水产学报2013，Vol.37Issue(1)：109-116,8.DOI:10.3724/SP.J.1231.2013.38170
草鱼呼肠孤病毒GCRV-GD108株VP5蛋白功能及免疫原性分析
Analysis of function and immunogenicity of GCRV-GD108 VP5
摘要
Abstract
A strain of grass carp reovirus,named GCRV-GD108 was isolated from sickened grass carp with symptoms of haemorrhage in Guangdong Province. Its whole genomic sequence was obtained. This reovirus consisted of 11 dsRNA segments which encoded 11 proteins instead of 12 proteins that belonged to Aquareovirus (AQRV). Sequence comparison showed that it possessed only 7 homologous proteins to grass carp reovirus ( GCRV) ( with 17. 6% - 45. 8% identities) , but 9 homologs to MRV ( with 15% -46% identities). The virus structure protein VP5 , which was encoded by M5 gene of GCRV-GD108 ,shared a high homology and a conserved motif for NTP binding with the μ2 protein of mammalian reovirus (MRV), suggesting that the VP5 protein is functional homologue of μ2,and possesses NTPase activity. A prokaryotic expression vector of M5 gene had been constructed previously, and the engineering bacteria pET30c-M5/ BL21 ( DE3 ) was selected. According to the requirements of experience, we had chosen appropriate purification method of VP5 protein. The NTPase activity of the purified recombinant VP5 protein was analyzed, using the method of malachite green/ammonium molybdate reagent for quantification of the released Pi of NTP. The result showed that the recombinant protein possessed NTPase activity. The activity of VP5 is dependent on the cations Mg2 + or Na+ /K+ , but its activity was inhibited with the presence of Ca2 +. DNAstar software was used to predict the antigenicity of M5 gene encoded protein. Online prediction based on the analysis results of the three indexes, hydrophilicity, surface probability and antigenic index, showed that 86 regions in the M5 encoding protein potentially form epitopes, suggesting that VP5 possesses strong immunogenicity. The purified recombinant VP5 protein was used to immunize the healthy grass carp, and its protection role was tested by artificial infection of the immunized fish. The serum of the immunized grass carp was analyzed by ELISA and the mRNA level of IgM from the head kidney of grass carp was analyzed by qRT-PCR. These results indicated that recombinant protein VP5 could induce the immunized grass carp to produce high titer antibodies and the expression levels of IgM were significantly improved. However,this protein could not provide protection for GCRV infection. The NTPase activity of GCRV-GD108 VP5 protein was confirmed for the first time,but this protein could not provide immune protection for grass carp. A strain of grass carp reovirus,named GCRV-GD108 was isolated from sickened grass carp with symptoms of haemorrhage in Guangdong Province. Its whole genomic sequence was obtained. This reovirus consisted of 11 dsRNA segments which encoded 11 proteins instead of 12 proteins that belonged to Aquareovirus (AQRV). Sequence comparison showed that it possessed only 7 homologous proteins to grass carp reovirus ( GCRV) ( with 17. 6% - 45. 8% identities) , but 9 homologs to MRV ( with 15% -46% identities). The virus structure protein VP5 , which was encoded by M5 gene of GCRV-GD108 ,shared a high homology and a conserved motif for NTP binding with the μ2 protein of mammalian reovirus (MRV), suggesting that the VP5 protein is functional homologue of μ2,and possesses NTPase activity. A prokaryotic expression vector of M5 gene had been constructed previously, and the engineering bacteria pET30c-M5/ BL21 ( DE3 ) was selected. According to the requirements of experience, we had chosen appropriate purification method of VP5 protein. The NTPase activity of the purified recombinant VP5 protein was analyzed, using the method of malachite green/ammonium molybdate reagent for quantification of the released Pi of NTP. The result showed that the recombinant protein possessed NTPase activity. The activity of VP5 is dependent on the cations Mg2 + or Na+ /K+ , but its activity was inhibited with the presence of Ca2 +. DNAstar software was used to predict the antigenicity of M5 gene encoded protein. Online prediction based on the analysis results of the three indexes, hydrophilicity, surface probability and antigenic index, showed that 86 regions in the M5 encoding protein potentially form epitopes, suggesting that VP5 possesses strong immunogenicity. The purified recombinant VP5 protein was used to immunize the healthy grass carp, and its protection role was tested by artificial infection of the immunized fish. The serum of the immunized grass carp was analyzed by ELISA and the mRNA level of IgM from the head kidney of grass carp was analyzed by qRT-PCR. These results indicated that recombinant protein VP5 could induce the immunized grass carp to produce high titer antibodies and the expression levels of IgM were significantly improved. However,this protein could not provide protection for GCRV infection. The NTPase activity of GCRV-GD108 VP5 protein was confirmed for the first time,but this protein could not provide immune protection for grass carp.关键词
草鱼呼肠孤病毒GCRV-GD108株/重组VP5蛋白/核苷三磷酸酶/活性/免疫原性Key words
grass carp reovirus GD108( GCRV-GD108 ) /recombinant VP5 protein/nucleoside triphosphatase (NTPase)/ enzymatic activity/ immunogenicity分类
农业科技引用本文复制引用
王杭军,叶星,田园园,张莉莉,邓国成..草鱼呼肠孤病毒GCRV-GD108株VP5蛋白功能及免疫原性分析[J].水产学报,2013,37(1):109-116,8.基金项目
广东省重点科技项目(2008A020100016) （2008A020100016）
广东省海洋渔业科技项目(A200899F01,A201101G01) （A200899F01,A201101G01）
