Jilin Collaborative Innovation Center for Antibody Engineering, Jilin Medical University, Jilin, Jilin 132013, P.R. China.
Clinical Laboratory, 944th Hospital of People's Liberation Army, Jiuquan, Gansu 735000, P.R. China.
Int J Mol Med. 2021 Feb;47(2):708-718. doi: 10.3892/ijmm.2020.4822. Epub 2020 Dec 16.
A large human natural single‑chain fragment variable (scFv) phage library was constructed based on Cre‑LoxP recombination, and used to successfully identify antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9). The library was derived from 400 blood samples, 30 bone marrow samples, and 10 cord blood samples from healthy donors. Lymphocytes were isolated from each sample and cDNA was synthesized using reverse transcription‑quantitative PCR. Two‑step overlap PCR was then used for scFv synthesis using a LoxP peptide as the linker. The scFv gene was inserted into the phagemid vector pDF by enzymatic digestion and ligation, and then transformed into Escherichia coli (E. coli) SS320 to establish a primary antibody library in the form of scFvs. A primary antibody library consisting of 5x107 peripheral blood and umbilical cord blood sources, as well as a primary antibody library of 5x107 bone marrow samples were obtained. By optimizing the recombination conditions, the primary phage library was used to infect E. coli BS1365 strain (which expresses the Cre enzyme), and a human scFv recombinant library with a size of 1x1011 was obtained through Cre‑LoxP enzyme‑mediated heavy and light chain replacement and recombination. This constructed recombinant library was employed to screen for antibodies against recombinant PCSK9. After four rounds of selection, a fully human antibody (3D2) was identified with a binding affinity of 1.96±1.56ⅹ10‑10 M towards PCSK9. In vitro, the PCSK9/low‑density lipoprotein receptor (LDLR) pathway of Hep‑G2 cells was inhibited by 3D2 treatment, thereby increasing LDL uptake in these cells. In addition, combination treatment with 3D2 and statin was more effective at increasing LDLR levels than treatment with 3D2 or statin alone. Furthermore, 3D2 resulted in a 3‑fold increase in hepatic LDLR levels, and lowered total serum cholesterol by up to 61.5% in vivo. Taken together, these results suggest that the constructed human Cre‑LoxP scFv phage display library can be used to screen fully human scFv, and that 3D2 may serve as a candidate hypolipidemic therapy.
构建了基于 Cre-LoxP 重组的大型人源天然单链片段可变 (scFv) 噬菌体文库，并成功鉴定了针对前蛋白转化酶枯草溶菌素/柯萨奇蛋白酶 9 (PCSK9) 的抗体。该文库源自 400 份健康供者的血液样本、30 份骨髓样本和 10 份脐血样本。从每个样本中分离淋巴细胞，并使用反转录定量 PCR 合成 cDNA。然后使用两步重叠 PCR 用 LoxP 肽作为接头合成 scFv。通过酶切和连接将 scFv 基因插入噬菌粒载体 pDF 中，然后转化到大肠杆菌 (E. coli) SS320 中，以 scFv 的形式建立初级抗体文库。获得了由 5x107 外周血和脐血来源以及 5x107 骨髓样本组成的初级抗体文库。通过优化重组条件，使用初级噬菌体文库感染表达 Cre 酶的大肠杆菌 BS1365 菌株，并通过 Cre-LoxP 酶介导的重链和轻链替换和重组获得大小为 1x1011 的人 scFv 重组文库。该构建的重组文库用于筛选针对重组 PCSK9 的抗体。经过四轮筛选，鉴定出一种对 PCSK9 具有 1.96±1.56ⅹ10-10M 结合亲和力的全人抗体 (3D2)。在体外，3D2 处理抑制 Hep-G2 细胞的 PCSK9/低密度脂蛋白受体 (LDLR) 途径，从而增加这些细胞对 LDL 的摄取。此外，与单独使用 3D2 或他汀类药物相比，3D2 与他汀类药物联合治疗更能有效增加 LDLR 水平。此外，3D2 使肝 LDLR 水平增加 3 倍，并使体内总血清胆固醇降低高达 61.5%。总之，这些结果表明，构建的人 Cre-LoxP scFv 噬菌体展示文库可用于筛选全人 scFv，并且 3D2 可能作为一种潜在的降脂治疗药物。
