隐孢子虫AOX和TSP6核酸疫苗的制备及免疫保护性研究

Development of DNA Vaccine Encoding Cryptosporidium parvum AOX and TSP6 Genes and Study on Its Immunoprotection Effects

旨在制备微小隐孢子虫(Cryptosporidium parvum)线粒体蛋白(AOX)基因和微线体蛋白(TSP6)基因的DNA疫苗,评价其对贝氏隐孢子虫(Cryptosporidium baileyi)感染雏鸡的免疫保护性。根据GenBank库中AOX和TSP6基因的序列设计引物,扩增出编码2个保护性抗原基因的DNA片段,利用DNA重组技术将2个目的片段分别克隆到真核表达载体pVAX1,制成核酸疫苗pVAX1-AOX和pVAX1-TSP6。将制备的核酸疫苗肌肉注射免疫雏鸡,共分为4组,pVAX1组,pVAX1-AOX组,pVAX1-TSP6组,pVAX1-AOX+pVAX1-TSP6组,2周后检测雏鸡血清IgG抗体滴度和细胞因子(IL-4和IFN-γ)水平的变化。结果显示,免疫组中鸡的血清特异性IgG抗体水平、细胞因子IFN-γ都有明显的升高。攻卵囊试验结果表明,免疫组比对照组的排卵囊量明显减少,且排卵囊时间有所缩短。表明制备的pVAX1-AOX和pVAX1-TSP6核酸疫苗有较好的免疫保护作用。

In order to develop DNA vaccine encording mitochondrion(AOX)and microneme(TSP6)genes of Cryptosporidium parvum and evaluate the immunoprotective effect against Cryptosporidium baileyi infection,two DNA fragments of protective antigen genes were amplified by PCR with primers designed according to the genes in GenBank.Then the two DNA fragments were cloned into pVAX1 vector using recombinant DNA technology,and the two nucleic acid vaccines(pVAX1-AOX and pVAX1-TSP6)were obtained.The chicks were devided into 4 groups,and immunized intramuscularly with pVAX1,pVAX1-AOX,pVAX1-TSP6 and pVAX1-AOX+pVAX1-TSP6,respectively.The specific immune responses were analyzed by serum antibody and cytokine(IL-4 and IFN-γ)measurements two weeks later.The results showed that the levels of specific antibody IgG in sera and IFN-γall rose obviously in immune groups.The immune protective experiments showed that shedding oocysts became reduced obviously and the duration time of shedding oocysts became shorten in immune groups.The results indicated that the DNA vaccines of pVAX1-AOX and pVAX1-TSP6 have better immune protective effects to the infection of Cryptosporidium.