Phage display cloning and characterization of monoclonal antibody genes and recombinant Fab fragment against the CD98 oncoprotein.
Phage display cloning and characterization of monoclonal antibody genes and recombinant Fab fragment against the CD98 oncoprotein.
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DOI：
10.1111/j.1349-7006.2001.tb02155.x
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发表时间：
2001-12
期刊：
影响因子：
--
通讯作者：
Masuko T
中科院分区：
文献类型：
--
作者：
Itoh K;Inoue K;Hirooka K;Maruyama K;Ohkawa M;Matsui K;Tada H;Enomoto T;Hashimoto Y;Suzuki T;Masuko T
克隆了抗CD98重链单克隆抗体（mAb） HBJ127的Fab基因，并利用噬菌体展示系统将其表达为重组Fab （rFab）片段。HBJ127的重链和轻链可变基因分别来源于VOx‐1和IgVk8‐30种系。在重链互补决定区2发现了广泛的体细胞突变。rFab片段通过镍螯合层析从细菌裂解物中纯化，产率为71.4 μg。rFab片段与CD98阳性细胞的细胞表面有反应，而与CD98阴性细胞无反应。rFab片段的识别位点与mAb相同，因为过量的mAb预处理完全抑制了rFab片段与helas3细胞的结合。rFab片段和mAb的相对亲和力值分别为0.11x108和0.35X108M‐1。rFab片段的亲和力降低了3倍，这可能是由于抗体制备价的差异。与抗Fab预孵育的rFab片段对体外细胞生长的抑制作用表明，克隆的携带基因的大肠杆菌产生的rFab片段与HBJ127单抗的Fab部分相同。这些结果表明，利用噬菌体展示系统可以很容易地制备出具有与亲本单抗相似的抗原结合活性的小片段。据我们所知，这是首次报道产生抗CD98 h.c. rFab片段。
The Fab gene of anti‐CD98 heavy chain (h.c.) monoclonal antibody (mAb) HBJ127 was cloned and expressed as a recombinant Fab (rFab) fragment by means of a phage display system. The variable heavy and light chain genes of HBJ127 were found to be derived from VOx‐1 and IgVk8‐30 germline, respectively. Extensive somatic mutation was found in the heavy chain complementarity determining region 2. rFab fragment was purified homogeneously from crude bacterial lysates by Ni‐chelate chromatography in a yield of 71.4 μg from 100 ml of culture. rFab fragment was reactive with the cell surface of CD98‐positive cells irrespective of tissues of origin, but not with CD98‐neg‐ative cells. The recognition site of the rFab fragment was identical to that of mAb since the binding of rFab fragment to HeLaS3cells was completely inhibited by pretreatment with an excess of mAb. The relative affinity values of rFab fragment and mAb were found to be 0.11x108 and 0.35X108M‐1, respectively. Three‐fold lower affinity of rFab fragment may be due to the difference of valency of the antibody preparation. Cell growth inhibition in vitro by rFab fragment preincubated with anti‐Fab suggests that the rFab fragment produced by cloned gene‐bearing Escherichia coli was identical to the Fab part of HBJ127 mAb. These results show that a small fragment with antigen binding activity similar to that of the parent mAb can easily be prepared by using a phage display system. To our knowledge, this is a first report of the production of anti‐CD98 h.c. rFab fragment.
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DOI：
10.1073/pnas.97.16.9234
发表时间：
2000-08-01
影响因子：
11.1
作者：
Ditzel, HJ;Masaki, Y;Burton, DR
通讯作者：
Burton, DR
DOI：
10.1073/pnas.90.5.1746
发表时间：
1993-03-01
影响因子：
11.1
作者：
PLOWMAN, GD;CULOUSCOU, JM;SHOYAB, M
通讯作者：
SHOYAB, M
影响因子：
2.7
作者：
Itoh, K;Nakagomi, O;Suzuki, T
通讯作者：
Suzuki, T
影响因子：
4.4
作者：
Johnson, G;Wu, TT
通讯作者：
Wu, TT
DOI：
10.1073/pnas.92.7.2529
发表时间：
1995-03-28
影响因子：
11.1
作者：
BARBAS, SM;DITZEL, HJ;BURTON, DR
通讯作者：
BURTON, DR