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Ohtani, M.,Hikima, J.i.,Jung, T.S.,Kondo, H.,Hirono, I.,Takeyama, H.,Aoki, T. Academic Press 2013 FISH AND SHELLFISH IMMUNOLOGY Vol.34 No.2
Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 x His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.
Ohtani, M.,Hikima, J.i.,Kondo, H.,Hirono, I.,Jung, T.S.,Aoki, T. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 Developmental and comparative immunology Vol.35 No.5
Cytosolic pattern recognition receptors such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) play an important role in sensing viral RNAs. The receptor encoded by melanoma differentiation-associated gene 5 (MDA5), an RLR, recognizes viral RNA in the cytoplasm and enhances antiviral response in host cells. The full-length MDA5 gene in Japanese flounder, Paralichthys olivaceus was cloned and found to have 11,251 nucleotides. MDA5 transcript abundance was significantly increased in whole kidney infected with viral hemorrhagic septicemia virus (VHSV) as well as whole kidney and peripheral blood leukocytes stimulated with poly I:C in vitro. Hirame natural embryo (HINAE) cells overexpressing MDA5 showed a lower cytopathic effect (CPE) against VHSV, hirame rhabdovirus (HIRRV) and infectious pancreatic necrosis virus (IPNV) infection. When infected with VHSV, MDA5-overexpressing HINAE cells had 24-75 fold lower virus titer than normal HINAE cells. These results suggest that Japanese flounder MDA5 is involved in the induction of antiviral response.
Jang, H B,Kim, Y R,Cha, I S,Noh, S W,Park, S B,Ohtani, M,Hikima, J,Aoki, T,Jung, T S Blackwell Publishing Ltd 2011 Journal of fish diseases Vol.34 No.7
<P><B>Abstract</B></P><P>Although the major capsid proteins (MCPs) of lymphocystis disease virus (LCDV) have been characterized, little is known about the host‐derived immune response to MCPs and other LCDV antigenic proteins. To identify antigenic proteins of LCDV that could be used as vaccine candidates in olive flounder, <I>Paralichthys olivaceus</I>, we analysed the viral proteins responsible for its virulence by applying immuno‐proteomics. LCDV proteins were separated by one‐dimensional gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with homogeneous <I>P.?olivaceus</I> antisera elicited by LCDV natural infection and vaccination with formalin‐killed LCDV. Four immune‐reactive proteins were obtained at 68‐, 51‐, 41‐ and 21 kDa using antisera collected from natural infection while two proteins at 51‐ and 21 kDa exhibited response to antisera from vaccinated fish, indicating that the latter two proteins have vaccine potential. Using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and nanoelectrospray MS/MS, the 51 and 21 kDa proteins were identified as MCP and an unknown protein, respectively.</P>