Development And Application Of Bivalent Nucleic Acid Vaccine For Cryptosporidium Parvum CP405/CP585 In Animals

CP405 and CP585 surface proteins on the sporozoites of C.parvum,which screened from T7 phage display Iibrary of C.parvum with IECs by our laboratory, were confirmed major protective antigens,could used to be study neotype vaccine of C.parvum.In this paper,using DNA recominbinated technique,we cloned two genes into eukaryotic expression vector for constructing bivalent nucleic acid vaccine. And immuned animals with eukaryotic expression vector.The results demonstrated animals genetate specific imrnune response,and eukaryotic expression vector had protective effect to animals.Two DNA fragment of pretective antigen genes were amplified by PCR.with primers according to GenBank.Then the Three DNA fragment were inserted into PMD18-T vector and obtained the recombinant plasmids: pMD18-T-CP405,pMD18-T-CP585 and pMD18-T-IRES. The ligation products were transformed to competent cells of E.coli host strainTOP10.lndividual clone was cultured and the recombinant plasmids were prepared as template for automatic sequencing.The nucleotide sequence were analysed by DNAsis and PROsis sequence soflware.The results indicated that the gene fragment encoding CP405,CP585 and IRES were 405,585 and 552bp in length.Compared with PIR,SWISS-PROT data base in GenBank,all of the segenes shared 100% DNA sequence homology and overall deduced amino acids identity of 100%.The amino acids substitutions couldn't change antigen epitopes.The recombinant plasmids pVAX1-CP585-IRES-eGFP,pVAX1-CP405-IRES-CP585 and pVAX1-CP405-IRES-eGFP were contructed by cloning CP405,CP585 and IRES genes into eukaryotic expression vector pVAXI and expressed in Hela cells strain. After transfected them into Hela cells. The specific recombinant proteins were detected in three Hela cell strains by indirect immuneofiuorescence assay.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 the ratio of CD4~+/CD8~+ and the specific antibodies responses.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 experimental mice excreted far less oocysts than the control mice.The average duration of oocysts excretion in the experiment mice was shorted 3-5days compared to the control mice.The decreased worm rate of the best immunity group is 65.5%.