新疆出血热病毒核蛋白NP的原核表达及其单克隆抗体的研制  

Expression of NP protein of crimean-congo hemorrhagic fever virus in E.coli and preparation of monoclonal antibodies against NP protein

作　　者：师永霞[1] 相大鹏[1] 孟逊 李小波[1] 洪烨[1] 黄吉城[1] 郑夔[1] 

机构地区：[1]广东出入境检验检疫局检验检疫技术中心,广州510700 [2]上海艾比玛特生物医药有限公司,上海200032

出　　处：《中国卫生检验杂志》2009年第6期1309-1311,共3页Chinese Journal of Health Laboratory Technology

基　　金："十一五"国家科技支撑计划项目(2006BAK10B07)

摘　　要：目的：利用原核表达系统表达新疆出血热病毒（CCHFV）核衣壳蛋白NP,并通过杂交瘤细胞技术制备其单克隆抗体。方法：利用pET大肠杆菌系统表达具有抗原性的重组NP蛋白,经包涵体纯化后免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0融合,间接ELISA筛选阳性的杂交瘤细胞,并结合Western blot和免疫荧光试验对抗体的特异性进行鉴定,筛选与NP蛋白有强结合能力且配对结果较好的一对单克隆抗体。结果：经间接ELISA筛选阳性的杂交瘤细胞,获得2株能稳定分泌抗NP蛋白抗体、配对效果较好的杂交瘤细胞株,将其命名为10B12和3G5。Western blot试验证明它们免疫小鼠后腹水中的单克隆抗体能特异性识别NP蛋白,免疫荧光试验显示两个单克隆抗体均与表达NP蛋白的杆状病毒感染的昆虫细胞抗原片有特异性荧光。双抗体间接ELISA表明它们可检测的重组NP蛋白的线性范围为1～20 ng,检测灵敏度为1 ng。结论：成功获得了针对NP蛋白的特异性单克隆抗体,为进一步研究新疆出血热免疫胶体金的研发奠定必要的物质基础。Objective ： To express NP protein of crimean - congo hemorrhagic fever virus （CCHFV） in Escherichia coli and ob- tain monoclonal antibodies （MABs） against NP protein. Methods：The recombinant NP protein was expressed in E. coli and purified. BALB/C mice were immunized with recombinant NP Protein. Hybridoma cell lines secreting MABs against NP protein were screened by regular cell fusion and indirect ELISA approach. The specificities of these MABs were determined by Western blot and immunofluorescecence assay. Results ：Two hybridoma cell lines （10B12 and 3G5 ） stable in secreting specific MABs were successfully obtained. Western blot showed that two MABs from mouse ascites could bind specifically to NP protein. Indirect immunofluorescence assay indicated that they could specifically bind to NP protein expressed in the recombinant baculovirus and the specific fluorescene could be observed in infected insect cells. ELISA with 10B12 MAB as the first antibody and HRP -3G5 MAB as the second antibody showed that the linear range of the detected NP protein was from 1 to 20 ng. Conclusion： MABs of high specificity against NP protein have been successfully prepared, which lay the foundation for colloidal gold immunoassay of diagnosing CCHFV.

关 键 词：新疆出血热病毒 核衣壳蛋白 单克隆抗体 

分 类 号：R512.8[医药卫生—内科学]