c mice were immunized three times before challenged with larvae(H.as and H.an),the anti-tick effects were analyzed according to the number of engorged ticks,and molting rate.We found that using r Mus-IFN-?as an adjuvant to r Has CPL vaccine promoted specific antibody Ig G(Ig G1>Ig G2a)(P<0.001)and increased production(P<0.001)of IFN-?and IL-4.The percentages of T cell subsets CD4~+and CD8~+in the spleen of immunized mice were evaluated using flow cytometry after vaccination,CD4~+T cell levels in CPL+IFN-?(35.19%)was significantly higher than that in CPL groups(30.87%)(P<0.05),compared with PBS group(27.40%;6.24%).For all of immune groups,CPL+IFN-?reduced the number of larva and molting rate could engorge compared to the CPL or PBS,respectively.The effect of intraperitoneal immunity is better than that of subcutaneous immunity.In the intraperitoneal immune experiment,the protected rate of immunized mice from H.as challenge were significantly higher after immunization with CPL+IFN-?(85.11%)than with CPL(63.28%);In the intraperitoneal immune experiment,the protected rate of immunized mice from H.as and H.an challenge were significantly higher after immunization with CPL+IFN-?(96.20%,83.41%)than with CPL(62.46%,61.33%).Conclusion:In the study,the biological characteristics and morphological characterization on H.as and H.an at different stages were studied by establishing a laboratory feeding,and the key points of identification between H.as and H.an are“cicatricle”occurrence time,platformparaproct/hypopygium,and spiracular plates;Has CPL and Mus-IFN-?were cloned and expression with high efficiency;Has CPL was proved to be a potential cross anti-tick candidate antigen,and immunization with Has CPL can also effectively against H.as and H.an;in BALB/c mice co-immunized with IFN-?and Has CPL,not only the immune response was enhanced,but also the anti-tick effect was enhanced.Has CPL was expected to be used as a effective antigen of anti-tick subunit vaccine. |
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