[Objective] To isolate three strains of avian leukosis virus subgroup J（ALV -J） , and then amplify and sequence the gp85 gene. [ Method] Three strains of ALV- J were isolated from Hubei Province, which were identifi...[Objective] To isolate three strains of avian leukosis virus subgroup J（ALV -J） , and then amplify and sequence the gp85 gene. [ Method] Three strains of ALV- J were isolated from Hubei Province, which were identified by pathological anatomy, DF- 1 cell culture and RT- PCR. And then they were named HB1002, HB1003 and HB1009, respectively. [ Result] Test sequence analysis showed, the length of gp85 gene was 921 bp, consistent with expect result; the nucleotide homology between the three isolates was in 97.7% -99.7% ,the homology of amino acid was in 95.1% - 99%. The nucleotide homology between HPRS - 103 and the three isolates was in the 94.1% - 94.8% ; and the nucleotide homolo- gy between other ALV -J and the three isolates was in 87.6% -97.3%. The phylogenetic trees analysis showed that the homology of JS09GY6 vi- rus and the three isolates was nearest, in 95.2% -97.3%. [ Conclusion] In the test, the three strains of virus which were isolated were ALV- J.展开更多
The envelope gene gp85 of ev/J, a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian leukosis virus. This repor...The envelope gene gp85 of ev/J, a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian leukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC10200 strain).展开更多
Subgroup J Avian leucosis virus (ALV-J) strain NX0101 was inoculated into chicken embryo fibroblasts (CEF) monolayers in 6-well plates. The six wells of CEF inoculated with NX0101 were divided into groups A (without a...Subgroup J Avian leucosis virus (ALV-J) strain NX0101 was inoculated into chicken embryo fibroblasts (CEF) monolayers in 6-well plates. The six wells of CEF inoculated with NX0101 were divided into groups A (without anti-ALV-J serum in the medium); B (with anti-ALV-J serum in the medium), then viruses from each well of both groups were separately passed in CEF every 6 d; formed their independent passage lineages. For each lineage of both groups, gp85 genes of the viruses in the 10th, 20th; 30th passages were amplified, cloned; sequenced. The sequence data indicated that the homologies of gp85 at aa level between the primary virus; the passed viruses of different passages of 3 lineages in group A were 97.7%–99.7%;; the homologies of gp85 between the primary virus; the passed viruses of different passages of 3 lineages in group B were 93.8%–96.1%. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S in 3 highly variable (hr-) regions at aa#110–120, aa#141–151; aa#189–194 of gp85 in 3 lineages of group A were 2 (8/4), 1(3/3); 1.3 (4/3), however, NS/S in the same 3 hr-regions of group B were 4.1 (13/3), 4.7 (14/3); 3.3 (11/3). This study is the first demonstration of influence of immune selective pressure on evolution of ALV-J gp85 by specific antibodies under the controlled in vitro experiments.展开更多
为了解禽白血病病毒(ALV)gp85基因的特点,采用Primer Premier 5.0设计一对特异性引物,对gp85基因进行扩增、克隆及序列测定,用相关生物学软件对贵州流行株gp85基因进行序列分析,并对其编码蛋白进行二级结构、B细胞表位、保守结构域、跨...为了解禽白血病病毒(ALV)gp85基因的特点,采用Primer Premier 5.0设计一对特异性引物,对gp85基因进行扩增、克隆及序列测定,用相关生物学软件对贵州流行株gp85基因进行序列分析,并对其编码蛋白进行二级结构、B细胞表位、保守结构域、跨膜结构域和信号肽预测。测序获得921 bp长的序列,将此流行株命名为ALV-GZ-16。分析发现,流行株ALV-GZ-16的gp85基因核苷酸序列与J亚群中国分离毒株的gp85基因序列同源性为97.5%~99.9%,其编码的氨基酸序列与J亚群中国分离毒株的同源性为96.8%~99.7%;系统进化分析显示,该流行株与J亚群原型毒株处于同一分支,与贵州分离株GZN49处于同一小分支;其二级结构中无规则卷曲和β-折叠所占比例较大;预测此蛋白存在4个B细胞优势抗原表位,无跨膜结构和信号肽区域。研究结果进一步完善了贵州省禽白血病病毒流行株的生物学特性,为gp85蛋白特异性抗体的制备提供一定的理论基础。展开更多
