中国农业科学2012，Vol.45Issue(22)：4705-4712,8.DOI:10.3864/j.issn.0578-1752.2012.22.017
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
摘要
Abstract
[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 of humoral and cellular immune responses. [Method] NcSAGI gene of bovine N. caninum was amplified by PCR. pMD18-T-NcSAGl and pCR259- NcSAGI were constructed. The correct pCR259-NcSAGl was transformed into HighQ-1 Transpose-AdTM 294 and HighQ-1TM competent cells to construct transpose-Ad-NcSAGl recombinant adenovirus. Coated with liposome, Transpose-Ad-NcSAGl, linearized by PacI, was transfected into QBI- HEK293 cells to package recombinant adenovirus Ad5-NcSAGl. The recombinant adenovirus Ad5-NcSAGl was detected by PCR. The expression of NcSAGI gene in QBI-HEK293 cells 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 of NcSAG1 gene was 982 bp. The nucleotide sequence of the gene shared 99.2% homology with that in GenBank (AF132217). The recombinant adenovirus Ad5-NcSAGl was successfully packaged in 293 cells. The protein expressed by Ad5-NcSAGl was 33kD and had good reactogenicity. The titer of the recombinant adenovirus Ad5- NcSAGl was 1010TCID50/mL. The Ad5-NcSAGl recombinant adenovirus induced Balb/c mice to produce strong humoral and cellular immune responses. [Conclusion] The recombinant adenovirus Ad5-NcSAGl 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.关键词
牛/犬新孢子虫/NcSAG1/重组腺病毒/动物免疫Key words
bovine/ Neospora Caninum/ NcSAGl/ recombinant adenovirus/ animal immunization引用本文复制引用
贾立军,张守发..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[J].中国农业科学,2012,45(22):4705-4712,8.基金项目
国家自然科学基金(31160501)、吉林省青年科研基金(201201076)、吉林省自然科学基金(201115230)、延边大学科技发展计划(延大科合字2011第37号) （31160501）
