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目的:制备人源HBsAb重链,初步鉴定其亲和力和特异性。方法:利用流式单细胞分选技术获得单个抗体分泌细胞,单细胞RT-PCR筛选单菌落,挑选其中有IgG保守区域的克隆全长,构建IgG(H)基因的表达质粒,转染COS-7细胞并诱导表达。间接ELISA测定检测亲和力,Western blot鉴定抗体特异性。结果:成功构建表达质粒,间接ELISA显示亲和力Ka值达到2.39×109 L/mol,Western blot鉴定重链有较高的特异性。结论:人源HBsAb重链的体外制备对乙肝防治具有重要价值。
Objective: To prepare human HBsAb heavy chain and preliminary identify its affinity and specificity. METHODS: Single antibody-secreting cells were obtained by flow cytometry. Single colonies were screened by single cell RT-PCR. The full-length clone with IgG conserved region was selected to construct IgG (H) gene expression plasmids and transfected into COS- 7 cells and induce expression. Indirect ELISA assay was used to detect affinity and Western blot to identify antibody specificity. Results: The expression plasmid was successfully constructed. The indirect ELISA showed that the Ka value reached 2.39 × 109 L / mol and the specificity of heavy chain was identified by Western blot. Conclusion: The preparation of human HBsAb heavy chain in vitro has important value in prevention and treatment of hepatitis B infection.