基金Plan of Hubei Province Science and Technology Research and Development Project:the Research and Demonstration of Comprehensive Prevention and Control Technology on Poultry Avian Influenza and other Major Diseases YJN0065the Construction Fund of Modern Agriculture Industry Technology System CARS-42-G11
文摘[Objective] To isolate three strains of avian leukosis virus subgroup J（ALV -J） , and then amplify and sequence the gp85 gene. [ Method] Three strains of ALV- J were isolated from Hubei Province, which were identified by pathological anatomy, DF- 1 cell culture and RT- PCR. And then they were named HB1002, HB1003 and HB1009, respectively. [ Result] Test sequence analysis showed, the length of gp85 gene was 921 bp, consistent with expect result; the nucleotide homology between the three isolates was in 97.7% -99.7% ,the homology of amino acid was in 95.1% - 99%. The nucleotide homology between HPRS - 103 and the three isolates was in the 94.1% - 94.8% ; and the nucleotide homolo- gy between other ALV -J and the three isolates was in 87.6% -97.3%. The phylogenetic trees analysis showed that the homology of JS09GY6 vi- rus and the three isolates was nearest, in 95.2% -97.3%. [ Conclusion] In the test, the three strains of virus which were isolated were ALV- J.
基金Natural Science Foundation of China (30460098)China Postdoctoral Science Foundation funded project (2005038585)
文摘The envelope gene gp85 of ev/J, a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian leukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC10200 strain).
文摘Subgroup J Avian leucosis virus (ALV-J) strain NX0101 was inoculated into chicken embryo fibroblasts (CEF) monolayers in 6-well plates. The six wells of CEF inoculated with NX0101 were divided into groups A (without anti-ALV-J serum in the medium); B (with anti-ALV-J serum in the medium), then viruses from each well of both groups were separately passed in CEF every 6 d; formed their independent passage lineages. For each lineage of both groups, gp85 genes of the viruses in the 10th, 20th; 30th passages were amplified, cloned; sequenced. The sequence data indicated that the homologies of gp85 at aa level between the primary virus; the passed viruses of different passages of 3 lineages in group A were 97.7%–99.7%;; the homologies of gp85 between the primary virus; the passed viruses of different passages of 3 lineages in group B were 93.8%–96.1%. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S in 3 highly variable (hr-) regions at aa#110–120, aa#141–151; aa#189–194 of gp85 in 3 lineages of group A were 2 (8/4), 1(3/3); 1.3 (4/3), however, NS/S in the same 3 hr-regions of group B were 4.1 (13/3), 4.7 (14/3); 3.3 (11/3). This study is the first demonstration of influence of immune selective pressure on evolution of ALV-J gp85 by specific antibodies under the controlled in vitro experiments.
文摘为了解禽白血病病毒(ALV)gp85基因的特点,采用Primer Premier 5.0设计一对特异性引物,对gp85基因进行扩增、克隆及序列测定,用相关生物学软件对贵州流行株gp85基因进行序列分析,并对其编码蛋白进行二级结构、B细胞表位、保守结构域、跨膜结构域和信号肽预测。测序获得921 bp长的序列,将此流行株命名为ALV-GZ-16。分析发现,流行株ALV-GZ-16的gp85基因核苷酸序列与J亚群中国分离毒株的gp85基因序列同源性为97.5%~99.9%,其编码的氨基酸序列与J亚群中国分离毒株的同源性为96.8%~99.7%;系统进化分析显示,该流行株与J亚群原型毒株处于同一分支,与贵州分离株GZN49处于同一小分支;其二级结构中无规则卷曲和β-折叠所占比例较大;预测此蛋白存在4个B细胞优势抗原表位,无跨膜结构和信号肽区域。研究结果进一步完善了贵州省禽白血病病毒流行株的生物学特性,为gp85蛋白特异性抗体的制备提供一定的理论基础。
