新型抗KPC-2型碳青霉烯酶纳米抗体的筛选与鉴定  

Screening and identification of novel anti-KPC-2 carbapenemase-specific nanobodies

作　　者：张鑫 王辉 许剑锋 ZHANG Xin;WANG Hui;XU Jianfeng(College of Food Science and Technology,Shanghai Ocean University,Quality and Safety Risk Assessment Laboratory of Aquatic Products Storage and Preservation,Ministry of Agriculture(Shanghai),Shanghai 201306,China;Qidong Camel Nano-biomedical Co.,Ltd,Qidong 226200,China)

机构地区：[1]上海海洋大学食品学院,农业部水产品贮藏保鲜质量安全风险评估实验室(上海),上海201306 [2]启东凯默纳米生物医药有限公司,启东226200

出　　处：《中国免疫学杂志》2024年第6期1259-1264,共6页Chinese Journal of Immunology

摘　　要：目的:产KPC-2型肺炎克雷伯菌可引起β-内酰胺类抗生素、碳青霉烯类抗生素产生耐药性。通过噬菌体展示技术从抗KPC-2纳米抗体文库中筛选与KPC-2特异性结合的纳米抗体,为产KPC-2型肺炎克雷伯菌耐药性的检测和诊断提供技术支持。方法:利用重组KPC-2免疫双峰骆驼,从骆驼的外周血淋巴细胞中提取RNA,逆转录为cDNA,通过两轮嵌套式PCR扩增出纳米抗体片段,构建抗体文库,并通过噬菌体展示技术筛选特异性纳米抗体。通过HPLC和OCTET分别进行表位分析和亲和力测定。结果:构建了一个库容为5.47×10^(8)cfu/ml且插入有效片段不低于81.25%的纳米抗体文库;并建立抗KPC-2纳米抗体的免疫淘选方法;获得了2个不同CDR3区的纳米抗体K2和K5,亲和力分别为6.0 nmol/L和4.8 nmol/L。且在KPC-2上具有2个非竞争性结合表位。结论:成功淘选出2个不同表位的特异性纳米抗体,有望替代传统抗体用于肺炎克雷伯菌耐药性疾病的检测和诊断。Objective:KPC-2-producing Klebsiella pneumoniae is able to cause drug resistance toβ-lactam antibiotics and car-bapenems antibiotics.Phage display technology was used to screen nanobodies that specifically bind to KPC-2 from the anti-KPC-2 nanobodies library,which provided technical supports for the detection and diagnosis of drug resistance of KPC-2-producing Klebsiella pneumoniae.Methods:A bimodal camel was firstly immunized by the recombinant KPC-2,then,RNA was extracted from peripheral blood lymphocytes of the bimodal camel and reverse transcribed into cDNA.Nanobody fragments were amplified by two rounds of nested PCR,and the antibody library were constructed.The specific nanobodies were screened by the phage display technology.Lastly the epitope analysis and affinity determination were performed by HPLC and OCTET,respectively.Results:A nanobody library was con-structed with a capacity of 5.47×108 cfu/ml and the inserting effective fragments of not less than 81.25%;the immune elutriation method of anti-KPC-2 nanobody was established.Two nanobodies,K2 and K5,with different CDR3 regions were obtained,and their affinities were 6.0 nmol/L and 4.8 nmol/L,respectively.Moreover,K2 and K5 were non-competitive for binding epitopes on KPC-2.Conclu-sion:Two specific nanobodies with different epitopes were successfully panned out,which expect to replace traditional antibodies for detection and diagnosis of Klebsiella pneumoniae drug-resistant diseases.

关 键 词：纳米抗体 噬菌体展示技术 KPC-2型碳青霉烯酶 肺炎克雷伯菌 

分 类 号：R392-33[医药卫生—免疫学]