Development And Application Of Nucleic Acid Vaccine For Cryptosporidium Parvum In Animals

The CP15,CP15/60 and CP23 on the sporozoites of Cryptoporidium parvum are major surface protein which are studied in the new pattern vaccine and developed a serologic detection method for cryptosporidiosis.In the experiment,prokaryotic and eukaryotic expression vectors of three genes were constructed by recombinant DMA technique and expressed in E.coli and Hela cells respectively.The nucleic acid vaccine could induced protective immune responses against C.parvum after BALB/c mice and goats were inoculated intranasally.Cloning and sequencing of protective antigen genes Three DNA fragment of pretective antigen genes were amplified by PCR with primers according to GenBank.The recombinant plasmids pMD18-T-15 pMD18-T-15/60 and pMDl8-T-23 were constructed respectively.The ligation products were transformed to competent cells of E.coli host strain DH5 a .Individual 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 CP15,CP15/60 and CP23 were 413,456 and 346bp in length.Compared with PIR,SW1SS-PROT database in GenBank, CP15,CP 15/60 and CP23 genes shared 99.2%,98.9% and 97.3% DNA sequence homology and overall deduced amino acids identity of 100%,99.3% and 98.2%.The amino acids substitutions couldn't change antigen epitopes.Construction and expression of prokaryotic expression vector of Cp The recombinant plasmids pET-28a-15,pET-28a-15/60 and pET-28a-23 were constructed by cloning CP15,CP15/60 and CP23 genes into prokaryotic expression vector pET-28a(+) 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.Three recombinant proteins were highly expressed by growing individual clone 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 ofrecombinant CP15,CP15/60 and CP23 were up to 38%,42% and 36% of the total bacterial protein in the cell lysate.They were specificity of Cp by Western blotting.Development of indirect ELISA with recombiant proteins as antigen The indirect ELISA for the detection of serum antibodies against Cp was established with the recombinant fusion protein expressed highly in E.coli as antigen and goat anti-mice IgG horse-radishperoxidase(HRP) conjugate as the second antibody.The best conditions were that coating antigen was 0.5 u g per well for recombiant CP15 protein(1.0u g per well for recombiant CP15/60 and CP23 protein),blocked with 10% blood serum of rabbit and the sample diluted with the supernatant of normal E.coli lysate.The assay was characterized by specificity,simplicity,rapidity and economical cost.Construction and expression of eukaryotic expression vectors The recombiant plasmids pCR3.1-15,pcDNA3-15/60 and pCR3.1-23 were contructed by cloning CP15,CP15/60 and CP23 genes into eukaryotic expression vector pCR3.1(+) and pCDNA3(+) and expressed in Hela cell strain.After transfected them into Hela cells,three expression cell strains were selected with G418.The specific recombiant proteins were detected in three Hela cell strains by indirect immunofluorescence assay.The protective immune response induced by nucleic acid vaccine in mice All the BALB/c mice were immunized with the vaccine plasmids by different vaccines,adjuvant,inoculation pathway,chemical preparation form,dosage.The responses of specific systemic and mucosal immunity were elevated by the immunological methods such as E rosstte assay,the specific lymphocyte transforming test,the ratio of CD4+/CDg+,the specific antibodies responses and the CTL activity.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.The results also indicated the female eider mice immunized with nucleic acid vacci...