Study On Immune Protection And Subcellular Localization Of VPS29 Protein Of Eimeria Tenella

Eimeria tenella as one of the most pathogenic coccidiosis in chicken,has caused serious harm to the global aviculture industry every year.At present,the use of anticoccidial drugs is the most common measure to control chicken coccidiosis,but with the long-term use of anti-coccidial drugs,drug-resistant strains have been found continuously.On the other hand,live coccidial vaccines have the risk of causing chicken coccidiosis outbreaks.Therefore,the research of new anti-coccidial vaccines has become the focus of attention.As a kind of key protein in eukaryotic cells,VPS29 protein are involved in retrograde endosome-to-Golgi and endosome-to-Plasma membrane transport,as well as maintaining other physiological activities.It has been found in yeast cells,animal cells and plant cells,and there are few studies on Apicomplexa parasites.By studying the subcellular localization and immunological function of VPS29 protein of E.tenella,we can understand the distribution of VPS29 protein in E.tenella and determine whether VPS29 protein can be used as a candidate antigen for a new type of anti-coccidial vaccine.In this study,the sporozoite c DNA of E.tenella was used as a template,and primers were designed according to the VPS29 predicted sequence of E.tenella in Gen Bank.Then the VPS29 gene sequence was amplified by RT-PCR,predicted and analyzed by bioanalysis software.Expression vector VPS29-p ET32a(+)and overexpression location vector Et H4-VPS29-EYFP-Actin were constructed.The expression of VPS29 gene was induced by IPTG,and purified by Ni-NTA affinity column.In order to evaluate the protective effect of VPS29 protein,three groups were divided: immune group,positive group and negative group.Seven-day-old chicks were immunized subcutaneously with 100 μg VPS29 recombinant protein and Freund’s adjuvant at 1:1,followed by booster immunization with the same dose twice every 2 weeks.Challenge test was carried out after three times of immunization.The weight gain,death,oocyst output and other data were monitored during the challenge test period,with the cecal lesions observed after challenge.Finally,the protective effect of VPS29 recombinant protein was evaluated by comprehensive data analysis.In order to subcellular localization of VPS29,polyclonal antibodies were prepared by immunizing mice with recombinant VPS29 protein,and the sporozoite localization of E.tenella was carried out by immunofluorescence analysis.At the same time,the constructed expression vector Et H4-VPS29-EYFP-Actin was transferred into sporozoite by electro-transfection.After the sporozoite was inoculated with chicken,the transgenic strain of E.tenella was obtained,and the VPS29 was localized by yellow fluorescence.In this study,the homology between the PCR amplified VPS29 sequence and the predicted sequence is 100%.The sequence encodes 183 amino acids with many kinds of structures in the secondary structure,such as α helix,β sheet and rotation angle,and 9 antigenic determinants predicted.The VPS29 of E.tenella nucleotide sequence had 81.6% similarity with Eimeria mitis and 71.24% similarity with Toxoplasma gondii.In this study there was no chicken death.In terms of weight gain,the immune group was lower than the negative group with no difference significant(P> 0.05),however the the positive group was significantly lower than the negative group(P<0.05).In terms of cecal lesions,the pathological changes in the immune group were slighter than those in the positive group with no difference significant(P > 0.05).Finally,the immune group ACI is 144.2.The Western-blot and ELISA results of chicken serum showed that chickens immunized with VPS29 recombinant protein could produce specific antibodies.According to the results of subcellular localization,we found that VPS29 protein existed widely in the cytoplasm of sporozoite of E.tenella.As the results,the VPS29 sequence was conserved in the Apicomplexa parasites;the recombinant VPS29 protein could produce partially protective effect after immunization in chickens.It is revealed that VPS29 protein can not be used as an excellent vaccine candidate antigen;It was found VPS29 is widespread in E.tenella,which indicates that VPS29 protein may be an important functional protein in E.tenella,and its biological function needs to be further studied.