登革病毒非结构蛋白NS1群特异性单克隆抗体的制备及初步应用

目的制备登革病毒NS1群特异性单克隆抗体,建立可检测登革病毒1～4型NS1抗原的ELISA检测法,为登革热的早期快速诊断奠定基础。方法应用毕赤酵母表达系统分泌表达登革病毒2型重组非结构蛋白NS1,以此为抗原免疫BABL/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,经HAT选择培养、间接ELISA筛选和亚克隆,获得能稳定分泌登革病毒NS1群特异性单克隆抗体的杂交瘤细胞株;用所获得的单克隆抗体建立可检测1～4型登革病毒NS1抗原的双抗体夹心ELISA法。结果从登革病毒2型NS1重组毕赤酵母（Pichia Pastoris-NS1）中获得了大量纯化的登革病毒重组NS1蛋白;经免疫小鼠、细胞融合、间接ELISA筛选及3次亚克隆后,最终获得2株能高效分泌抗登革病毒NS1单克隆抗体的杂交瘤细胞株2D7B6B4和2D10E2F6,间接ELISA显示抗体效价高达1∶8000～1∶16000;ELISA及免疫荧光检测证实,其所分泌的抗体与1～4型登革病毒及其重组NS1蛋白均有特异性免疫反应,为登革病毒NS1群特异性单克隆抗体;两株单克隆抗体均为IgG2a亚类;初步建立了检测4个血清型登革病毒NS1抗原的双抗体夹心ELISA法。结论成功研制出两株能高效分泌抗登革病毒NS1群特异性单克隆抗体的杂交瘤细胞株。初步建立了可检测1～4型登革病毒NS1抗原的双抗体夹心ELISA检测法。

The preparation and preliminary application of the group-specific monoclonal antibodies against the nonstructural glycoprotein 1 of dengue virus

Objective Prepare the group specific monoclonal antibodies（McAbs） against DENV NS1 antigens, to establish a double antibody sandwich ELISA which could detect the NS1 antigen of DENV 1-4. Methods BABL/c mice were immunized with recombinant DENV2-NS1 protein, which is expressed in Pichia Pastoris. Then, the splenocyte were collected and fused with mouse myeloma cell SP2/0 by PEG. After the indirect ELISA screening and sub-cloning, hybridoma cell lines which could stably secrete group specific anti-NS1 mouoclonal antibodies were prepared.A double antibody sandwich ELISA to detect the DENV 1-4 NS1 protein was established. Results Plenty of purified recombinant DENV2-NS1 proteins were expressed and 2 hybridoma cell lines, 2D7B6B4 and 2D10E2F6 were obtained after traditional hybridomas technology.It was detected that both of them could co-react with DENV 1-4 and its NSls. The titer which was detected by indirect ELISA was 1：8 000-1：16 000. The subtype of immunoglobulin was IgG2a. An early rapid testing method was initially established. Conclusion Successfully prepared two cross-serotype monoclonal antibodies that co-reactive with DENV 1-4 and the NSls. A double antibody sandwich ELISA was established, making the great foundation of early rapid diagnosis to dengue fever.