人流感病毒核蛋白的重组表达及其抗原性分析

Recombinant expression of human influenza A virus nucleocapsid protein and its antigenicity analyses

目的 探索用原核表达的人甲3型流感病毒核蛋白（NP）免疫小鼠所获得的抗重组蛋白抗体检测甲型流感病毒的可行性。方法 用Clustal X，Antheprot等软件，对IFV-A3 NP进行分析，确定保守区并分析其抗原性。RT-PCR获得NP基因，分3段克隆NP基因到PET-28（C），并在BL21中诱导表达。经Ni-Agarose亲和层析柱纯化后，免疫BALB／c鼠制备抗重组蛋白抗体。用Western Blot和免疫组化法检测抗重组蛋白抗体与病毒抗原的反应性。结果 重组质粒在BL21中高效表达，表达量为15～20mg／L菌液。Western Blot检测显示，抗NP1、NP2、NP3多克隆抗体（1：2000）均对流感病毒NP具有反应性。免疫组化检测也显示，抗NPI、NP2、NP3多克隆抗体对IFV-A3、-Al感染的MDCK细胞有特异性染色，抗体滴度从1：640到1：1280。结论 重组表达的NP能够诱导产生高效价多克隆抗体，所产生的抗体与IFV-A3、Al具有交叉免疫反应性。通过生物信息学分析预测抗原性并制备多克隆抗体是可行的，并可望用于流感病毒感染的早期快速诊断的研究中。

Objective To recombinant nucleocapsid protein （NP） prepare anti-recombinant protein of human influenza A3 （ IFV-A3 ） antibody from immunized mice with virus expressed in prokaryotic cell, and to explore the feasibility of utilizing anti-recombinant protein antibody to detect influenza A virus. Methods NP genes of human influenza A virus were analyzed with computer softwares of ClustalX, Antbeprot, et al. to determine the antigenicity in conserved regions. Three different partial NP genes were harvested and cloned into pET-28（c） plasmid, the recombinant plasmids were induced to express partial NP segments in BL21 cells. The recombinant proteins were purified with Ni-agarose by affinity chromatography and immunized BALB/c mice. The polyclonal antisera harvested from mice were analyzed with Western Blot and immunohistocbemistry assays to detect the reactions with IFV-A. Results Three recombinant plasmids were expressed with high yield in BL21 cells, about 15-20 mg/L. Western Blot results indicated that the three prepared antisera （1：2000） positively reacted with NP from IFV-A3-infected cells. And immunohistochemistry assays suggested that anti-NP1, anti-NP2, anti-NP3 antisera positively reacted with IFV-A3 or IFV-Al-infected MDCK cells, with titers of 1 ： 640 to 1 ： 1280. Conelusion The recombinant NP of IFV-A3 would induce polyclonal antibodies with high titers in mice. The polyclonal antibodies would cross-react with IFV-A3 and IFV-A1. It is feasible to predict the antigenicity with systematical bioinformatics analyses and then induce anti-IFV antibodies with high dilutions, and it is possible to be utilized in the early detection and subtyping analyses of IFV-infections.