抗人3型副流感病毒核衣壳蛋白单克隆抗体的制备及其活性鉴定

The preparation and evaluation of the monoclonal antibodies against nucleocapsid protein of human parainfluenza virus type 3

目的制备抗人3型副流感病毒核衣壳蛋白特异性单克隆抗体,为研发3型副流感病毒新型诊断试剂提供基础。方法利用重组克隆表达技术,获得可溶性的重组核衣壳蛋白;采用纯化的重组核衣壳蛋白免疫小鼠,采用杂交瘤技术制备抗人3型副流感病毒核衣壳单克隆抗体;采用ELISA及间接免疫荧光法筛选并鉴定可识别天然3型副流感病毒核衣壳蛋白单克隆抗体。结果获得9株分泌抗重组核衣壳蛋白的特异性单克隆抗体的细胞株,其中2株杂交瘤细胞分泌的单克隆抗体能识别天然的3型副流感病毒核衣壳蛋白。结论利用重组核衣壳蛋白作为免疫原制备出了识别天然核衣壳蛋白的单克隆抗体,并且具有较高的反应性。

Objective To prepare the monoclonal antibodies （MAbs） against nucleocapsid protein （NP） of human parainfluenza virus type 3 （ HPIV - 3 ） and evaluate the possible use in the development of early diagnosis reagent for HPIV -3 infection. Methods Recombinant nucleocapsid protein from HPIV -3 were induced for expression in BL21 cells and purified with GST affinity agarose column. BALB/C mice were immunized with purified recombinant NP and the antisera were preliminarily tested for sifting the mice with high immune response. The hybridoma cells were constructed from immunized mice spleen ceils and SP2/0 cells with PEG1500. The supernatant fluid of hybridoma cells was tested for specific monoclonal antibodies by Enzyme -Linked Immunosorbent Assay （ELISA） and indirect immunofluorescence assay （IFA）. Then large yields of the MAbs were induced via cell culture or peritoneal inoculation with hybridoma. The MAbs were evaluated for specific and cross reaction with HPIV - 3 and other common respiratory virus in pediatrics, respectively. Results Nine hybridoma cell lines were constructed for excreting specific monoclonal antibodies against recombinant NP. Two of them could recognize natural HPIV - 3 at titrations of 1 ： 1600 and 1 ： 6400 respectively by IFA. Conclusions The recombinant NP can elicit monoclonal antibodies against natural human parainfluenza virus type 3, and it has the potential to be used in the diagnosis of HPIV -3 infection.