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目的建立乙肝表面抗体(抗HBs)阳性者EBV永生化的B淋巴母细胞系(B-LCLs),筛选、获得人源抗HBs及其分泌细胞。方法常规技术获得乙肝疫苗强化免疫后志愿者的特异性外周血单核细胞(PBMC),EBV转染,加入CpGDNA免疫调节基序诱导B淋巴细胞增殖,环孢菌素A(CyA)抑制T淋巴细胞活性。雅培法定量动态检测永生化细胞培养上清中的抗HBs滴度,取高滴度抗HBs孔的B-LCLs细胞有限稀释法培养,用免疫组化SP法观察细胞分化状况及抗HBs表达。结果成功构建了分泌抗HBs的永生化B-LCLs;培养上清抗HBs滴度维持较高水平3周左右,之后逐渐降低;免疫组化鉴定B-LCLs细胞培养3个月时,永生化细胞仍然为抗HBs合成细胞。结论采用改进的EBV永生化技术转化强化接种后获得的特殊PBMC,可高效获得永生化的B-LCLs,并有效表达抗HBs。
Objective To establish an EBV-immortalized B lymphoblastoid cell line (B-LCLs) with positive HBsAg positive (HBsAg) -binding and to screen for human anti-HBs and its secreted cells. Methods The specific peripheral blood mononuclear cells (PBMCs) of volunteers after hepatitis B vaccine immunization were obtained by conventional techniques. EBV transfection was performed. The CpG DNA immunomodulatory motif was added to induce the proliferation of B lymphocytes. Cyclosporin A (CyA) Cell activity. Abbott method was used to quantitatively detect anti-HBs titer in immortalized cell culture supernatant. B-LCLs with high titer anti-HBs were cultured in a limited dilution method. The differentiation and anti-HBs expression were observed by immunohistochemical SP method. Results The immortalized B-LCLs secreting anti-HBs were successfully constructed. The anti-HBs titer in the culture supernatant was maintained at a high level for about 3 weeks and then gradually decreased. Immunohistochemistry showed that when the B-LCLs were cultured for 3 months, the immortalized cells Still for anti-HBs synthetic cells. Conclusion The improved EBV immortalization technique can transform immortalized B-LCLs efficiently and express anti-HBs efficiently by using the special PBMC obtained after vaccination.