Expression In Prokaryotic And Eukaryocyte Of λ Mzp5-7 Gene For Eimeria.tenella And Immunoprotecive To Chicken

The λ Mzp5-7 on the sporozoites of Eimeria.tenella is major surface protein which is studied in the new pattern vaccine and developed a serologic detection method for E.tenella.In the experiment,prokaryotic and eukaryotic expression vectors of the gene was constructed by recombinant DNA technique and expressed in E.coli and Hela cells respectively.The nucleic acid vaccine could induce protective immune responses against E.tenella after chicken were inoculated .Cloning and sequencing of λ. Mzp5-7 genes One cDNA fragment of λ Mzp5-7 genes was amplified by RT-PCR with primers according to GenBank.The recombinant plasmids pMD-Mzp5-7 was constructed .The ligation products were transformed to competent cells of E.coli host strain DH5 .The clone was cultured and the recombinant plasmids were prepared as template for automatic sequencing.The nucleotide sequence were analysed by DNAsis and PROsis sequence software.The results indicated that the gene fragment encoding λ. Mzp5-7 was 936bp in length.Compared with database in GenBank, λ Mzp5-7 shared 98%,DNA sequence homology and overall deduced amino acids identity of 95.6%.Construction and expression of prokaryotic expression vector of λ. Mzp5-7 The recombinant plasmids pET-Mzp5-7 was constructed by cloning λ. Mzp5-7 gene into prokaryotic expression vector pET-28b(+) and expressed in E.coli host cells BL21(DE3).The expression levels were optimized by manipulating the host strain,the media,the time of harvest.The recombinant proteins were highly expressed in normal LB media,then induced by the Immol/L IPTG for 4h before harvesting cells.The recombinant proteins were inclusion bodies in the cytoplasm.The yield of recombinant λ Mzp5-7 were up to 18% of the total bacterial protein in the cell lysate.lt was specificity of E.t by Western blotting.Development of indirect ELISA with recombiant proteins as antigen The indirect ELISA for the detection of serum antibodies against E.t was established with the recombinant fusion protein expressed in E.coli as antigen and mice anti-chicken IgG HRP conjugate as the second antibody .The best conditions were that coating antigen was 1 g per well for . Mzp5-7 recombiant protein.blocked with 10% serum of foetus Cattle and the sample diluted with the supernatant of normal E.coli lysate.The assay was characterized by simplicity.rapidity and economical cost.The Immunoprotection induced by recombinant antigen in chicken The chicken were inoculated with recombinant antigen three times,then were challenged with E.t oocysts.The results showed the inoculation with live E.coli expressing E.t recombinant antigen can protect against E.t infection partial.Oocyst number and time shedding of experiment groups were shorter,and the increase of body weight (BW) were swifer,and the cecum Lesion were slighter than those of control groups.Construction and expression of eukaryotic expression vectors The recombiant plasmids pVAXl-Mzp5-7 were contructed by cloning X Mzp5-7 genes into eukaryotic expression vector pVAXl and expressed in Hela cell strain.After transfected them into Hela cells.the specific recombiant proteins were detected in supernatant of Hela cell by Wester blot. The immunoprotection response induced by nucleic acid vaccine in chicken All the chicken were immunized with the vaccine plasmids by different inoculation pathway.dosage three times,then were challenged with E.t oocysts.The responses of specific humoral and cell immunity were elevated by the immunological methods such as the specific antibodies responses,the ratio of CD4+/CD8+. The results showed the nucleic acid vaccines can induce the immune response The specific immune responses were strengthened with the increase of immunization times.The immunity indexes among experiment groups were not significant,while those between experiment and control groups were significant.At the same time, the results showed the nucleic acid vaccines can protect against E.t infection.Oocyst number and timeshedding of experiment groups were significantly shorter...