The Characterization Of CaPKR-like Expression

The double-stranded-RNA-dependent protein kinase (PKR) is an important component in an antiviral defence pathway that is mediated by interferon (IFN) in vertebrates. CaPKR-like, an antiviral-relevant gene of crucian carp, was the first identified fish gene most similar to mammalian PKR. In this dissertation, the total RNA was extracted from tissues of crucian carp treated with Poly I:C or not, and analysis tissue specific expression of CaPKR-like by RT-PCR. Then the C-terminal half of CaPKR-like, representing catalytic domain, amplified by PCR and the recombinant expression plasmid, pGEX-KG/CaPKR-like-C, was constructed. Expression plasmid was transformed into BL21.The positive colonies, selected by PCR, were induced with IPTG and identified by 10% SDS-PAGE electrophoresis. To acquire multiclonal antibodies, the expressed proteins were purified and applied to immunize rabbit. With multiclonal antibodies, we analyse the expression of CaPKR-like in tissues of crucian carp by Western blots.Like mammalian PKR, CaPKR-like had a low level of constitutive expression in normal CAB(Carassius auratus blastulae embryonic )cells and tissues of crucian carp without treated.With poly I:C induced, the protein expression of CaPKR-like is increasing, which was confirmed by Western blots. The result suggested that CaPKR-like gene possesses the activated and induced signal pathway that is most similar to mammalian PKR.