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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 compare the immune effect of CP23 eukaryotic expression vector pcDNA3.0-23 with different immunization routes. Methods Genomic DNA of Cryptosporidium parvum was extracted. The CP23 gene fragment was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.0. The recombinant plasmid pcD-NA3.0-23 was constructed and immunized by intramuscular injection and intranasal (mucosal) BALB / c mice were immunized three times with immunization. After two weeks, anti-CP23 specific antibody IgG titer, mouse spleen, serum CD4 + and CD8 + T cells and IFN- Mice challenged with Cryptosporidium parvum were immunized with mice and feces were collected and the amount of oocysts released from mice was calculated. Results The serum anti-CP23 IgG antibody titer of mice with intramuscular injection and nasal drops significantly increased with the number of immunization, which was higher than that of the control group and the empty plasmid group (P <0.05) (P <0.05). The ratio of CD4 + T cells and CD4 + / CD8 + in mice intramuscularly and intranasal group were higher than that in phosphate buffered saline (PBS) control group and pcDNA3.0 empty plasmid group (P <0.05) There was no significant difference in the two immunological pathways (P> 0.05). IFN-γ in the supernatant of spleen cells in intramuscular injection group and intranasal injection group was significantly higher than that in control group and empty plasmid group (P <0.05) After the mice were attacked by the worms, the amount of oocysts released by the two immunization routes was significantly less than that of the control group, and the discharge time was shortened. There was no significant difference between the two routes. Conclusion Recombinant plasmids pcDNA3.0-23 can produce good cellular and humoral immune responses as a gene vaccine. Different immune pathways can produce different immune responses.