Development And Application Of Bivalent Nucleic Acid Vaccine For Cryptosporidium Parvum CP12/CP21 In Animals

CP12 andCP21 surface proteins on the sporozoites of C.parvum,which screened from T7 phage display library 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 univalent and bivalent nucleic acid vaccine.Based on the above,we inserted CpG-ODN immunological facilitation sequence,and immuned animals with eukaryotic expression vector.The results demonstrated animals genetate specific immune response,and eukaryotic expression vector had protective effect on animals.Cloning and sequencing of protective antigen genes.Five DNA fragment of pretective antigen genes were amplified by PCR with primers according to GenBank. Then the five DNA fragment were inserted into pMD18-T vector and obtained the recombinant plasmids:pMD18-T-CP12^a,pMD18-T-CP21^a,pMD18-T-C-CP12, pMD18-T-CP12^b and pMD18-T-CP21^b.The ligation products were transformed to competent cells of E.coli host strain DH5α.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 CP12^a,CP21^a,C-CP12,CP12^b and CP21^b were 315,543,332,312 and 558bp in length.Compared with PIR,SWISS-PROT database in GenBank,all of these genes shared 100%DNA sequence homology and overall deduced amino acids identity of 100%.The amino acids substitutions couldn't change antigen epitopes.Construction and expression of eukaryotic expression vectors.The recombiant plasmids pVAX1-CP12^a,pVAX1-CP12^a,pVAX1-CP12^b-CP21^b and pVAX1-C-CP12-CP21^b were contructed by cloning CP12^a,CP21^a,C-CP12,CP12^b and CP21^b genes into eukaryotic expression vector pVAX1 and expressed in Hela cell strain. After transfected them into Hela cells,four 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 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 3-5 days compared to the control mice.The decreased worm rate of the best immunity group is 84.1%.The protective immune response induced by nucleic acid vaccine in mice.The adult pregnant goats were inoculated intranasally with pVAX1-C-CP12-CP21^b plasmid. Their offspring were challenged with C.parvum oocysts.The results showed the nucleic acid vaccines can induce the immune response of goats and the vaccinated goats can tranfer the immunity to offspring conferring protection against C.parvum infection. Oocyst number and time shedding by the kids of experiment groups were shorter than those of control groups.(the experiment group is 12.5±1.13d,the control group is 14.4±1.27d).The decreased worm rate of the experiment is 65.4%.