微小隐孢子虫pcDNA3.0-23重组质粒免疫效果评价

Immune effect of Cryptosporidium parvum CP23 gene vaccine

目的比较微小隐孢子虫表面抗原CP23真核表达载体pcDNA3.0-23经不同免疫途径产生的免疫效果。方法提取微小隐孢子虫基因组DNA,PCR扩增CP23基因片段,克隆至真核表达载体pcDNA3.0,构建pcD-NA3.0-23重组质粒,分别通过肌肉注射和滴鼻(粘膜)免疫2种途径免疫BALB/c小鼠,免疫3次,2周后检测抗CP23特异性抗体IgG滴度、小鼠脾脏、血清中CD4+和CD8+T细胞、细胞因子γ干扰素(IFN-γ);用微小隐孢子虫攻击感染被免疫小鼠,收集小鼠粪便,计算小鼠排出的卵囊量。结果肌注组与滴鼻组小鼠血清抗CP23特异性抗体IgG滴度随免疫次数增加均明显升高,高于对照组及空质粒组(P<0.05),肌注组高于滴鼻组(P<0.05);肌注组与滴鼻组小鼠的CD4+T细胞、CD4+/CD8+比值均高于磷酸缓冲液(PBS)对照组及pcDNA3.0空质粒组(P<0.05),但2种免疫途径的差异无统计学意义(P>0.05);肌注组和滴鼻组脾细胞培养上清中IFN-γ明显高于对照组及空质粒组(P<0.05);微小隐孢子虫攻击小鼠后,2种免疫途径的小鼠排出卵囊量明显少于对照组,且排出时间缩短,2种途径的差异无统计学意义。结论 pcDNA3.0-23重组质粒作为基因疫苗,可产生较好的细胞及体液免疫反应;不同的免疫途径可产生不同的免疫反应。

Objective To construct Cryptosporidium parvum（C.parvum）CP23 eukaryotic expression vector pcDNA3.0-23 and to identify different immune responses caused by different immune route.Methods Genomic DNA of C.parvum was extracted from oocysts of C.parvum.CP23 gene fragment was amplified from genomic DNA of C.parvum with PCR and cloned to eukaryotic expression vector pcDNA3.0 under restriction enzyme.Eighty mice were divided into muscle injection,intranasal,PBS control,and pcDNA3.0 group.The mice in each group were immunized with recombinant pcDNA3.0-23,PBS and pcDNA3.0 intramuscularly or intranasally,respectively.All mice were immunized for three times with an interval of two weeks.Spleen and blood were taken for the detection of CD4＋,CD8＋ T cell,interferon（IFN）-γ and IgG against CP23 two weeks after the final immunization.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 pcDNA3.0-23 was successfully constructed.The number of CD4＋T cells,the ratio of CD4＋/CD8＋ and IFN-γ in muscle injection group and intranasal immune group were significantly higher than those of other two groups（P0.05）.There was no significant difference between the two immune route groups（P0.05）.However IgG titer against CP23 of muscle injection mice wase significantly higher than those of other three groups（P0.05）.Conclusion Cryptosporidium parvum CP23 eukaryotic expression vector pcDNA3.0-23 was established for the construction of C.parvum gene vaccine and can induce strong cellular and humoral responses.Different immune responses could be produced by different immune route.