| Avian coccidiosis is caused by genus Eimeria parasitizing the epithelial cells of the intestinal mucosa of chickens.There are seven Eimeria species of chicken,among which E.necatrix is one of the most pathogenic coccidia,mainly damages eight to eighteen-week-old chickens,and causes severe morbidity and mortality.Eimeria is a single-host parasite,and its development undergoes three stages of sporogony,schizogony and gametogony.The sporozoites and merozoites are two key stages to invade intestinal epithelial cells.Surface antigen（SAG）is a kind of cysteine-rich membrane proteins which are expressed on the surface of parasites by the SAG gene family of apicomplexan in the invasion stage.SAG is anchored on the surface of parasites through its carboxyl-terminal glycophosphatidylinositol（GPI）,and is a good target for the innate and acquired immune responses of the host and plays an important role in mediating the interaction between the parasite and the host immune system.To date,there are no reports on the SAG gene of E.necatrix and its protein function.In our previous study,a total of 53 SAG gene from 68 differentially expressed genes that involve in cellular invasion were identified by the transcriptome and proteome correlation analysis of E.necatrix unsporulated oocysts（UO）,sporozoites（SZ）and second-generation merozoites（MZ-2）.In the present study,two E.necatrix SAG genes were cloned,expressed and immunolocalization analysed,the preparation of monoclonal antibody were studied,and the immune protection of the recombinant EnSAG proteins were evaluated.Our results may lay the foundation for the research of coccidia subunit vaccine and further elucidate the role of SAG in coccidia invasion.1.Cloning,expression and immunolocalization analysis of EnSAG30 and EnSA G-CAP gene from E.necatrixThe gene EnSAG30 and EnSAG-CAP were cloned from the total RNA of MZ-2 of E.necatrix（Yangzhou strain）by RT-PCR,and inserted to pGEM-T-Easy vector by TA cloning.After sequencing analysis,EnSAG30 cDNA and EnSAG-CAP cDNA were subcloned to pET-28a（+）vector to obtain a recombinant prokaryotic plasmid.After transformed into E.coli BL21,the recombinant plasmid pET28a（+）-EnSAG30 and pET28a（+）-EnSAG-CAP were induced to express by IPTG,which were identified and purified.BALB/c mice were immunized with purified recombinant protein and the anti-rEnSAG30 and anti-rEnSAG-CAP polyclonal antibodies were prepared,which were used to detect the native protein and their localization in SZ and MZ-2 of E.necatrix by Western blot and indirect immunofluorescence assay,respectively.Finally,the transcriptional level of EnSAG30 and EnSAG-CAP in SZ and MZ-2 of E.necatrix were analyzed by qRT-PCR.The results showed that the EnSAG30 gene was 768 bp,coding 255 amino acids.It was predicated that the first 23 amino acids formed a signal peptide;EnSAG30 had a molecular weight of 26.99 kDa and no transmembrane region,and its isoelectric point（PI）was 4.83.Genetic evolution analysis showed that the EnSAG30 was in the same branch as E.tenella EtSAG30,and belonged to the SAGA sub-family;The EnSAG-CAP gene was 813 bp,coding 270 amino acids.It was predicated that the first 25 amino acids formed a signal peptide;EnSAG-CAP protein had a molecular weight of 28.86 kDa and two transmembrane regions at the N-terminal and C-terminal,and its isoelectric point（PI）was 6.7.Genetic evolution analysis showed that the EnSAG-CAP belonged to the SAGA sub-family,Conserved domain analysis showed that the EnSAG-CAP of E.necatrix contained CAP domain.The recombinant protein rEnSAG30 was about 30 kDa and predominately expressed in inclusion body,the recombinant protein rEnSAG-CAP was about 32 kDa and predominately expressed in the solubility.Western blot analysis indicated that the recombinant protein rEnSAG30 and rEnSAG-CAP could be specifically recognized by mouse anti-His monoclonal antibodies and the convalescent serum of chicken infected with E.necatrix,but not with E.tenella,E.acervulina or