抗禽流感病毒NS1蛋白单克隆抗体的制备及其抗原表位的初步分析

Preparation of monoclonal antibodies against NS1 of avian influenza virus and the antigenic epitope identification

为制备抗禽流感病毒(AIV)NS1蛋白的单克隆抗体(MAb)及鉴定其抗原表位鉴定,本研究以原核表达并纯化的NS1重组蛋白免疫BALB/c小鼠,采用淋巴细胞杂交瘤技术制备杂交瘤细胞,通过间接ELISA进行筛选,制备的D7和D9 2株能够稳定分泌抗NS1蛋白MAb的杂交瘤细胞,亚型鉴定均为IgG1型,轻链均为κ链。Western blot鉴定表明,这2株MAbs均能够识别NS1重组蛋白。间接免疫荧光鉴定表明,这2株MAbs均能够识别真核表达的NS1蛋白。利用噬菌体展示技术得到D9对应的短肽WNLNTV,与NS1蛋白aa 182～aa 187基本匹配,提示182WNDNTV187为NS1蛋白的一个线性表位。该结果为进一步研究NS1蛋白的结构和功能以及建立诊断方法奠定了基础。

To prepare monoclonal antibodies （MAb） against NS1 of avian influenza virus （AIV）, BALB/c mice were immunized with purified recombinant NS1 expressed in E.coli. The immunized mice spleen ceils were.fused with SP2/0, and then 2 MAbs （D7 and D9） against NS1 were screened by indirect ELISA. The 2 MAbs were belonging to IgG1 subclass and K light chain. Western blot assay showed that the MAbs were reacted with recombinant NS1. Indirect immunofluorescence assay indicated that the MAbs reacted with NS1 expressed in SF9 cells transfected with eukaryocyte recombinant plasmids. Furthermore, screening a phage display random 7-mer peptide library with the 2 MAbs, the MAb D9 recognized phages displaying peptides with the consensus peptide WNLNTV, which was substantially matched with IWWNDNTV^7 of AIV NS1. The result suggested that the peptide is a linear epitope of NS1, which facilitate further study on the function of NS1 and diagnostic method development for AIV detection.