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目的快速克隆全人源乙型肝炎病毒表面抗体可变区序列,为构建乙肝病毒表面抗体表达载体、筛选并制备治疗性全人源乙型肝炎单克隆抗体提供基础。方法 20μg乙肝疫苗(GSK公司)强化免疫1名乙肝表面抗体阳性的健康志愿者,7d后,检测其乙肝表面抗体滴度大于1 000 mIU/ml,采集30 ml肝素抗凝血,采用流式细胞仪分选出CD19+/CD27+/IgG+细胞群,挑选96个单细胞,置于20μl单细胞裂解液中。采用混合引物单细胞RT-PCR获得重链、轻链可变区序列,将PCR产物纯化后克隆至pMD19-T载体,进行测序。对测序结果采用DNA Star 5.0 MegAlign和IgBLAST进行相似性和同源性分析。结果成功筛选出12个阳性浆细胞,12个阳性细胞的轻链和重链PCR产物序列具有很高的同源性。结论成功筛选出单个浆细胞;获得了可能的HBsAb重链、轻链可变区序列,为后期开发乙肝治疗性抗体并建立一套快速制备抗原特异性全人源单克隆抗体的方法打下了坚实基础。
OBJECTIVE: To rapidly clone the variable region sequence of human antibody against hepatitis B virus (HBV), and to provide a basis for screening and preparing monoclonal antibodies against hepatitis B virus (HBV). Methods A healthy volunteer with a positive hepatitis B surface antigen (HBsAg) was immunized with 20 μg of Hepatitis B vaccine (GSK). After 7 days, HBsAg titer greater than 1 000 mIU / ml was measured and 30 ml heparin was collected for anticoagulation. Flow cytometry The CD19 + / CD27 + / IgG + cell populations were sorted and 96 single cells were picked and placed in 20 μl of single cell lysate. The heavy chain and light chain variable region sequences were obtained by mixed-primer single-cell RT-PCR. The PCR product was purified and cloned into pMD19-T vector for sequencing. Similarity and homology analysis was performed using DNA Star 5.0 MegAlign and IgBLAST. Results 12 positive plasma cells were successfully screened, and 12 positive cells showed high homology with the light and heavy chain PCR products. Conclusion The single plasma cells were successfully screened out. The possible sequences of HBsAb heavy chain and light chain variable regions were obtained, which laid a solid foundation for the development of a therapeutic antibody against hepatitis B in the later stage and the establishment of a set of rapid preparation of antigen-specific whole human monoclonal antibodies basis.