抗HCMV Pp65蛋白单克隆抗体的制备及性质研究

人类巨细胞病毒（Human cytomegalovirus,HCMV）是一种机会性感染疱疹病毒,在人群中感染比较常见,但在免疫缺陷个体与新生儿中可引起严重的疾病。HCMV Pp65是HCMV活动感染的主要标志,也是临床检验检测HCMV感染的重要靶标。为研发抗HCMV Pp65蛋白单克隆抗体作为临床免疫检测HCMV感染的关键原料,本研究采用重组表达的HCMV Pp65蛋白免疫BALB/c小鼠,将免疫小鼠淋巴结细胞与sp2/0细胞融合,采用间接ELISA法筛选阳性克隆与效价测定,再用Western blotting进行抗体特异性鉴定,最后用免疫捕获PCR和免疫荧光法评价其应用前景。最终获得了1株能稳定分泌高效价的抗HCMV Pp65单克隆抗体的杂交瘤细胞株,命名为8D6。Western blotting及间接ELISA检测其效价分别达1∶4 000和1∶105;细胞免疫荧光与免疫捕获PCR实验结果说明,该杂交瘤细胞株分泌的单克隆抗体具有良好的亲和力和特异性,具有作为外周血细胞免疫荧光细胞化学和免疫捕获PCR检测HCMV临床感染的关键原料,也可作为双抗体夹心法检测HCMV临床感染的关键原料,为建立HCMV感染快速灵敏的临床诊断试剂打下了重要的基础。

Preparation and Characterization of Monoclonal Antibodies against HCMV Pp65 Protein

Human cytomegalovirus（ HCMV） is a kind of opportunistic infection of herpes virus,and a common infection among people,which can cause severe disease in immunodeficiency individuals and neonates. Pp65 protein is the main biomarker and important target for clinical detection of active HCMV infection. To develop the key raw material for immunological detection of HCMV infection,the recombinant expression of HCMV Pp65 protein were used to immunize BALB / c mice,and the lymph node cells of immunized mice were fused with sp2 /0 cells. First of all,the positive clones were screened and the antibody titers were determined by indirect ELISA. And then the monoclonal antibody specificity for HCMV were identified by Western blotting. Finally,immunocapture PCR and immunofluorescence assay for HCMV detection were used to evaluate application prospect of the prepared monoclonal antibody. One hybridoma cell line was achieved,and it can stably secrete monoclonal antibody against HCMV Pp65 with high titers,named 8D6. The antibody titers reached to 1∶ 4 000 and 1∶ 105 when detected by Western blotting and indirected ELISA,respectively. The results of immunofluorescence assay and immunocapture PCR showed that the monoclonal antibody secreted by 8D6 had excellent affinity and specificity. All experimental results indicate that the antibody can be used as the key raw material to establish immunofluorescence cytochemistry and immunocapture PCR for detecting HCMV in peripheral blood cell,and also can be used as the key raw material of double antibody sandwich method for detection of HCMV in clinical infection.which lay an important basis for the studies of the development of the rapid and sensitive clinical diagnosis reagent for HCMV infection.