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| 猪流感病毒（A／swine／Jiangsu／2／2006（H3N2）NS1基因表达产物分析 | |
| 引用本文： | 郭林,王晓杜,刘庆伟,沈阳,邱亚峰,李向东,罗满林,马志永.猪流感病毒（A／swine／Jiangsu／2／2006（H3N2）NS1基因表达产物分析[J].中国兽医寄生虫病,2009,17(1):22-28. |
| 作者姓名： | 郭林 王晓杜 刘庆伟 沈阳 邱亚峰 李向东 罗满林 马志永 |
| 作者单位： | 1. 中国农业科学院上海兽医研究所,上海,200241;华南农业大学兽医学院,广州,510642 2. 中国农业科学院上海兽医研究所,上海,200241 3. 华南农业大学兽医学院,广州,510642 |
| 基金项目： | 上海市浦江人才计划，中央级公益性科研院所基本科研业务费专项基金 |
| 摘 要： | 采用RT—PCR方法克隆猪流感病毒H3N2亚型NS1全长基因，构建NS1基因原核表达质粒pET-28a—NS1和真核表达质粒p3XFLAG—CMV—NS1，在大肠杆菌和真核细胞内表达NS1基因，制备抗NS1多克隆抗体。用所制备的抗体分析p3xFLAG—CMV—NS1转染表达NS1蛋白和病毒感染细胞内的NS1蛋白。经终浓度为1mmol／L的IPTG诱导后，重组蛋白NS1在大肠杆菌中得到表达。表达蛋白纯化后免疫Wistar大鼠制备抗NS1蛋白多克隆抗体，Western—blot分析表明抗NS1多克隆抗体可以识别大肠杆菌表达的NS1蛋白、转染Veio细胞表达的NS1蛋白和病毒感染细胞内的NS1蛋白。间接免疫荧光发现NS1蛋白主要定位于细胞核。结果显示，本试验成功构建了NS1基因原核和真核表达系统，获得了NS1特异性多克隆抗体，分析了NS1细胞定位，为进一步研究NS1蛋白在病毒复制过程中的生物学功能和猪流感病毒的复制机理奠定基础。 |
| 关 键 词： | 猪流感病毒 NS1基因 基因克隆 基因表达 抗体制备 |
ANALYSIS OF NS1 GENE PRODUCTS OF SWINE INFLUENZA VIRUS (A/SWINE/JIANGSU/2/2006(H3N2)) |
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| GUO Lin,WANG Xiao-du,LIU Qing-wei,SHEN Yang,QIU Ya-feng,LI Xiang-dong,LUO Man-lin,MA Zhi-yong.ANALYSIS OF NS1 GENE PRODUCTS OF SWINE INFLUENZA VIRUS (A/SWINE/JIANGSU/2/2006(H3N2))[J].Chinese Journal of Veterinary Parasitology,2009,17(1):22-28. | |
| Authors: | GUO Lin WANG Xiao-du LIU Qing-wei SHEN Yang QIU Ya-feng LI Xiang-dong LUO Man-lin MA Zhi-yong |
| Institution: | GUO Lin, WANG Xiao-du, LIU Qing-wei, SHEN Yang, QIU Ya-feng, LI Xiang-dong, LUO Man-lin, MA Zhi-yong (1. Shanghai Veterinary Research Institute,CAAS, Shanghai 200241, China;2. College of Veterinary Medicine, South China Agricultural University ,Guangzhou 510624,China) |
| Abstract: | To construct recombinant plasmids encoding NS1 gene of swine influenza virus （SIV） for prokaryotic and eukaryotic expression and to analyze the NS1 gene products using the produced NS- specific antibodies,NS1 gene of SIV H3N2 subtype was amplified by RT-PCR using SIV genome RNAs extracted from the allantoic fluid of SlV- infected chicken embryo as template. The amplified product was cloned into the expression vector pET- 28a（ ＋ ） and p3xFLAG-CMV- 7. 1 to construct the recombinant plasmid pET- 28a- NS1 and p3xFLAG- CMV - NS1, respectively. The expression of NS1 was induced and purified for production of polyclonal antibodies against it. The NS1 gene products were detected by Western- blot and immunofluorescence analysis with the produced antibodies. The expression of NS1 protein was induced by IPTG and purified for generation of polyclonal antibodies specific to NSI. Western -blot analysis showed that the produced antibodies were able to react with NS1 proteins expressed in E. coli as well as synthesized in SIV - infected cells. The NS1 proteins were predominantly localized in the nuclei as detected by immunofluorescence analysis. The results indicated that the prokaryotic and eukaryotic expression plasmids encoding NS1 gene of SIV H3N2 subtype were constructed and expressed and the NS1 - specific polyclonal antibodies were produced. The expression and intracellular localization of NS1 protein in virus- infected cells were also analyzed using the NS1 - specific polyclonal antibodies in this study, also. |
| Keywords: | Swine influenza virus NS1 gene gene cloning gene expression production of antibodies |
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