Davis J A, Peen E, Williams R C, Perkins S, Malone C C, McCormack W T, Csernok E, Gross W L, Kolaskar A S, Kulkarni-Kale U
Immunology and Laboratory Medicine, University of Florida, Gainesville, Florida, 32610, USA.
Clin Immunol Immunopathol. 1998 Oct;89(1):35-43. doi: 10.1006/clin.1998.4582.
Transformed B cells making monoclonal IgM-lambda anti-PR3 antibody WGH1 from a patient with Wegener's granulomatosis were used to prepare mRNA and synthesize cDNA. PCR primers for human micro and lambda chains were then employed to amplify heavy- and light-chain V-regions followed by cloning into pCR2-1 vector and sequencing. Molecular modeling of VH regions employed knowledge-based homology modeling to obtain minimum energy conformation. The VH sequence was subgroup III with marked overall homology to VH1.9III. The VHCDR3 region of WGH1 was unique, consisting of 21 amino acid residues which included seven tyrosines as well as three negatively charged aspartic acid residues. The VL region was subgroup II with a negatively charged glutamic acid at position 100 in CDR3. Molecular modeling of VH revealed a major conformational difference in the shape of CDR3 compared with other antibodies for which three-dimensional structures have been determined. Monoclonal antibody WGH1 reacting with PR3 (a highly positively charged molecule) shows a unique reactive cassette within VHCDR3 with a number of negatively charged aspartic acid residues. WGH1 VHCDR3 contains a loop which shows a major projection not usually recorded in other previously studied antibody molecules.
从一名韦格纳肉芽肿患者身上获取产生单克隆IgM-λ抗PR3抗体WGH1的转化B细胞，用于制备mRNA并合成cDNA。然后使用人μ链和λ链的PCR引物扩增重链和轻链V区，随后克隆到pCR2-1载体中并进行测序。VH区的分子建模采用基于知识的同源建模来获得最低能量构象。VH序列属于III亚组，与VH1.9III具有显著的总体同源性。WGH1的VHCDR3区是独特的，由21个氨基酸残基组成，其中包括7个酪氨酸以及3个带负电荷的天冬氨酸残基。VL区属于II亚组，在CDR3的第100位有一个带负电荷的谷氨酸。VH的分子建模显示，与已确定三维结构的其他抗体相比，CDR3的形状存在主要构象差异。与PR3（一种高度带正电荷的分子）反应的单克隆抗体WGH1在VHCDR3内显示出一个独特的反应盒，其中有许多带负电荷的天冬氨酸残基。WGH1 VHCDR3包含一个环，该环显示出一个主要突起，这在其他先前研究的抗体分子中通常未被记录。
