Expression In Prokaryotic And Eukaryocyte Of ET-Rho-2 Gene For Eimeria Tenella And Immunoprotecive To Chicken

Coccidiosis which is caused by several species of the genus Eimeria is animportant disease which harmed severly to modern chick-keeping and exists inwidely. E.tenella is an important specie among those protozoans. Until now manyproblems need to be solved such as drug-resistance and so on. Signal transduction ofEGFR and EGF is a hot spot and ahead field in nowadays.The rhomboid,EGFR andEGF are related the adhesion and invasion of E.tenella to host cells had beenfound.The ET-Rho-2 gene of Eimeria tenella is a gene encoding romboid proteinwhich discovered by the researchers in my lab in 2004. In the experiment,prokaryotic and eukaryotic expression vectors of ET-Rho-2 was constructed andexpressed in E.coli and Hela cells respectively.The nucleic acid vaccine couldinduce protective immune responses against E.tenella after chicken wereinoculated .Cloning and sequencing of ET-Rho-2 gene The cDNA fragment of ET-Rho-2genes was amplified by RT-PCR with primers according to the sequence whichdiscovered by the teachers in my lab.The recombinant plasmids pMD-ET-Rho-2 wasconstructed .The ligation products were transformed to E.coli host strain DH5α.Theclone was cultured and the recombinant plasmids were prepared as template forautomatic sequencing.The nucleotide sequence were analysed by DNAsis andPROsis sequence software. The results indicated that the gene fragment encodingrhomboid was 774bp in length which encoding 257 deduced amino acid residues.The molecular weight of the unknown putative protein was 28.3 Kda.Construction and expression of pET-ET-Rho-2 The recombinant plasmidspET-ET-Rho-2 was constructed by cloning ET-Rho-2 gene into prokaryoticexpression vector pET-28a(+) and expressed in host cells BL21(DE3).Therecombinant proteins were highly expressed induced by IPTG.The recombinantproteins were inclusion bodies in the cytoplasm.The yield of recombinant ET-Rho-2were up to 18% of the total bacterial protein in the cell lysate.It was specificity ofE.tenella by Western blotting.Development of indirect ELISA with recombiant proteins as antigen Theindirect ELISA for the detection of serum antibodies against E.tenell was establishedwith the recombinant fusion protein expressed in E.coli as antigen and miceanti-chicken IgG HRP conjugate as the second antibody.The best conditions werethat coating antigen was 1μg per well for ET-Rho-2 recombiant protein,blockedwith 10% fetal calf serum and the sample diluted with the supernatant of normalE.coli lysate.The assay was characterized by simplicity,rapidity and economicalcost.The Immunoprotection induced by recombinant antigen in chicken Thechicken were immuned with recombinant antigen three times,then were inoculatedwith E.tenella oocysts.The results showed that the chicken immuned with live E.coliexpressing E.tenella recombinant antigen can protect against E.tenella infectionpartially. The numerber of oocysts and the time shedding of immuned groups wereshorter than the groups haven't been immuned,and the increase of body weightwere swifer,the cecum lesion were slighter than control groups.The highest rate ofimmunoprotection of immuned group is 34.3%.Construction and expression of pVAX1-ET-Rho-2 The recombiant plasmidspVAX1-ET-Rho-2 were contructed by cloning ET-Rho-2 genes into eukaryoticexpression vector pVAX1 and expressed in Hela cell strain.After transfected intoHela cells,the specific recombiant proteins were detected in supernatant of Hela cellby Wester blotting.The immunoprotection response induced by nucleic acid vaccine in chickenAll the chicken were immunized with the vaccine plasmids by different inoculationpathway and dosage three times,then were challenged with E.tenella oocysts.Theresponses of specific humoral immune were elevated by the immunologicalmethods.The results showed the nucleic acid vaccines can induce the immuneresponse.The specific immune responses were strengthened with the increase ofimmunization times.The immunity indexes among experiment groups were notsignificant,while those were significant between experiment and control groups.The numerber of oocyst and time shedding of experiment groups were significantlyshorter,and the increase of body weight were significantly swifer,and the cecumlesion were slighter than those of control groups. The highest immunoprotection ofexperiment group is 83.5%. The results showed the nucleic acid vaccines can protectagainst E.tenella infection.