抗SARS抗体独特型单链抗体的筛选及鉴定

Screening and identification of anti-idiotypic antibody to anti-SARS Ig

目的从人源噬菌体抗体库中筛选抗SARS独特型抗体为SARS的诊断及疫苗的研究奠定基础。方法以混合SARS病人恢复期血清免疫球蛋白(Ig)作为筛选分子,从噬菌体抗体库中筛选出能与病人血清IgG(IgM)结合的抗体,并通过与混合正常人血清Ig作负性筛选,从而获得抗SARS病毒的独特型抗体(AId)。通过4轮“吸附-洗涤-扩增”,从最后一轮所得单克隆菌株中分别通过ELISA筛选出能和SARS病人恢复期血清Ig结合的菌株,提取质粒DNA,并对PCR扩增产物进行DNA序列测定,最后用所筛选的克隆通过与不同人群血清反应,证实其特异性。结果随机挑选经第4轮筛选后的200个单克隆菌落,其中有10个与SARS病人恢复期血清Ig结合的OD值明显高于其与正常人Ig结合的OD值;以上10个克隆经PCR扩增,其中有6个克隆于1 000 bp处有电泳条带;其DNA序列经与BLAST数据库和IMGT/QUEST软件比对有5个均与人的重链可变区(VH)和轻链可变区(VL)高度同源,且与其同源性最高的人Ig的VH属VHI亚群,VL属k链,属VKⅡ亚群;最后确认有3个克隆其噬菌体抗体和SARS病人血清Ig结合的OD值均明显高于与正常人血清Ig结合的OD值。结论利用抗体库技术可不经免疫制备特异性抗SARS抗体独特型抗体,为SARS诊断及疫苗的研制奠定基础。

Objective To screen and identify anti-idiotypic antibody （AId） to SARS-CoV by phage display technique, for the diagnosis and vaccine research of severe acute respiratory syndrome （SARS）. Methods The human synthetic VH ＋ VL ScFv library was screened by mixed serums （containing IgG and IgM） from SARS patients, and then negatively screened by mixed serums from healthy individuals. The obtained anti-idiotypic antibodies to anti-SARS Ig could specially bind to IgG （IgM） from SARS patients. After 4 cycles of ＂absorption, elufion, and application＂, some clones which could specially bind to SAPS Ig were obtained by using ELISA. The clones screened from the above steps were identified by PCR, and their speciticities of the products were demonstrated by their reactions to serums from different people. Results Of the 200 clones from the fourth cycle, there were 10 clones whose OD492nm value binding to SARS Ig were distinctly higher than that of binding to normal human Ig. Six out of the 10 clones had the products whose length was 1000 bp detected by PCR. Compared with IMGT and BLAST data bank, there were 5 clones whose DNA sequence was highly congenetic to that of human VH and VL, respectively. Three positive clones that could specially bind to serums of SARS patients were confirmed. Conclusion Special anti-idiotypic antibody to anti-SARS Ig can be obtained by using the phage display technique, which paves the way for the study of the diagnosis and vaccine research of SARS.