利用单个细胞RT-PCR法克隆全人源性HBV单克隆抗体的探讨  

Preparation of human hepatitis B virus monoclonal antibodies using single cell RT-PCR

作　　者：李特特[1] 吴静[1] 吕信萍 周豪[2] 陈京涛[1] 

机构地区：[1]吉林大学白求恩第一医院转化医学研究院,长春130061 [2]吉林大学白求恩第一医院肝胆胰内科,长春130021

出　　处：《临床肝胆病杂志》2016年第12期2312-2316,共5页Journal of Clinical Hepatology

基　　金：科技部"十二五"艾滋病和病毒性肝炎等重大传染病防治项目子课题(2013ZX10004609-004);吉林省发展与改革委员会项目(2014N147)

摘　　要：目的建立外周血单个抗体分泌细胞分离和单个细胞RT-PCR系统,高效快速克隆高度特异性、全人源性的HBV单克隆抗体。方法选择3例HBV感染恢复期患者,从外周血中富集B淋巴细胞,加入HBcAg Peptide pool活化B淋巴细胞,流式分选出记忆性抗体分泌细胞(CD19^+CD10^-IgG^+CD27^+),通过有限稀释法获得单个细胞,单细胞RT-PCR反转出cDNA,巢式PCR扩增抗体重链恒定区序列片段,将PCR产物纯化并克隆至pEASY~-T1 simple克隆载体,进行测序鉴定。结果 ELISA结果显示,3例HBV感染恢复期患者血浆中均检测到较高水平IgG,与健康志愿者相比,差异均有统计学意义(P值均<0.05)。流式结果显示,B淋巴细胞比例均高于94%,且均可分离出记忆性抗体分泌细胞。从分离的抗体分泌细胞中挑选出24个含有1个形态完好的细胞,经鉴定,其中有14个扩增成功,且条带在200 bp左右,与预期片段大小相符。对抗体重链序列分析显示成功扩增出抗体恒定区片段。结论成功构建了单个抗体分泌细胞分离和单个细胞RT-PCR系统,获得了抗体重链序列,为后期全人源性乙型肝炎单克隆抗体的高通量生产打下了坚实的基础。Objective To establish the system for the isolation of peripheral single antibody- secreting cells（ ASCs） and single cell RT-PCR system,and to clone highly specific human hepatitis B monoclonal antibody efficiently and rapidly. Methods Three patients with HBV infection in the recovery stage were enrolled. Peripheral B cells were collected and activated by HBc Ag Peptide pool. Flow sorting was performed to obtain memory ASCs（ CD19~＋CD10^-IgG~＋CD27~＋）,the limiting dilution method was used to obtain single cells,single cell RT-PCR was used to obtain cDNA,and nested PCR was used for the amplification of the fragment in the constant region of the heavy chain of antibody. PCR product was purified and cloned to pEASY~- T1 simple cloning vector for sequencing and identification. Results The results of ELISA showed that a high level of IgG was observed in all three patients with HBV infection in the recovery stage. There were significant differences in peripheral IgG level between patients and healthy volunteers（ both P〈0. 05）. The results of flow sorting showed that the proportion of B cells was higher than 94% and memory ASCs were isolated. A total of 24 ASCs containing at least one cell with normal morphology were selected,and after identification,14 were successfully amplified,with a size of about 200 bp,which was consistent with expected results. The analysis of the sequence of the heavy chain of antibody showed successful amplification of the fragment in the constant region of the antibody. Conclusion The system for the isolation of single ASCs and single cell RT- PCR system are successfully established and the sequence of the heavy chain of antibody is obtained,which lays a solid foundation for the high- throughput production of human hepatitis B monoclonal antibodies in future.

关 键 词：肝炎病毒 乙型 抗体生成细胞 抗体 单克隆 逆转录聚合酶链反应 

分 类 号：R512.62[医药卫生—内科学] Q785[医药卫生—临床医学]