Research Center for Applied Medical Engineering, Oita University, Dan-noharu 700, Oita-shi, Oita 870-1192, Japan.
J Biol Chem. 2013 Jul 5;288(27):19558-68. doi: 10.1074/jbc.M113.454579. Epub 2013 May 15.
Human antibody light chains belonging to subgroup II of germ line genes were amplified by a seminested PCR technique using B-lymphocytes taken from a human adult infected with influenza virus. Each gene of the human light chains was transferred into the Escherichia coli system. The recovered light chain was highly purified using a two-step purification system. Light chain 22F6 showed interesting catalytic features. The light chain cleaved a peptide bond of synthetic peptidyl-4-methyl-coumaryl-7-amide (MCA) substrates, such as QAR-MCA and EAR-MCA, indicating amidase activity. It also hydrolyzed a phosphodiester bond of both DNA and RNA. From the analysis of amino acid sequences and molecular modeling, the 22F6 light chain possesses two kinds of active sites as amidase and nuclease in close distances. The 22F6 catalytic light chain could suppress the infection of influenza virus type A (H1N1) of Madin-Darby canine kidney cells in an in vitro assay. In addition, the catalytic light chain clearly inhibited the infection of the influenza virus of BALB/c mice via nasal administration in an in vivo assay. In the experiment, the titer in the serum of the mice coinfected with the 22F6 light chain and H1N1 virus became considerably lowered compared with that of 22F6-non-coinfected mice. Note that the catalytic light chain was prepared from human peripheral lymphocyte and plays an important role in preventing infection by influenza virus. Considering the fact that the human light chain did not show any acute toxicity for mice, our procedure developed in this study must be unique and noteworthy for developing new drugs.
采用半巢式 PCR 技术,从感染流感病毒的成人 B 淋巴细胞中扩增人抗体轻链,属于胚系基因亚群 II。将人轻链的每个基因转入大肠杆菌系统。回收的轻链使用两步纯化系统高度纯化。轻链 22F6 表现出有趣的催化特征。该轻链切割合成肽基-4-甲基香豆酰-7-酰胺(MCA)底物(如 QAR-MCA 和 EAR-MCA)的肽键,表明具有氨肽酶活性。它还水解 DNA 和 RNA 的磷酸二酯键。通过氨基酸序列分析和分子建模,22F6 轻链在近距离内具有两种作为氨肽酶和核酸酶的活性位点。在体外试验中,22F6 催化性轻链可抑制 Madin-Darby 犬肾细胞中甲型流感病毒(H1N1)的感染。此外,在体内试验中,通过鼻腔给药,催化性轻链明显抑制 BALB/c 小鼠流感病毒的感染。在实验中,与未感染 22F6 的小鼠相比,感染 22F6 轻链和 H1N1 病毒的小鼠血清中的滴度明显降低。值得注意的是,该催化性轻链是用人外周血淋巴细胞制备的,在预防流感病毒感染方面发挥着重要作用。考虑到人轻链对小鼠没有任何急性毒性,我们在本研究中开发的程序必须是独特的,值得开发新药。