抗银环蛇毒素人源单链抗体的筛选及鉴定

Screening and Identification of Human Anti-bungarotoxin ScFv

目的从大容量天然噬菌体抗体库中筛选抗银环蛇毒素的人源单链抗体(ScFv),并进行鉴定。方法以银环蛇毒素为靶抗原,通过"吸附-洗脱-扩增"过程从大容量天然噬菌体抗体库中筛选特异性抗体,采用ELISA法检测其结合活性;与抗蛇毒素马血清进行竞争抑制ELISA,检测其中和活性;DNA指纹图谱分析和序列测定确定其抗体类型。结果经4轮筛选,获得了5株对银环蛇毒素具有结合活性的阳性克隆;其中1株结合活性高的单克隆抗体中和活性抑制率可达8.5%;DNA指纹图谱显示为3个不同的ScFv基因,序列分析表明其重链可变区均属于VHⅠ型,轻链可变区分别为VκⅡ、VκⅢ和VλⅡ。结论利用噬菌体抗体库技术获得了具有中和活性的抗银环蛇毒素人源单链抗体。

Objective To screen human anti-bungarotoxin ScFv from large natural phage antibody library then identify.Methods Specific antibody was screened from large natural phage antibody library by a course of ＂adsorption-elution-amplification＂ using bungarotoxin as target antigen,then determined for binding activity by ELISA,for neutralizing activity by competitive inhibition ELISA with horse antiserum against bungarotoxin,and typed by DNA fingerprint analysis and sequencing.Results Five positive clones with binding activities to bungarotoxin were obtained after four rounds of panning,of which the highest inhibiting rate to neutralizing activity reached 8.5%.Three different DNA fingerprints were observed.Sequencing result proved that all the heavy chain variable regions of the three ScFv genes belonged to VH Ⅰ,while the light chain variable regions were VκⅡ,VκⅢ and VλⅡ.Conclusion The anti-bungarotoxin ScFv with neutralizing activity was obtained from large natural phage antibody library.