Park Sung Jae, Seol Sang Yong, Jee Sam Ryong, Park Eun Taik, Lee Youn Jae, Lee Sang Hyuk, Chung Jung Myung, Cho Hyun Dae, Jeong Young-Ju, Choi In Hak, Park Sae Gwang
Department of Internal Medicine, Inje University College of Medicine, Busanjin-Gu, Busan, Korea.
Korean J Gastroenterol. 2007 Feb;49(2):85-92.
BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy.
Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1 x 10(10), human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing.
pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1 micro g of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15 x 10(-8) M to 1.75 x 10(-6) M.
Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.
背景/目的：为开发一种针对乙型肝炎病毒（HBV）感染的新型治疗方法，我们旨在利用噬菌体展示技术制备一种抑制P蛋白逆转录酶（RT）活性的人单克隆抗体，该蛋白在HBV复制中起重要作用。因此，我们分析了人单克隆抗体作为基于蛋白质的基因治疗方法的可行性。
将HBV的P蛋白逆转录酶/聚合酶（RT/POL）功能基序克隆到pMAL-c载体中，并以麦芽糖结合融合蛋白形式表达。通过直链淀粉树脂柱纯化RT/POL重组蛋白（pMRT/POL）。使用含有1.1×10¹⁰的人单链Fv噬菌体抗体库，通过BIAcore淘选筛选出针对pMRT/POL的人抗体。分析所选抗体片段的RT抑制活性。最后，通过BIAcore分析其亲和力，并通过核苷酸测序分析互补决定区。
在大肠杆菌中表达的pMRT/POL重组蛋白具有RT活性，1μg重组蛋白的活性相当于5单位的莫洛尼鼠白血病病毒逆转录酶（MMLV RT）。通过BIAcore淘选，我们筛选出3个克隆：POL-A5、POL-B8和POL-B12。每个克隆的RT抑制活性为52%-82%，对抗原的亲和力为8.15×10⁻⁸M至1.75×10⁻⁶M。
本研究制备的人单克隆抗体亲和力较低，但在体外能有效抑制RT活性。如果POL-A5、POL-B8和POL-B12能够转化为细胞内抗体形式，通过中和HBV聚合酶蛋白来抑制复制，可用于基于蛋白质的基因治疗。
