表达牛源犬新孢子虫NcSRS2基因重组腺病毒的构建及免疫原性分析

Construction of a Recombinant Adenovirus Expressing NcSRS2 Protein of Bovine Neospora Caninum and Immunogenicity Analysis of the Protein

为构建牛源犬新孢子虫NcSRS2基因重组腺病毒,并分析其免疫原性,PCR扩增牛源犬新孢子虫Nc-SRS2基因,构建克隆质粒pMD18-T-NcSRS2、重组腺病毒穿梭质粒pCR259-NcSRS2及表达质粒Transpose-Ad-NcSRS2,脂质体介导转染QBI-HEK293细胞,包装重组腺病毒Ad5-NcSRS2,PCR检测重组腺病毒NcSRS2基因,IFAT和Western blotting检测NcSRS2基因在QBI-HEK293细胞中的表达,测定病毒滴度后,收集病毒液免疫BALB/c小鼠,间接ELISA检测小鼠血清IgG抗体水平。结果显示,扩增的牛源犬新孢子虫NcSRS2基因大小为1 227bp,与GenBank中发表的NcSRS2(AF061249)核苷酸序列相似性为99%;重组腺病毒Ad5-NcSRS2在293细胞中包装成功,表达蛋白的相对分子质量为43ku,具有较好的反应原性;测得重组腺病毒Ad5-NcSRS2滴度为109 TCID50.mL-1,间接ELISA检测二免后3周BALB/c小鼠血清中IgG抗体效价达1∶2 048。本研究成功构建了具有良好免疫原性的重组腺病毒Ad5-NcSRS2,为牛源犬新孢子虫NcSRS2基因重组腺病毒载体疫苗的研制奠定了基础。

In order to construct a recombinant adenovirus expressing NcSRS2 protein of bovine Neospora Caninum, NcSRS2 gene of bovine Neospora Caninum was amplified by PCR, pMD18 T-NcSRS2, pCR259-NcSRS2 and Transpose-A＆NcSRS2 were constructed in this research. Coated with liposome, Transpose-Ad-NcSRS2 was transfected into QBI-HEK293 cells to package recom binant adenovirus Ad5-NcSRS2. Recombinant adenovirus NcSRS2 gene was detected by PCR. The expression of NcSRS2 gene in QBI-HEK293 cells was detected by IFAT and Western blot- ting. After the virus titer was determined, the virus fluid was collected to inoculate BALB/c mice and IgG antibody levels in the sera were measured by indirect ELISA. The size of NcSRS2 gene was 1 227 bp. The nucleotide sequence of the gene shared 99Y0 homology with that in GenBank （AF061249）. Recombinant adenovirus AdS-NcSRS2 was successfully packaged in 293 cells. The protein expressed by Ad5-NcSRS2 was 43 kD and had good reactogenicity. The titer of recombi nant adenovirus AdS-NcSRS2 was 109TCID50 ·mL^-1. Three weeks post the second immuniza- tion, IgG antibody titers in the sera o{ BALB/c mice were up to 1：2 048, measured by indirectELISA. A recombinant adenovirus AdS-NcSRS2 which have good immunogenicity was success- fully constructed. This settles a solid foundation for the development of recombinant adenovirus vaccine against Neospora Caninum based on NcSRS2 gene.