1 Institute of Pharmacy and Pharmacology, University of South China, Hunan, Hengyang, 421001, China; 2 Hunan Center for Disease control and prevention,Hunan Changsha,410005,China
Abstract：Objective To clone rapidly the variable region sequence of hepatitis B virus surface antibody (HBsAb), and to provide a basis for constructing the HBsAb expression vector, screening and manufacturing the therapeutic monoclonal antibodies for HBV. Methods A volunteer who is HBsAb positive was injected with 20 μg of hepatitis B vaccine (GSK Company). Seven days later, his serum HBsAb titer was greater than 1000 mIU/ml. Thirty ml of heparin anticoagulant blood were obtained for sorting CD19+/CD27+/IgG+ cells by flow cytometry. And 96 single cells were picked from CD19+/CD27+/IgG+ positive cells and lysated into 20 μl lysis buffer for single cell. Reverse transcript PCR was used to obtain the variable region of heavy and light chain of each single cell with mixed primers. PCR products were purified and cloned into pMD®19-T vector. Six clones were picked and sequenced. The similarity and homology of sequence were analyzed with DNA Star 5.0 MegAlign and IgBLAST softwares. Results Twelve positive plasma cells were screened. And they showed high similarity and homology in the variable region of heavy and light chain. Conclusions Plasma cells are selected and the variable region sequence of the heavy chain and light chain of HBsAb may be acquired. This study will provide a solid foundation for the future developing the therapeutic antibodies of HBV and establishing a method of manufacturing monoclonal antibody.
唐亚华,蔡亮,罗芳贞,黄红林. 单细胞筛选全人源乙型肝炎病毒表面抗体可变区序列[J]. 实用预防医学, 2015, 22(7): 816-819.
TANG Ya-hua,CAI Liang,LUO Fang-zhen,HUANG Hong-lin. Screening for the variable region sequence of hepatitis B virus surface antibody by single cell. , 2015, 22(7): 816-819.
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2015, Vol. 22 
