Recombinant Expression Of Eimeria Necatix EnApiAP2 Protein And Its Combined Immune Protective Effect With REtGAM

Chicken coccidiosis caused by Eimeria is one of the most common parasitic diseases in intensive chicken farms,which causes an annual economic loss of 30 million US dollars to the global chicken industry.ApiAP2（Apicomplexan AP2）protein is a kind of specific transcription factor of Apicomplexa.Previous studies have found that the ApiAP2 protein of Plasmodium spp.can bind to DNA sequences containing specific motifs to regulate the growth and development of Plasmodium parasites,which has been the target for drug development and vaccine design.However,there is no report on the study about ApiAP2 protein of Eimeria.Our research group sequenced the transcriptome of the second generation merozoites（MZ-2）,third generation merozoites（MZ-3）and gametocytes of E.necatrix,and obtained five mRNA sequences encoding ApiAP2 hypothetical protein.In this paper,one of the gene sequences was cloned and expressed.Next,the location of natural protein in the merozoites（MZ）was analyzed.Finally,chickens were immunized with recombinant protein rEnApiAP2 alone or co-immunized with rEtGAM（recombinant gametocyte protein）to evaluate the immune protective effect against E.necatrix or E.tenella.These research results laid a foundation for explaining the mechanism of ApiAP2 protein regulating the growth and development of coccidiosis and for the development of chicken coccidiosis subunit vaccines.1.Cloning,prokaryotic expression of EnApiAP2 gene and the localization analysis of EnApiAP2 proteinE.necatrix MZ-3 were isolated and purified,and the total RNA was extracted.Its cDNA was synthesized by reverse transcription and used to template.The sequence of EnApiAP2 was amplified with PCR,and was cloned into pMD18-T vector,and the positive clones were selected for sequencing.The gene was cloned into the pET28a（+）expression vector,and the recombinant plasmid was transformed into the expression strain BL21（DE3）.After identification of the expression vector by sequencing,the recombinant strain was induced by IPTG,and expression products were identified by SDS-PAGE and Western blot,respectively.The purified and renatured recombinant protein was used to immunize BALB/c mice to prepare mouse anti-rEnApiAP2 polyclonal antibody.With the polyclonal antibody,the native protein in MZ was identified with Western blot,and the location of native protein in MZ was detected with indirect immunofluorescence staining.At last,the transcription level of EnApiAP2 in MZ-2 and MZ-3 was analyzed by qRT-PCR.The results showed that the full length of EnApiAP2 gene is 1830 bp,which has 99.8%homology with the sequence of EnApiAP2 gene（XM₀13583028,1）in Genbank.EnApiAP2 gene encodes 610 amino acids,which the molecular weight is about 67.69 kDa.The software predicted that the EnApiAP2 protein contain an AP2 domain and 25 antigenic determinants.The recombinant protein is about 74 kDa,and mainly existed in the form of inclusion bodies,and could be specifically recognized by the 6×HIS tag monoclonal antibody,the mouse anti-recombinant protein polyclonal antibody and rehabilitation serum obtained from E.necatrix or E.tenella.The native protein was about 85 kDa in MZ-3 and localized in the nucleus of MZ-3.EnApiAP2 gene was up-regulated in MZ-3.2.Analysis of immune protective effect of recombinant protein rEnApiAP2 against E.necatrixFifty yellow feather chickens were divided into five groups:the group immunized with high dose（200μg）,the group immunized with medium dose（100 μg）,the group immunized with low dose（50 μg）,the group unimmunized and challenged（UC）,the group unimmunized and unchallenged（UU）.The immunized groups were immunized at 7 and 14 days old,respectively,and all groups except the UU group were challenged at 21 days old.The survival rate（SR）,bloody stools（BS）,weight gain（WG）,relative weight gain rate（RWG）,lesion score（LS）,oocyst reduction（OR）,anti-coccidial index（ACI）and serum antibody level（SAL）were used as indexes to evaluate the immune protective effect of rEnApiAP2 against E.necatrix.The results showed that the group immunized with with low dose（50 μg）had the best protective effect,and its ACI was the highest（155.25）,which was close to the moderate anti-coccidial level;Compared with the UC group,in the immunized groups the LS was significantly lower,and the WG was significantly higher（P<0.05）,and oocyst reductions were reduced.The level of SAL in the immunized groups were significantly higher than that in the UC group and UU group（P<0.05）.3.Comparison of immune protective effect of three recombinant gametocyte proteins rEtGAMThe yellow feather broilers were used as experimental animals.The recombinant gametocyte proteins rEtGAM22,rEtGAM56 and rEtGAM59 were used to immunize chickens alone or combination in pairs.The indexes,same as in Chapter 2,were used to evaluate the immune protective effect against E.tenella or E.necatrix.The results showed that among the groups infected with E.tenella,in the group co-immunized with EtGAM22 and rEtGAM59 the RWG and the ACI was the highest（86.71%,163.71）,in the rEtGAM59 group the LS was the lowest and the OR was the highest（59.61%）.Among the groups infected with E.necatrix,the RWG in the group co-immunized with rEtGAM22 and rEtGAM59 was the highest（83.06%）,the OR in the rEtGAM56 group was the highest（66.97%）,in the group co-immunized with rEtGAM56 and rEtGAM59 the LC was the lowest and the ACI was the highest（158.23）.The SAL of all immunized groups was significantly higher than that of the control group（P<0.05）.The SAL in the rEtGAM56 group was highest,and significantly higher than that in all other immunized groups（P<0.05）.4.Combined immune protective effect of recombinant protein rEnApiAP2 and rEtGAMThe yellow feather broilers were used as experimental animals.The rEnApiAP2,rEtGAM56 and rEtGAM59 were used to immunize chickens alone or combination in pairs.The indexes,same as in Chapte 2,were used to evaluate the immune protective effect against E.tenella or E.necatrix.The results showed that among the groups infected with E.tenella,the RWG（84.88%）,the OR（51.16%）and the ACI（151.88）in the rEtGAM59 group was the highest respectively,but the LS in the group co-immunized with rEnApiAP2 and rEtGAM59 was the lowest.Among the groups infected with E.necatrix,the RWG in the rEtGAM59 group was the highest（91.26%）.The LC in the rEtGAM56 group was the lowest,and the OR was the highest（64.36%）.The ACI in the group co-immunized with rEnApiAP2 and rEtGAM56 was the highest（169.83）.The SAL of all immunized groups was significantly higher than that of the control group（P<0.05）.The SAL in the rEtGAM56 group was the highest,and significantly higher than in all other immunized groups（P<0.05）.In conclusion,EnApiAP2 gene was cloned and prokaryotic expressed,and the EnApiAP2 protein was located in the nucleus of MZ-3.The rEnApiAP2 has an immune protective effect against E.necatrix.The rEnApiAP2 combined with rEtGAM56 can enhance the immune protective effect against E.necatrix.The rEtGAM59 alone or co-immunization with rEtGAM22 had a good protective effect against E.tenella,and rEtGAM56 alone or co-immunization with rEtGAM59 had a good protective effect against E.necatrix.