| Histomoniasis is a protozoiasis caused by Histomonas meleagridis(H.meleagridis),which can infect Galliformes birds.The main lesion features are cecal swelling,liver necrosis and sulfur-like feces.It is also known as infectious enterohepatitis.The disease mostly infects turkeys,but also prevalent in chickens,quail,keets and peacocks.The mortality rate of some chickens reaches 20-30%,which seriously affects the development of poultry industry.At present,there is no effective vaccine for the disease.Chemical drugs such as organic arsenic and nitroimidazole play important roles in the prevention and treatment of Histomoniasis.However,it has been banned for public safety and health considerations due to drug residue and environmental pollution,resulting in severe prevalence.Through differential proteome analysis in previous study,ME 1 was found highly expressed in virulent strains,and the expression level in attenuated strains was significantly lower than that in virulent strains.Therefore,in this study,ME 1 gene was cloned and expressed by genetic engineering technology,and the antigenicity of the protein encoded by the ME 1 gene was analyzed.The mouse polyclonal antibody and monoclonal antibody were prepared to analyze the distribution of ME 1 protein in H.meleagridis,and immunoprotective effect has been evaluated,aiming to lay a basis for the development of H.meleagridis subunit vaccine.1.Cloning,expression and enzyme activity identification of ME 1 of H.meleagridisPrimers were designed according to the ME 1 gene sequence in the differential proteome of H.meleagridis to amplified HmME 1 gene by RT-PCR from the cDNA of H.meleagridis.The target gene and pET28a(+)were digested with BamH I and Not I to constructthe recombinant plasmid pET28a(+)-HmME 1 which is transformed into BL21(DE3)to express induced by IPTG.Following from identifying by HIS tag monoclonal antibody,the enzyme activity of the recombinant protein was identified.The results showed that the HmME 1 gene(MW400608.1)was 1182 bp,coding 394 amino acids.The amino acid sequence analysis showed that the homology was 100%98.5%compared with ME 1 of H.meleagridis in GenBank.,and it was closely related to the ME of Trichomonas fetalis,with a homology of 81.9%.The recombinant protein mainly expressed in supernatant and the molecular weight was about 46kDa,which can be specificantlly recognized by HIS tag monoclonal antibody.The purified recombinant protein band was single,and the rHmME 1 protein had enzyme activity.2.Antigenicity and immunefluorescence localization analysis of recombinant protein rHmME 1 of H.meleagridisThe positive serum(with polyclonal antibody)was prepared by immunizing mice with purified and non-enzyme activity rHmME 1.The reactivity of rHmME lwas detected with mouse anit-rHmME 1 positive serum and chicken anitH.meleagridis positive serum by western-blot,and the reactivity of the native HmME 1 protein expressed in H.meleagridis with mouse anti-rHmME 1 positive serum is also detected.Monoclonal antibodies against rHmME 1 protein were prepared by cell fusion,positive hybridoma cell screening and subcloning with rHmME 1 as immunogen.The localization of rHmME 1 protein was analyzed by indirect immunefluorescence assay(IFA)with mouse polyclonal antibody and monoclonal antibody,respectively.The results showed that the rHmME 1 protein could be specifically recognized by both chicken anit-H.meleagridis positive serum and mouse anti-rHmME 1 positive serum.After three times of subcloning,six hybridoma cell lines stably secreting monoclonal antibodies were obtained,which were named as 3D12,1A6 and 1C10 classified as IgG1,and 3G12,1E12 and 4B5 classfied as IgG2b.The mouse anti-rHmME 1 positive serum and monoclonal antibody could both react with the native HmME 1 protein and rHmME 1 recombinant protein.The IFA results showed that HmME 1 was widely present in the cytoplasm of H.meleagridis,and the expression level in virulent strains was significantly higher than that in attenuated strains.3.Immunoprotective effect evaluation of recombinant protein rHmME 1 of H.meleagridisThe recombinant protein was mixed with adjuvant and then emulsified to prepare subunit vaccine.The recombinant protein was devided into three dose immunization groups of 200,100 and 50μg/per chicken,and unimmunized challenged group,unimmunized unchallenged group and adjuvant control group were set up,with 10 SPF chickens in each group.Each immunization group was immunized at 5 d and 12 d.Each challenged group was challenged at 19 d via cloaca with 3×105 H.meleagridis.Then put down all groups after 12 d.The immunoprotective effect of was analyzed by morbidity,mortality,average weight gain,lesion score,humoral and cellular immunity level.The results showed that within immunization groups,the morbidity of recombinant protein 100 μg group was the lowest.There was no death of chickens in each group.The average weight gain of the recombinant protein 100 μg group was significantly higher than unimmunized challenged group(p<0.05).The lesions in 100 μg immunization group were relatively mild while the cecal lesion score was significantly lower than the unimmunized challenged group(p<0.05).Both the 200 μg and 100 μg immunization group can stimulate the high level of antibody.IFN-γ and IL-2 of the 200 μg and t100μg immunization group were both increased significantly,and IL-4 showed a downward trend.Based on all above,the rHmME 1100μg group has the best immunoprotective effect.Conclusion:The HmME 1 gene was cloned and expressed,and the rHmME 1 recombinant protein was about 46 kDa.It was located in the cytoplasm of H.meleagridis.The expression level in the virulent strain was significantly higher than that in the attenuated strain.The recombinant protein rHmME 1 has a certain immunoprotective effect. |
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