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目的:制备特异性的抗人类微小病毒B19单克隆抗体(mAb)杂交瘤。方法:采用PCR从B19病毒感染患者的血清标本中扩增编码VP2蛋白的基因,经克隆、测序确认后,再分别亚克隆至原核表达载体和真核表达载体。以从SDS-PAGE凝胶中回收的原核表达产物免疫BALB/c小鼠,制备可分泌特异性B19mAb的杂交瘤细胞。以真核表达载体转染CHO细胞后,筛选单细胞表达克隆,将真核表达产物与用原核表达产物为免疫原制备的mAb反应。结果:从患者血清标本中扩增出了1665bp长的DNA,测序结果证实为vp2基因。将该基因克隆至原核表达载体中后,在大肠杆菌中获得了表达。表达产物经SDS-PAGE后,在Mr约为6000处可见1条明显的表达带。Westernblot的结果表明,该电泳带与B19患者的血清呈特异性免疫反应。以从SDS-PAGE凝胶中回收的表达产物为免疫原,免疫BALB/c小鼠,经细胞融合、克隆化,获得2株分泌特异性的抗VP2mAb的杂交瘤细胞。以这2株杂交瘤细胞制备的腹水可与SDS-PAGE凝胶Mr6000的蛋白条带也出现很强的免疫反应。用ELISA证明以Vp2基因真核表达载体转染的CHO培养上清,也与筛选到的mAb呈阳性反应。结论:成功地制备抗VP2的mAb,为进一步研制可用于B19病毒感染早期免疫诊断试剂盒奠定了基础。
Objective: To prepare a specific anti-human parvovirus B19 monoclonal antibody (mAb) hybridoma. Methods: The gene encoding VP2 protein was amplified by PCR from the serum of patients with B19 virus infection. After cloning and sequencing, the gene was subcloned into prokaryotic and eukaryotic expression vectors respectively. BALB / c mice were immunized with the prokaryotic expression product recovered from the SDS-PAGE gel to prepare a hybridoma cell secreting a specific B19 mAb. After transfection of CHO cells with the eukaryotic expression vector, the single cells are screened to express the eukaryotic expression product and the mAb prepared by using the prokaryotic expression product as an immunogen. Results: 1665bp DNA was amplified from the serum samples of patients and the sequencing result was confirmed as vp2 gene. After cloning this gene into prokaryotic expression vector, expression was obtained in E. coli. After SDS-PAGE of the expression product, a clear expression band was found at Mr about 6000. The results of Western blot showed that this band was specifically immunoreactive with the serum of B19 patients. BALB / c mice were immunized with the expression product recovered from the SDS-PAGE gel as an immunogen. After cell fusion and cloning, two hybridoma cells secreting specific anti-VP2 mAb were obtained. Ascites prepared from these two hybridoma cells also showed strong immunoreactivity with the protein bands of SDS-PAGE gel Mr6000. The culture supernatant of CHO cells transfected with the eukaryotic expression vector Vp2 was also proved to be positive by ELISA. Conclusion: The successful preparation of anti-VP2 mAb lays the foundation for the further development of an immunodiagnostic kit for B19 virus infection.