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Generation and Immunity Testing of a Recombinant Adenovirus Expressing NcSRS2-NcGRA7 Fusion Protein of Bovine Neospora caninum

Li-Jun Jia1, Shou-Fa Zhang1, Nian-Chao Qian1, Xue-Nan Xuan2, Long-Zheng Yu1, Xue-Mei Zhang1, Ming-Ming Liu1
The Korean Journal of Parasitology 2013;51(2):247-253.
Published online: April 25, 2013

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Corresponding author (lijunjia1015@sohu.com)
• Received: September 26, 2012   • Revised: December 19, 2012   • Accepted: February 17, 2013

© 2013, Korean Society for Parasitology and Tropical Medicine

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Generation and Immunity Testing of a Recombinant Adenovirus Expressing NcSRS2-NcGRA7 Fusion Protein of Bovine Neospora caninum
Korean J Parasitol. 2013;51(2):247-253.   Published online April 25, 2013
DOI: https://doi.org/10.3347/kjp.2013.51.2.247
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Generation and Immunity Testing of a Recombinant Adenovirus Expressing NcSRS2-NcGRA7 Fusion Protein of Bovine Neospora caninum
Korean J Parasitol. 2013;51(2):247-253.   Published online April 25, 2013
DOI: https://doi.org/10.3347/kjp.2013.51.2.247
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Generation and Immunity Testing of a Recombinant Adenovirus Expressing NcSRS2-NcGRA7 Fusion Protein of Bovine Neospora caninum
Image Image Image Image Image Image Image
Fig. 1 Results of PCR and SOE-PCR amplification. Lane M, DNA marker DL2,000; Lane 1, PCR products of the NcSRS2 gene; Lane 2, PCR products of the NcGRA7 gene; Lane 3, PCR products of the NcSRS2-NcGRA7 fusion gene; Lane 4, water control.
Fig. 2 QBI-HEK 293 cells packaging Ad5-NcSRS2-NcGRA7. (A) QBI-HEK 293 cells packaging Ad5-NcSRS2-NcGRA7 (×1,000). (B) normal QBI-HEK 293 cells (×1,000).
Fig. 3 PCR identification of Ad5-NcSRS2-NcGRA7. Lane M, DNA marker is DL10,000; Lane 1, PCR products of Ad5-NcSRS2-NcGRA7; Lane 2, empty plasmid control.
Fig. 4 IFA detection of the expression of NcSRS2-NcGRA7 fusion gene. (A) Ad5-NcSRS2-NcGRA7-infected 293 cells (×400). (B) empty plasmid-infected 293 cells (×400).
Fig. 5 Western blotting analysis of the expression of the NcSRS2-NcGRA7 fusion gene. Lane M, Low molecular weight marker; Lane 1, expression products of Ad5-NcSRS2-NcGRA7-infected 293 cells; Lane 2, empty plasmid control.
Fig. 6 IgG antibody titer induced by Ad5-NcSRS2-NcGRA7 in mice as determined by indirect ELISA. BALB/c mice were inoculated at 3-week intervals 3 times, and sera were collected at week 2 after each immunization for antibody titer determination. The IgG antibody titers in sera were measured by indirect ELISA. Data were analyzed by 1-way ANOVA. w.p.p.i., week post-prime immunization; w.p.b.i., week post-boost immunization.
Fig. 7 Levels of IFN-γ (A) and IL-4 (B) in sera of immunized BALB/c mice. BALB/c mice were inoculated and the levels of IFN-γ (A) and IL-4 (B) were measured using IFN-γ and IL-4 ELISA kits. Data were analyzed by 1-way ANOVA. w.p.p.i., week post-prime immunization; w.p.b.i., week post-boost immunization.

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Generation and Immunity Testing of a Recombinant Adenovirus Expressing NcSRS2-NcGRA7 Fusion Protein of Bovine Neospora caninum
Primer Primer sequences (5´-3´) Enzyme sites P1 CGGAATTCGCCACCATGCTGCTATTCGGCGTCGTT EcoR I P2 CATCTCCCGCCAACTTGAGGAGGTCGAAGTTCAGAA GTGACGCAGCGGATAGTG P3 CTCAAGTTGGCGGGAGATGTTGAGTCCAACCCTGGG CCCGGCTCTACCTCTTTGG P4 CGTCTAGATTAGCTGCCTTTCTGAGTCGTG Xba I
Table 1. Primers for amplifying the NcSRS2 and NcGRA7 genes of N. caninum

Table 1.

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