Abstract: Objective To construct Crypto sporidium parvum(C.parvum)CP23 eukaryotic expression vector pcDNA 3.0-23 and to identify different mimune responses caused by different mimune route.Methods Genomic DNA of C.parvum was extracted fromoocysts of C.parvum.CP23 gene fragment was amplified from genomic DNA of C.parvum with PCR and cloned to eukaryotic expression vector pcDNA 3.0 under restriction enzyme.Eighty mice were divided into muscle injection,intranasal,PBS control,and pcD NA 3.0 group.The mice in each group were immunized with recom binant pcDNA 3.0-23,PBS and pcDNA 3.0 in tramuscularly or intrana sally,respectively.Allmice were mimunized for three times with an interval of two weeks.Spleen and blood were taken for the detection of CD4⁺,CD8⁺ T cell,interferon(IFN)and IgG ag ainst CP23 two weeks a fter the final mimunization.All mice were challenged with C.parvum for further observation.Results The CP23 gene fragment was amplified correctly.The size of the gene was about 340 bp.The C.parvum CP23 eukaryotic expression vector pcDNA 3.0-23 was successfully constructed.The number of CD4⁺ T cells,the ratio of CD4⁺/CD8⁺ and IFN in muscle in jection group and intranasal mimune group were significantly higher than those of other two groups(P<0.05).There was no significant difference between the two mimune route groups(P>0.05).However IgG titer against CP23 of muscle in jection mice wase significantly higher than those of other three groups(P<0.05).Conclusion Crypto sporidium parvum CP23 eukaryotic expression vector pcDNA 3.0-23 was established for the construction of C.parum genevaccine and can induce strong cellular and humoral responses.Different mimune responses could be produced by differen timmune route.