E.maxima.Native protein EnSAG30 was detected in MZ-2 and had a molecular weight of 27 kDa.Native protein EnSAG-CAP was detected in MZ-2 and had a molecular weight of 17 kDa.EnSAG30 protein were located in the membrane of MZ-2.The transcription level of EnSAG30 in MZ-2 was extremely significantly higher than that in SZ（P<0.01）.EnSAG-CAP protein were located in the membrane of SZ and MZ-2.The transcription level of EnSAG-CAP in MZ-2 was significantly higher than that in SZ（P<0.05）.2.Preparation and preliminary application of monoclonal antibodies against recombinant proteins rEnSAG30 and rEnSAG-CAPTo prepare monoclonal antibodies（McAbs）against the surface antigen EnSAG30 and EnSAG-CAP of E.necatrix,BALB/c mice were immunized with purified recombinant proteins rEnSAG30 and rEnSAG-CAP.After three times of immunization,the spleen cells of the immunized mice were fused with myeloma cells SP2/0.Hybridoma culture supernatant were examined by the pre-established ELISA assay.Three positive hybridoma cells stably secreting specific monoclonal antibodies were obtained after four serial subclones by limiting dilution method and continuous passage of positive hybridoma cells.Monoclonal antibodies against rEnSAG30 prokaryotic protein was named 2E12,and Monoclonal antibodies against rEnSAG-CAP prokaryotic protein were named 2H12 and 4C6.The subclasses and specificities of McAbs were identified and the recognition of McAbs to natural proteins and the localization of McAbs in E.necatrix were studied.The results showed that the subclasses of 2E12 and 4C6 were IgG1,the subclasses of 2H12 was IgG2a.The titer of purified ascites were 1:204800.The McAb 2E12 could recognize rEnSAG30 and the native protein of SZ-2.The McAb 2H12 and 4C6 could recognize rEnSAG-CAP and the native protein of SZ-2.The indirect immunofluorescence assay with McAb 2E12 showed that EnSAG30 was located in the MZ-2 of E.necatrix.The indirect immunofluorescence assay with McAbs 2H12 and 4C6 showed that EnSAG-CAP were located in the SZ and MZ-2 of E.necatrix,respectively.3.The immune protective effects of recombinant antigen rEnSAG30 and rEnSAG-CAPThe chickens were immunized with recombinant rEnSAG30 or rEnSAG-CAP at three doses（200 μg,100 μg,50 μg）,respectively.At the same time,the two groups,unimmunized and challenged（UC）,and unimmunized and unchallenged（UU）,were used as controls.The immune protective effects were evaluated based on the survival rate,bloody stool,lesion score,weight gain（WG）,relative weight gain（RWG）,oocyst reduction（OR）,anti-coccidial index（ACI）and serum antibody levels（SAL）.The results showed that the survival rate of all groups were 100%,the numbers of bloody stools in the immunized groups were less than those in the UC group,the lesion score of the immunized groups were significantly lower than those of UC group（P<0.05）,the WG and the SAL of the immunized groups were significantly higher than those of UC group（P<0.05）.The RWG of rEnSAG-CAP group with middle dosage were the highest（90.49%）,followed by rEnSAG30 group with low dosage（86.19%）;The OR of rEnSAG30 group with high dosage were the highest（61.54%）,followed by rEnSAG-CAP group with high dosage（51.76%）;The ACI of rEnSAG-CAP group with high dosage were the highest（164.99）,followed by rEnSAG30 group with high dosage（160.03）.The results showed that both recombinant protein rEnSAG30 and rEnSAG-CAP could produce certain immune protection against E.necatrix.Conclusion:EnSAG30 and EnSAG-CAP genes were cloned and expressed;EnSAG30 and EnSAG-CAP genes were differentially expressed in SZ and MZ-2;Native protein EnSAG30 were located in the membrane of MZ-2 by the anti-rEnSAG30 polyclonal antibody and McAb 2E12;Native protein EnSAG-CAP were located in the membrane of SZ and MZ-2 by the anti-rEnSAG-CAP polyclonal antibody and McAb 2H12 and McAb 4C6;The recombinant proteins rEnSAG30 and rEnSAG-CAP have an immune protective effect against E.necatrix. |
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