| Canine adenovirus type I (CAV-l) and type 2 (CAV-2) are the two major infectious agents that cause Canine Infectious Hepatitis and Canine Infectious Laryngotrachitis, respectively, in dogs. CAy-i can also cause encephalitis in foxes and bears. CAV-2 bears the same antigenicity with CAV- I and confers cross- protective immunity to CAy-i, so it has been developed as an effective commercial vaccine in clinics. Recombinant live vectors based on various sources of adenoviruses have been successfully constructed and some of these have been confirmed to be effective in inducing immunities to infectious agents from which the foreign antigen(s) derived. However, the study on Canine adenovirus as a live vaccine vector has not yet been succeeded due to th lack of full understanding of its genetic background and its expression control. In this paper, the El and E3 regions of a CAV-2 vaccine strain designated as SY were firstly cloned, sequenced and studied intensively. EcoR I C fragment containing El region of CAV-2 strain SY and Kpn I B fragment containing E3 region were cloned into pGEM-3zf and pBluescript II KS, respectively, resulting in plasmids pG-El and pBE3-2. Both fragments were sequenced and were simultaneously compared with that of strains Toronto A26/6 1 ,Vaxitas and Manhattan. Results showed that the sequence of the El region of CAV-2 strain SY was completely identical to that of Toronto A26/6 1, and the maximum homology between the E1A region of SY strain and that of Vaxitas strain was 99.8%. The sequence of the E3 region of CAV-2 strain SY was 100% consistent with that of Toronto A26/61 and Manhattan. The plasmid pBE3-2 containing E3 region was deleted by cutting a fragment out between two Bgl II sites that produced by in vitro mutagenesis. It was then re-ligated to form a recombinant plasmid pB A E3, into which a polylinker was introduced to 98 form pBE3L. By a series of recombinations, GFP and Rgp expression vectors with and without a foreign CMV promotor were constructed and named as pBE3LGFP, pBE3LCGFP, pBE3LRgp and pBE3LCRgp, respectively. The reading frames of GFP in pBE3LGFP and Rgp in pBE3LRgp were in accordance with that of E3. Transfection of the 4 recombinant plasmids into DK cells showed that GFP in pBE3LCGFP and Rgp in pBE3LCRgp were expressed as determined by Flourescent Microscope and immunoflourescent assay, respectively. The expression of Rgp and GFP was not observed in pBE3LRgp and pBE3LGFP. This demonstrated that a foreign prornotor was necessary to express an interest protein within the E3 region of CAV-2. As the cloned E3 fragment of the expression vector has a 1.6kb and a 1.7kb flanking region at each end, and an unique restriction site within each flanking region exists, the expression vector can therefore be used for both in vitro recombination and homologous recombination in cells. To establish a helper cell line, pG-El was cut by BstX I . The ends were blunted and the 3ne of them was re-ligated to its upstream Sma I end. The l3amH I -EcoR I fragment containing the El region was subcloned into the downstream of CMV promotor of pcDNA3, preducing a p]asmid pc-E It. DK cells were transfected by this recombinant plasmid, and selected in I mg/mi G418 medium. 26 clones were obtained, purified and amplified. Out of which 6 clones were confirmed to be El DNA integration and EIA transcription positive. Western blotting analysis showed that one of these clones could express the l9kD protein of EIB. This El tran...
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