Grass carp (Ctenopharyngodon idella) is an important species of freshwater aquaculture fish in China. However, grass carp reovirus (GCRV) can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic capacity of the virus's structural proteins for preparing an effective vaccine has not been available. In this study, some single-chain variable fragment antibodies (scFv), which could specifically recognize grass carp IgM, were selected from a constructed mouse na ve antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from virus-infected grass carp was more than two times higher than that of the control group at 5-7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.
Myostatin is a member of the transforming growth factor-β(TGF-β) super-family and functions as a negative regulator of muscle growth.Binding of the specific receptor,Activin receptor IIB(Act RIIB),with myostatin or other related TGF-β members,could be inhibited by the activin-binding protein follistatin(Fst) in mammals.Overexpressing Fst in mouse skeletal muscle leads to muscle hypertrophy and hyperplasia.To determine if Fst has similar roles in fish,we generated transgenic zebrafish expressing high levels of zebrafish Fst1 using the promoter of the zebrafish skeletal muscle-specific gene,myosin,light polypeptide 2,skeletal muscle(Mylz2).Independent transgenic zebrafish lines exhibited elevated expression levels of myogenic regulatory genes MyoD and Pax7 in muscle cells.Adult Fst1 overexpressing transgenic zebrafish exhibited a slight body weight increase.The high level of Fst1 expression dramatically increased myofiber numbers in skeletal muscle,without significantly changing the fiber size.Our findings suggest that Fst1 overexpression can promote zebrafish muscle growth by enhancing myofiber hyperplasia.
Grass carp reovirus(GCRV),a double stranded RNA virus that infects aquatic animals,often with disastrous effects,belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses,genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies,also called viral factories. Sequences analysis revealed the nonstructural protein NS80,encoded by GCRV segment 4,has a high similarity with μNS in MRV(Mammalian orthoreoviruses) ,which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication,the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region(335-742) was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28oC. In addition,serum specific rabbit antibody was obtained by using super purified recombinant NS80(335-742) protein as antigen. Moreover,the expressed protein was able to bind to anti-his-tag monoclonal antibody(mouse) and NS80(335-742) specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.
