| Chicken coccidiosis is a parasitic disease caused by Eimeria spp.（Apicomplexa）parasitizing in the intestinal epithelial cells of chickens.There are seven Eimeria species that infect chickens,and E.necatrix is the one of most virulent species pathogenic to chickens,which is the most harmful to 8 to 18 weeks old chickens causing mass morbidity and mortality.Eimeria have a complex life cycle that includes three developmental stages:sporogony,schizogony and gametogony.The sporozoites from sporogony and merozoites from schizogony are the invasion stage of Eimeria parasites.Therefore,studies on invasion-related proteins and their roles will help to discover therapeutic drug targets or to screen vaccine candidate antigens against coccidiosis,and may provide new strategies for the prevention and control of coccidiosis.Previous studies have showed that heat shock proteins（Hsps）are involved in the invasion of Apicomplexan protozoa including E.tenella,Toxoplasma gondii and Babesia bovis.However,studies on the Hsp of E.necatrix have not been reported.In our previous study,a total of 68 differentially expressed genes associated with invasion was found in the transcriptome and proteome correlation analysis of different developmental stage of E.necatrix.The Hsp gene（XM013582099.1）was one of them.In the present study,EnsHsp gene was cloned and expressed,and its protein localized in parasites.The recombinant protein rEnsHsp20 was used to immunize chickens to evaluate the immunoprotective effect against E.necatrix infection.The efficiency of anti-rEnsHsp20 antibody inhibiting sporozoite invasion host cell was evaluated in vitro.1.Cloning,expression and identification of the E.necatrix EnsHsp20 geneThe sporozoites of E.necatrix were isolated and purified to extract the total RNA to amplify the gene（EnsHsp20）of E.necatrix by RT-PCR.After being inserted into pGEM-T Easy vector and sequence analysis,EnsHsp20 was subcloned into pET-28a（+）vector and transformed into Escherichia coli BL21（DE3）.Following from the double enzyme digestion identification and sequence analysis,the positive recombinant E.coli was induced to express successfully.In order to prepare the anti-rEnsHsp20 polyclonal antibodies,the recombinant protein was identified by Western-blot then purified,renatured and immunized to BALB/c mice.The results demonstrated that the target gene was 549 bp,coding 183 amino acids with a predicated molecular weight of 20.3 kDa.The protein formed an alpha-crystallin domain（ACD）.The recombinant protein mainly expressed in inclusion body,and could be specifically recognized by anti-6×His tag mouse monoclonal antibodies and the convalescent serum of chicken infected with E.necatrix,E.tenella,E.cervuline and E.maxima,respectively.2.The subcellular localization of EnsHsp20 protein and its transcription levels in different developmental stagesWith the anti-rEnsHsp20 polyclonal antibodies as the primary antibody,the native EnsHsp20 protein in SZ and MZ-2 of E.necatrix were detected by Western-blot while the subcellular localization was detected by indirect immunofluorescence assay.The total RNA of MZ-2,UO and SZ of E.necatrix were extracted respectively to analyze the transcriptional level of EnsHsp20 by qRT-PCR.The result showed that the native protein of EnsHsp20 can be detected in MZ-2 with a molecular weight about 36 kDa,and localized in the cytomembrane or cytoplasm of SZ and MZ-2.The transcription level of EnsHsp20 gene was significantly higher in SZ than that in MZ-2 and in UO（P<0.01）.3.The effect of rEnsHsp20 polyclonal antibody to sporozoites invasion to MDBK cells in vitroThe recombinant protein rEnsHsp20 polyclonal antibody and E.necatrix SZ was collected and purified,respectively.With MDBK cells as the host cells to design the invasion model of MDBK cell by E.necatrix sporozoites.The invasion rate peaked at the optimal inoculation time and dosage of 6 h and 4×105.After 4×105 SZ incubating different concentrations of rEnsHsp20 polyclonal antibody（100 μg/mL,200μg/mL,300 μg/mL,400μg/mL and 500 μg/mL）for 2 h in 37℃,the SZ were inoculated to MDBK cells for 6 h to calculate the invasion rates.The mouse IgG groups with the corresponding concentrations and serum-free incubation group（the concentration of antibody is 0）were also set as the control agents to calculate the invasion inhibitory rates.The results showed that the invasion inhibitory rates of SZ rised with the increase of antibody concentration at 100～300μg/mL and reached the peak of 33.05%at 300 μg/mL.Then the invasion inhibition rate tended to be stable while the mouse IgG control groups was consistently blow 0.4.The analysis of immunoprotective effect of rEnsHsp20 against E.necatrixThe recombinant protein high dose（200μg）,medium dose（100 μg）and low dose（50 μg）immunization group were set and immunized at 7 d and 14 d,respectively.With unimmunized and challenged group（positive）and unimmunized and unchallenged group（negative）as controls,all groups except the negative group were challenged at 21 d.The immunoprotective effect of rEnsHsp20 was evaluated by anticoccidial index（ACI）with survival rate,number of blood stools,mean weight gain,relative weight gain rate,lesion score,oocyst reduction rate,humoral immunity level and the transcriptional level of IL-2、IL-4、IL-10 and IFN-y in chicken spleens.The results demonstrated that the 200 μg immunized group showed the best protective effect with its highest ACI of 161.60,reaching moderate anti-coccidial level.Serum antibody levels of chicken anti-rEnsHsp20 were significantly elevated and rEnsHsp20 promoted the expression of IL-2,IL-4,IL-10 and IFN-γ.Conclusion:The small heat shock protein gene（EnsHsp20）is present in E.necatrix genome,encoding a protein with a molecular weight of 36 kDa,approximately.The protein can be localized in the cytomembrane or cytoplasm of SZ and MZ-2,and showed differentially expressed at different developmental stages.EnsHsp20 may be involved in the invasion procedure of E.necatrix and has immunoprotective effect against E.necatrix infection.All these results lay the basis for elucidating the mechanism of Eimeria invasion and preparing chicken coccidiosis subunit vaccine. |
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