Immunoprotective Analysis Of Gametocyte Antigen Genes Of Eimeria Necatrix

Chicken is an important economic species of poultry,characterized by fast growth and precocity with a short generation interval,giving customers an opportunity to access to a larger number of poultry products within a short period of time.Poultry products are important sources of protein throughout the world.Coccidiosis is an economically significant and most prevalent parasitic disease of livestock,particularly for chicken.This disease is worldwide in occurrence which not only hazards animal health,but also limits the development of the global chicken industry.Eimeria necatrix is one of the most pathogenic coccidial species in chickens.It may cause very severe intestinal coccidiosis in 8-18 weeks old chickens.In recent years,yellow-feathered broilers are widely raised and they are mostly raised on the ground or net,resulting the cases of intestinal coccidiosis caused by E.necatrix gradually increase.It is essential to carry out new research on the prevention and treatment of coccidiosis.It has been reported that the monoclonal antibody against the natural gametocyte antigen of Eimeria tenella can effectively inhibits the development of gametocyte to oocyst.It firstly confirmed antigenicity of the sexual lifecycle stage of Eimeria.In this study,we analyzed the oocysts production at different infectious doses of E.necatrix.At the same time,the gametocyte protein encoding gene of E.necatrix,Engam56 was cloned and expressed successfully.The immunoprotective experiment showed that recombinant protein rEnGAM56(rEnGAM56-F)could protect chickens against E.necatrix infection at some level.The truncated recombinant protein rEnGAM56(rEnGAM56-T)also protected chickens against E.necatrix infection,and showed better protection than rEnGAM56-F.Three recombinant proteins,including rEnGAM56-T,rEnGAM59 and rEnGAM22,were selected to construct combined immunization groups.As results indicated that,three proteins immunized group showed better protection against E.necatrix than two proteins or one protein immunized groups.These results will provide important idea to develop multivalent coccidial vaccine.1.The effect of different infectious doses on the oocysts production of E.necatrixIn order to study the effect of different infectious doses on the oocyst production of E.necatrix,four infectious dose groups of 500,2 500,5 000 and 10 000 sporulated oocysts were orally infected to 23-day-old chickens.Each group had 5 chickens.After the infection,the oocysts production was calculated with McMasters' method from the day one oocyst appeared in the feces to sixteen days post-infection.On the sixteen days post-infection,the oocysts production was calculated in the ceca.As results indicated that,there was no death in each group,but the group infected with 10 000 sporulated oocysts had the worst clinical symptoms.Every group began to excrete oocysts at 7 days post infection.The groups being infected with 500,2 500 and 5 000 sporulated oocysts had two excretion peaks,but 10 000 sporulated oocysts group only had one excretion peak.Each group's oocysts production between 7 and 13 days post infection was above 94.74%.The analysis showed that the average oocysts productions increased with the higher infection dose,while the single oocyst production decreased with the higher infection dose.The oocysts in ceca were only 1%of the total number of oocysts both in feces and ceca.Given the above,when conducting clinical infection assay and live vaccine production,the suggested infection number is 10 000 sporulated oocysts,and the oocysts in feces should have been collected between 7-13 days post infection2.Cloning and sequence analysis of the Engam56 gene of E.necatrixEngam56 gene was amplified from the purified E.necatrix gametocyte.Because of high level of GC content,Engam56 gene was cloned in three fragments,which were inserted to pGEM-T easy vecter to do sequencing respectively.As results indicated that,Engam56 gene was 1 407 bp encoding a complete open reading frame with 468 amino acids and the predicted protein molecular weight(MW)was 58.0 kDa.The first 20 amino acids of the predicted protein was signal peptide.The antigenic determinants prediction analysis showed that the encoded protein contained 10 epitopes.Among them,the tyrosine-serine rich region(positions 246-323)located in a complete antigenic determinant,which is the longest;the proline-methionine rich region(positions 338-454)contained 1 epitope.The result of sequences alignment showed that Engam56 was highly homologous to other Eimeria gametophyte protein-encoding genes,ranging from 99.2%to 59.3%.Based on the above sequence analysis,the amplified Engam56 gene was indeed a gametophyte protein encoding gene and has with other species.3.Prokaryotic expression and immunogenicity analysis of the Engam56 gene of E.necatrixEngam56 gene without signal peptide was synthesized following the optimal amino acid codons in Escherichia coli.And then the synthetic sequence was inserted to pET28a(+)using BamH I and Xho 1 restriction sites.The constructed expression vector was named as pET28a(+)-Eng-am56.pET28a(+)-Engam56 was transferred to BL21(DE3)and expressed inducing by IPTG As results indicated that,the recombinantly expressed protein was about 58.0 kDa and mainly in inclusion bodies.Western blot analysis showed that the recombinant protein 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 infected chicken.All these showed that the recombinant protein in vitro is a specificity expression protein with good immunogenicity.4.Immunoprotective effect of the recombinant gametocyte antigen rEnGAM56 of E.necatrixThe yellow feather broiler was used as experimental animal.And the average weight gain,relative increase rate,lesion score and oocyst reduction rate were used to evaluate immunoprotective effect of full-length recombinant protein EnGAM56(rEnGAM56-F)and the truncated expressed recombinant protein EnGAM56(rEnGAM56-T).As results indicated that,the group immunized with low dose of rEnGAM56-T(50?g)had the best protection.In the humoral and cellular immunity,the groups immunized with middle dose of rEnGAM56-T(100?g)showed better protection than the group immunized with low dose of rEnGAM56-T.According to immune protection test,the groups immunized with rEnGAM56-F and rEnGAM56-T had of some immunogenicity against E.necatrix infection,while rEnGAM56-T had better protection.5.The combined immune protection effect of three recombinant gametophyte antigens E.necatrix infectionIn this research,three recombinant proteins were used to do immunoprotective assay.As results indicated that,the group immunized with three recombinant proteins rEnGAM(22+59+56-T)had better protection in lesion score,oocyst reduction and average weight gainthan rEnGAM22 and rEnGAM(22+56-T)immunized groups.In humoral and cellular immunity,the group immunized with rEnGAM(22+59+56-T)had the best protection,followed by the rEnGAM(22+56-T)and rEnGAM22 immunized group.In conclusion,the effect of combined immunization of proteins is better than that of protein alone,and the effect of combined immunization of three proteins is better than that of two proteins.This result lays a theoretical foundation for the development of multivalent subunit vaccines against coccidiosis.