用强化接种后的特殊PBMC和改良永生化技术制备人源抗HBs  

Preparation of human anti-HBs from volunteers with hepatitis B vaccine boost vaccination by modified B-cell immortalization technique

作　　者：娄海波[1] 周元平[1] 周福元[1] 张明霞[1] 魏君锋[1] 曾艳丽[1] 王俊洁[1] 王燕军[1] 吴爱华[1] 侯金林[1] 

机构地区：[1]南方医科大学南方医院感染内科,广东广州510515

出　　处：《南方医科大学学报》2010年第5期949-952,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30771899);广东省自然科学基金面上项目(7005179)

摘　　要：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 immortalized B lymphoblast cell lines （B-LCLs） from healthy anti-HBs antibody （anti-HBs）-positive volunteers and screen for human anti-HBs and the antibody-secreting cells. Methods The peripheral blood mononuclear cells （PBMC） isolated from 3 healthy volunteers positive for anti-HBs with hepatitis B vaccine boost vaccination were infected with Epstein-Barr virus （EBV） and incubated in the presence of CpG DNA motifs and cyclosporin A （CyA）. The anti-HBs in the culture supernatant of the immortalized B-cells was quantified by Architect anti-HBs assay with chemiluminescent microparticle technique. Immunocytochemistry was performed to identify the differentiation of the cell clones expressing anti-HBs. Results Immortalized B-cell culture was successfully established from the cell clones secreting anti-HBs with EBV infection and CpG DNA stimulation. The titer of anti-HBs in the culture supernatant was at its peak at 3 weeks of cell culture and then decreased gradually. At 3 months of cell culture, the cells still retained the capacity of anti-HBs production as verified by the results of immunocytochemistry for CD20 and CD138. Conclusion Immortalized B-cell culture secreting anti-HBs from volunteers receiving boost hepatitis B vaccination has been successfully established by modified EBV immortalization technique.

关 键 词：B细胞永生化 人源单克隆抗体 乙型肝炎表面抗体 

分 类 号：R512.62[医药卫生—内科学] R392.12[医药卫生—临床医学]