人源HBsAb重链体外表达和亲和力、特异性初步鉴定

Cloning,expression and immunological traits of humanized HBsAb heavy chain

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 establish a method for cloning humanized HBsAb heavy chain and identify its functional characterization. Methods： Single antibody -secreting cell was isolated by single -cell sorting from peripheral blood of HBV - vaccinated subjects. 20 colonies were screened by single - cell RT - PCR. The full - length coding HBsAh heavy chain conserved sequence was cloned into pEGFP -N1 vector. Then the vector was transfected into COS -7 cells. The binding affinity of IG（H） heavy chain in cell- free supernatant expressed by COS-7 cells was deteeed by western blot and ELISA. Results： The IG（H） heavy chain expression plasmid was eonstruced correctly. ELISA showed the value of Ka was 2.39 x 109 L/tool. Western blot showed the IG（H） heavy chain had a high specificity. Conclusion： The production of IG（H） heavy chain in vitro is of great value to prevention of hepatitis B.