人高致病性H5N1亚型禽流感病毒NS1蛋白多克隆抗体的制备及鉴定  

Preparation and characterization the polyclonal antibody of the nonstructural protein of human highly pathogenic H5N1 avian influenza viruses

作　　者：蒋培余[1] 黄惠莲[1] 周洪昌[1] 徐伯赢[1] 顾福萍[1] 闵丽姗[1] 钟婧[1] 戴利成[1] 

机构地区：[1]湖州师范学院医学院,湖州市分子医学重点实验室,浙江省湖州市313000

出　　处：《中华实验和临床病毒学杂志》2013年第2期138-140,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：浙江省湖州市一般科技计划项目（2009YS15）

摘　　要：目的制备高灵敏度和高特异性的人高致病性H5N1亚型禽流感病毒NS1蛋白抗体并对其效价进行初步评估。方法构建含有H5N1亚型禽流感病毒NS1序列的pET一28a（＋）重组载体的大肠埃希菌BL21（DE3），诱导表达NS1蛋白，并经Ni-NTA色谱柱亲和层析纯化获得NS1重组蛋白，并进行SDS-PAGE和WesternBlot鉴定。以纯化的蛋白为抗原免疫新西兰大白兔，获得兔抗NS1血清，亲和纯化获得多克隆抗体。应用ELISA和WesternBlot检测纯化抗体的效价和特异性。结果NS1融合蛋白得到高表达，且纯度〉90％，用该融合蛋白免疫新西兰大白兔后得到的抗NS1多克隆抗体，效价达1：80000，并特异性识别H5N1亚型禽流感病毒NS1蛋白。结论获得了NS1多克隆抗体，具有较好的效价和特异性。Objective Of this study was to prepare high sensitivity and high specificity of highly pathogenic H5N1 subtype avian influenza virus NS1 protein antibody and a preliminary assessment of its potency. Methods Construct pET-28a （ ＋ ） recombinant vector containing the H5N1 subtype of avian influenza virus NS1 sequences of E. coli BL21 （DE3） , induced expression of NS1 protein, NS1 recombinant protein was obtained by Ni-NTA column purified by affinity chromatography, and SDS-PAGE and Western Blot analysis. Purified protein antigen to immunize New Zealand white rabbits, obtained rabbit anti-NS1 serum, affinity-purified polyclonal antibodies. Using ELISA and Western Blot analysis of purified antibody titer and specificity. Results NS1 fusion protein was highly expressed in a purity of greater than 90% , with the fusion protein was used to immunize New Zealand white rabbits anti-NS1 polyelonal antibody titer of 1：80 000, and specific recognition of the H5N1 subtype of avian influenza virus NSI protein. Conclusions NS1 polyclonal antibodies to NS1 recombinant protein purified antigen, with better potency and specificity, and to prepare the conditions for the development of the H5N1 subtype of avian influenza virus deteetion kit.

关 键 词：流感 序列分析 蛋白质 抗体 病毒 克隆 分子 

分 类 号：R3[医药卫生—基础医学]