牛源犬新孢子虫NcSAG1基因重组腺病毒株的构建及动物免疫试验

Construction and Animal Experiment of a Recombinant Adenovirus Expressing NcSAG1 Protein of Bovine Neospora caninum

【目的】构建表达牛源犬新孢子虫NcSAG1基因的重组腺病毒株,并接种Balb/c小鼠,评价重组腺病毒株对Balb/c小鼠的体液免疫和细胞免疫应答水平。【方法】PCR扩增牛源犬新孢子虫NcSAG1基因,构建pMD18-T-NcSAG1克隆质粒和pCR259-NcSAG1重组腺病毒穿梭质粒;将鉴定正确的pCR259-NcSAG1重组腺病毒穿梭质粒依次转化HighQ-1 Transpose-Ad 294和HighQ-1感受态细胞,构建Transpose-Ad-NcSAG1重组腺病毒表达质粒;PacI酶切线性化后,脂质体介导转染QBI-HEK 293细胞包装重组腺病毒Ad5-NcSAG1,应用PCR和Westernblotting技术检测Ad5-NcSAG1及其表达蛋白产物。测定Ad5-NcSAG1重组腺病毒滴度后,接种Balb/c小鼠,测定小鼠血清中IgG特异性抗体和细胞因子IFN-γ、IL-4水平,以此评价重组腺病毒株的免疫应答效果。【结果】扩增的牛源犬新孢子虫NcSAG1基因大小为982bp,与GenBank中发表的NcSAG1(AF132217)核苷酸序列的同源性为99.2%;经PCR和Western blotting检测,重组腺病毒Ad5-NcSAG1在QBI-HEK 293细胞中包装成功,表达蛋白的分子量为33kD,具有较好的反应原性;测得重组腺病毒Ad5-NcSAG1滴度为1010TCID50/mL,Ad5-NcSAG1接种Balb/c小鼠后,能够诱导产生高水平的IgG特异性抗体和IFN-γ、IL-4细胞因子。说明Ad5-NcSAG1重组腺病毒对Balb/c小鼠产生了较强的体液免疫和细胞免疫应答。【结论】成功构建了牛源犬新孢子虫NcSAG1基因的重组腺病毒株,该毒株能够诱导Balb/c小鼠产生高水平的体液免疫和细胞免疫应答,为犬新孢子虫新型疫苗的临床试验奠定了基础。

[ Objective ] A recombinant adenovirus expressing NcSAG1 protein of bovine N. caninum was constructed. Balb/c mice were immunized with the recombinant adenovirus to evaluate the levels ofhumoral and cellular immune responses. [Method] NcSAG1 gene of bovine N. caninum was amplified by PCR. pMD18-T-NcSAG1 and pCR259- NcSAG1 were constructed. The correct pCR259-NcSAG1 was transformed into HighQ-1 Transpose-AdTM 294 and HighQ-1TM competent cells to construct transpose-Ad-NcSAG 1 recombinant adenovirus. Coated with liposome, Transpose-Ad-NcSAG1, linearized by PacI, was transfected into QBI- HEK293 cells to package recombinant adenovirus Ad5-NcSAG1. The recombinant adenovirus Ad5-NcSAG1 was detected by PCR. The expression of NcSAG1 gene in QBI-HEK293 ceils was detected by Western blotting. After the virus titer was determined, the virus fluid was collected to inoculate Balb/c mice and the levels of IgG antibody and cytokines IFN-γ, IL-4 in the sera were measured to evaluate the recombinant adenovirus effect. [Result] The size ofNcSAG1 gene was 982 bp. The nucleotide sequence of the gene shared 99.2% homology with that in GenBank （AF132217）. The recombinant adenovirus Ad5-NcSAG1 was successfully packaged in 293 cells. The protein expressed by Ad5-NcSAG1 was 33kD and had good reactogenicity. The titer of the recombinant adenovirus Ad5- NcSAG1 was 1010TCID50/mL. The Ad5-NcSAG1 recombinant adenovirus induced Balb/c mice to produce strong humoral and cellular immune responses. [ Conclusion ] The recombinant adenovirus Ad5-NeSAG 1 was successfully constructed. It could induce Balb/c mice to produce strong humoral and cellular immune responses. This study has laid a solid foundation for the clinical experiment of the novel vaccine against N. caninum.