Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (22): 4705-4712.doi: 10.3864/j.issn.0578-1752.2012.22.017

• VETERINARY SCIENCE • Previous Articles     Next Articles

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

 JIA  Li-Jun, ZHANG  Shou-Fa   

  1. Department of Veterinary Medicine, Agriculture College of Yanbian University, Yanji 133002, JiLin
  • Received:2012-05-09 Online:2012-11-15 Published:2012-09-28

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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】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-Ad™ 294 and HighQ-1™ competent cells to construct transpose-Ad-NcSAG1 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 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-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-NcSAG1 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.

Key words: bovine , Neospora Caninum , NcSAG1 , recombinant adenovirus , animal immunization

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