大容量天然人源单链抗体库的构建及抗乙型肝炎病毒PreS1单链抗体的淘选

Generation of human single-chain antibody against PreS1 of hepatitis B virus from large naive phage-display library

目的 :从大容量抗体库中淘选全人源化抗乙型肝炎病毒 Pre S1的单链抗体。方法 :以 6 0例健康供者的外周血淋巴细胞为来源 ,构建了 10 1 0人源天然单链抗体库 ,并以 Pre S1抗原肽进行淘选。 3轮淘选后 ,单克隆经 EL ISA检测 ,富集的特异性单克隆经 E.coli HB2 15 1表达后进行 Western检测并测序。结果 :3轮淘选之后 ,噬菌体的投入 /产出比提高了 10 0倍。抗原抗体反应结果显示 ,A4 1 0 =1.0的抗 Pre S1的单链抗体得到富集。Western结果显示其插入的单链抗体片段正确。该抗体基因序列的 DNAPL OT软件分析结果进一步表明该抗体是人抗体 ,其 VH 属于 VH1亚族 ,Vλ属于 Vλ1亚族。结论 :这一技术是获得全人源化抗 Pre S1的单链抗体的有效途径 ,同时为基因工程抗体用于临床治疗乙型肝炎奠定基础。

Objective To obtain the completely humanized single-chain antibody(scFv) against PreS1 of hepatitis B virus(HBV). Methods A 10 10 cfu naive combinatorial library of phage-display human scFv, genes of which were derived from peripheral blood lymphocytes(PBLs) of 60 healthy donors, was constructed and panned by PreS1 of HBV. After 3 rounds of panning, the activity of individual clone was examined by ELISA. The clone with the highest A 410 value was expressed in E. coli HB2151 then identified by Western blotting. Sequencing was also carried out. Results After 3 rounds of panning, a 100 folds increase in the phage yielding relative to the first panning was noted. Measurement of antibody-antigen reaction revealed that scFv mono-clone specific to PreS1 with A 410 =1.0 was enriched, which proportion of insert was verified by Western after being expressed. The sequencing result demonstrated further that it was a human scFv gene with which V H belonged to human V H1 family, and V λ belonged to V λ1. Conclusion The technique described in this paper is a powerful way to obtain humanized scFv gene against PreS1 of HBV, which may provide a more satisfactory therapy for hepatitis B.