Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprise...Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.展开更多
To overexpress EBNA-1 in E.coli and generate the specific antibody,in this study,the antigenicity,epitope and hydrolysis of EBNA-1 were analyzed using the computer design software Biosun.Based on the prediction by com...To overexpress EBNA-1 in E.coli and generate the specific antibody,in this study,the antigenicity,epitope and hydrolysis of EBNA-1 were analyzed using the computer design software Biosun.Based on the prediction by computer analysis,the sequence encoding EBNA-1385-557 was amplified by PCR with the specific primers.The expression vector containing EBNA-1385-557 coding sequence was constructed.His-tagged EBNA-1385-557 was expressed in E.coli.The soluble recombinant protein was purified using Ni-NTA chromatography.The purified protein was used as antigens to immunize mice and screen the antibodies,which will serve as an important tool for further studies.展开更多
PCR technique is used to amplify the mature peptide gene of human transforming growth factor β1(hTGFβ1); the gene is verified by full length sequence analysis. In DH5α/pBV220 expression system, hTGFβ1 attains expr...PCR technique is used to amplify the mature peptide gene of human transforming growth factor β1(hTGFβ1); the gene is verified by full length sequence analysis. In DH5α/pBV220 expression system, hTGFβ1 attains expression in the cytoplasm of \%E. coli\% up to 16%. The recombinant protein is proved to be the monomer of hTGFβ1 by N terminal amino acids analysis and immunoblotting. After refolding of the monomer protein \%in vitro\% in glutathione system or CHPAS/DMSO system, the dimeric protein accumulates to 30% in the refolding mixture. The recombinant protein is purified to homogeneity on silver staining, and is shown to have strong biological activity from MTT bioassay on Mv1Lu cells.展开更多
Through mRNA extract, RT and a series of PCR, phage antibody libraries were constructed from rP27kip1-immunized and non-immunized mice. After only one round of selection with rP27kip1, clones from each library were ch...Through mRNA extract, RT and a series of PCR, phage antibody libraries were constructed from rP27kip1-immunized and non-immunized mice. After only one round of selection with rP27kip1, clones from each library were chosen randomly and digested by Tag I and Hinf I. 11 of 64 clones from the immunized animal had consistent restriction pattern, while none of the 64 clones from the non-immunized animal had, except that one had the same fragments pattern as that of the 11 clones. The 12 fragments were expressed in E. coli BL2l(DE3)/pET-28b( + ) system. ELISA showed that some of the fragments could bind to rP27kip1 specifically. All these results implied that specific antibody can be obtained by genetic engineering without hybridoma or many rounds of growth and panning selection.展开更多
基金National Basic Research Program ofChina (973 Program) (2009CB118701)National NaturalScientific Foundation of China (30671615, 30871940)+1 种基金Innovation Project of the Chinese Academy of Sciences(KSCX2-YW-N-021)Science and Technology Foundation of Zhejiang Province (2007C22052)
文摘Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.
基金supposed by National Basic Research Program of China(973 Program)(2006CB504305)Beijing Natural Science Foundation(No.7051006).
文摘To overexpress EBNA-1 in E.coli and generate the specific antibody,in this study,the antigenicity,epitope and hydrolysis of EBNA-1 were analyzed using the computer design software Biosun.Based on the prediction by computer analysis,the sequence encoding EBNA-1385-557 was amplified by PCR with the specific primers.The expression vector containing EBNA-1385-557 coding sequence was constructed.His-tagged EBNA-1385-557 was expressed in E.coli.The soluble recombinant protein was purified using Ni-NTA chromatography.The purified protein was used as antigens to immunize mice and screen the antibodies,which will serve as an important tool for further studies.
文摘PCR technique is used to amplify the mature peptide gene of human transforming growth factor β1(hTGFβ1); the gene is verified by full length sequence analysis. In DH5α/pBV220 expression system, hTGFβ1 attains expression in the cytoplasm of \%E. coli\% up to 16%. The recombinant protein is proved to be the monomer of hTGFβ1 by N terminal amino acids analysis and immunoblotting. After refolding of the monomer protein \%in vitro\% in glutathione system or CHPAS/DMSO system, the dimeric protein accumulates to 30% in the refolding mixture. The recombinant protein is purified to homogeneity on silver staining, and is shown to have strong biological activity from MTT bioassay on Mv1Lu cells.
文摘Through mRNA extract, RT and a series of PCR, phage antibody libraries were constructed from rP27kip1-immunized and non-immunized mice. After only one round of selection with rP27kip1, clones from each library were chosen randomly and digested by Tag I and Hinf I. 11 of 64 clones from the immunized animal had consistent restriction pattern, while none of the 64 clones from the non-immunized animal had, except that one had the same fragments pattern as that of the 11 clones. The 12 fragments were expressed in E. coli BL2l(DE3)/pET-28b( + ) system. ELISA showed that some of the fragments could bind to rP27kip1 specifically. All these results implied that specific antibody can be obtained by genetic engineering without hybridoma or many rounds of growth and panning selection.
