抗二氟沙星单克隆抗体可变区基因的克隆及序列分析

CLONING AND SEQUENCING ANALYSIS OF VARIABLE REGION GENES OF ANTI-DIFLOXACIN McAb

采用RT-PCR技术,从抗二氟沙星单克隆抗体X1杂交瘤细胞株总RNA中扩增VH和VL基因片段,克隆入pUC18载体,测序进行(X1)可变区基因的克隆及序列分析。通过互联网检索发现VH和VL基因与Ig同源,分别符合小鼠IgVH和Igκ基因特征。VH基因全长363bp,编码121个氨基酸;VL基因全长为348bp,编码116个氨基酸。氨基酸序列分析结果显示,可变区含有明确的4个骨架区和3个抗原决定簇互补区。本试验成功获得X1株单抗的重、轻链可变区基因,为基因工程抗体的构建奠定了基础。

The variable region genes of heavy chain（VH） and light chain（VL） of anti-difloxacin monoclonal antibody（McAb） were amplified by RT-PCR from total RNA extracted from X1 hybridoma cell.PCR products were cloned into pUC18 vector and sequenced by Sanger＇s dideoxymediated chain-termination method.The results showed that VH gene consisted of 363bp encoding 121 amino acid residues;VL gene consisted of 348bp encoding 116 amine acid residues,which contain four FRs and three CDRs,respectively.Compared with mouse Ig database,the VH and VL regions are in accord with the characterization of DNA sequence present in the mouse Ig region.The successful clone of VH and VL genes of X1 McAb made it a solid foundation to the construction of engineering antibody against difloxacin.