Objective: To construct and identify recombinant human adenovirus type 5 with E2 glycoprotein gene of classical swine fever virus (CSFV). Methods The full-length E2 gene sequence was amplified from live CSFV vaccine by nested PCR and cloned to the polyclonal site of shuttle plasmid pacAd5 CMVK-NpA. The constructed recombinant shuttle plasmid pacAd5 CMVK-NpA-E2 was linearilized and co-transfected to 293AD cells with backbone plasmid pacAd59. 2-100 for pacakging. After appearance of CPE, the transcription of E2 mRNA was determined by RT-PCR. The recombinant adenovirus was determined for titer by CPE method, and for stability after subculture. The expression of E2 protein was determined by Western blot. Kunming mice were immunized i.p. with the recombinant adenovirus at a dosage of 10 5.82 TCID 50, and determined for antibody titer against CSFV in sera by indirect ELISA. Results: Restriction analysis and sequencing proved that recombinant shuttle plasmid pacAd5 CMVK-NpA-E2 was constructed correctly. Both transcription of E2 gene and expression of E2 protein were proved in 293AD cells transfected with recombinant adenovirus rAd5v-MxE2. The titer of rAd5v-MxE2 was 10 5.82 TCID 50/ml. Target gene fragments were amplified from rAd5v-MxE2 of passages 5 and 30, while the virus titer showed no significant change. The recombinant adenovirus induced antibody against CSFV in mice. Conclusion: Recombinant human adenovirus type 5 with E2 gene of CSFV was successfully constrcuted and showed high immunogenicity, which laid a foundation of further preparation of recombinant CSFV vaccine.
|Original language||English (US)|
|Journal||Chinese Journal of Biologicals|
|State||Published - Jul 20 2012|
- Classical swine fever virus (CSFV)
- E2 protein
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology