Construction and characterization of the chimeric antibody 8C11 to the hepatitis E virus.
Construction and characterization of the chimeric antibody 8C11 to the hepatitis E virus.
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DOI：
10.1111/j.1574-695x.2007.00253.x
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发表时间：
2007-10
影响因子：
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通讯作者：
Wen-xin Luo;Yingwei Chen;Li-feng Li-Li-feng-Li-2107913899;Chenyu Xu;J. Miao;J. Shih;Jun Zhang;N. Xia
中科院分区：
文献类型：
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作者：
Wen-xin Luo;Yingwei Chen;Li-feng Li-Li-feng-Li-2107913899;Chenyu Xu;J. Miao;J. Shih;Jun Zhang;N. Xia
截断的戊型肝炎病毒（HEV）衣壳蛋白E2和p239的同型二聚体符合模型HEV的主要抗原决定因素。以E2为免疫原，产生两种中和性单克隆抗体8C11和8H3。我们构建了由8C11衍生的鼠人嵌合抗体，并在中国仓鼠卵巢（CHO）细胞中表达。从杂交瘤细胞中分离编码重链和轻链可变区的cDNA，分别插入含有人γ -1和kappa恒定区cDNA的哺乳动物表达载体中。将载体共转染CHO细胞，建立稳定的细胞系。间接酶联免疫吸附试验（ELISA）和Western blot分析结果表明，嵌合抗体与天然IgG分子组装正确，可从细胞中分泌。与最初的单抗相似，表达的嵌合抗体显示出HEV抗原结合活性，并增强了8H3与HEV抗原的结合。嵌合抗体能特异性抑制p239与HepG2细胞的结合，并通过抗体竞争ELISA与阳性血清中的HEV IgG竞争。预计该嵌合抗体在人体内的免疫原性较低，更适合戊型肝炎的抗体治疗。
Homodimers of the truncated hepatitis E virus (HEV) capsid proteins, E2 and p239, were conformed to model the dominant antigenic determinants of HEV. Using E2 as an immunogen, two neutralizing monoclonal antibodies (mAbs), namely 8C11 and 8H3, were produced. We constructed a mouse-human chimeric antibody derived from 8C11 and its expression in Chinese hamster ovary (CHO) cells. cDNAs encoding variable regions of heavy and light chains were isolated from hybridoma cells and inserted into mammalian expression vectors containing cDNA of human gamma-1 and kappa constant regions, respectively. The vectors were then cotransfected into CHO cells, and a stable cell line was established. Results from indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the chimeric antibody was assembled correctly to the native IgG molecule and could be secreted from the cells. Similar to the original mAb, the expressed chimeric antibody displayed HEV antigen-binding activity and an enhancement effect on 8H3 binding to HEV antigen. The chimeric antibody could specifically inhibit the binding of p239 to HepG2 cells and compete with HEV IgG in positive serum by antibody-competitive ELISA. The chimeric antibody is expected to be less immunogenic in human and more suitable for antibody therapy of hepatitis E.