摘要
为了解羊口疮病毒（Orf virus，ORFV）单克隆抗体2E4的抗体可变区的分子特征，选择其轻链和重链可变区基因分别进行体外扩增和测序，并通过IgBLAST程序分析（http：／／www.ncbi．nlm．nih．gov／igblast／），确定了2E4抗体可变区的互补决定区（complementarity determining regions，CDRs）和骨架区（framework regions，FRs）氨基酸结构。继而，采用SWISS-MODEL同源建模方法，展示抗体分子轻、重链可变区的三维空间构型，并推测出可能的OR—FVB细胞抗原表位多肽结合部位（pocket）。目的基因测序结果显示：2E4轻链可变区（V L）基因扩增全长为324bp，编码108个氨基酸；2E4重链可变区（VH）基因扩增全长为345bp，编码115个氨基酸。IgBLAST结果显示：轻链与GenBank公布的GHP53（GI：197494）单抗轻链可变区序列基因序列一致性达到97．6％；重链与vHsAF34（GI：215263227）单抗重链可变区基因序列一致性达到98．6％。并且，2E4轻链CDRs（CDR1-CDR3）分别位于25～348a、52～54aa和91～98aa的序列位置；重链CDRs（CDR1-CDR3）分别位于25～32a8、50～57aa和96～104aa的序列位置。同源建模分析结果显示：这些CDRs区域共同形成的“pocket”极可能作为抗原表位多肽结合的位点。最终成功克隆2E4单抗可变区基因，分析了抗体轻、重链可变区CDRs构成形式，模拟了其空间构型，为进一步了解和验证“CDRs-表位”特异性结合的分子机制，并在Orf免疫保护中加以应用提供了可能。
The aim of this study was to characterize variable domains of the monoclonal antibody 2E4 against ORFV,the light and heavy chain variable domains of 2E4 were specifically amplified in vitro and sequenced. The amino acid sequences of the variable domains composed by complementarity determining regions （CDRs） and framework regions （FRs） were cautiously determined by IgBLAST software （http.//www. ncbi. nlm. nih. gov/igblast/） followed by molecular modeling for exhibition of the three dimensional spatial configuration of antibody light and heavy chain using available SWISS-MODEL homology modeling method,and the possible binding site （Pocket） of ORFV B cell antigen epitope polypeptide was supposed. Gene sequencing results showed that the full length of 2E4 light chain variable domain gene was 324 bp encoding 108 amino acid residues,and the heavy chain variable domain gene was 345 bp amounted to 115 amino acid residues. IgBLAST results showed 97.6% nucleotide sequence identity in light chain variable domain between 2E4 and GHP53 （GI： 197494） from GenBank, similarly, 98. 6% identity in heavy chain variable domain was observed when compared with VHSAF34 （GI. 215263227）. In addition, the CDRs （CDR1 to CDR3） aa,and the CDRs（CDR1 of light chain variable domain were located at 52-54 aa,91-98 aa and 25 34 to CDR3） of heavy chain were located at 25-32 aa,50-57 aa and 96-104 aa, respectively. The results of homology modeling analysis showed that these domains of CDRs spontaneously formed a ＂pocket＂ for ORFV epitope polypeptide binding. In brief,we have successfully cloned and analyzed the variable domain genes of 2E4, and simulated the composition and spatial configuration of the variable domains of antibody light and heavy chains. All the data will further facilitate understanding and confirmation of the molecular mechanism of ＇CDRs-Epitope＇ interaction,by which we will attempt to utilize their specific binding in the strategy of immune defense against ORFV infection.
作者
张雪
谭强
赵文博
段旭阳
冯振月
马金柱
崔玉东
于永忠
ZHANG Xue, TAN Qiang, ZHAO Wen-bo, DUAN Xu-yang, FENG Zhen-yue, MA Jin-zhu, CUI Yu- dong, YU Yong-zhong(College of Life Science and Technology, Heilongjiang Bayi Agricultural University ,Daqing , Heilongjiang 163000, Chin)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2018年第5期863-870,共8页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31172353)
黑龙江省教育科学“十二五”规划项目(GBC1212060)
黑龙江省自然科学基金资助项目(C2017052)
关键词
羊口疮病毒
单克隆抗体
可变区
基因克隆
序列分析
同源建模
Orf virus
monoclonal antibody
variable domains
gene cloning
sequence analysis
homology modeling
