分离人杀伤细胞抑制受体NKAT2特异的单链抗体  

Isolation of scFv-Fragments Specific for the Human Killer Cell Inhibitory Receptor NKAT 2

作　　者：乔旭刚[1] Colon.,M 

机构地区：[1]暨南大学医学院血液病研究室

出　　处：《暨南大学学报（自然科学与医学版）》1998年第6期51-52,共2页Journal of Jinan University(Natural Science & Medicine Edition)

摘　　要：人自然杀伤细胞表面的抑制受体（ＫＩＲ），是与表达在其他细胞表面的相应ＨＬＡＩ类分子相互作用的。许多ＫＩＲ受体已被克隆，小部分量的单克隆抗体也已产生。然而为区分不同位点及其等位基因的产物，并精确阐明其功能，需要更多的研究试剂。本研究探讨用噬菌体抗体库，分离ＫＩＲ单链抗体（ｓｃＦｖ）的可行性。方法：ＮＫＡＴ２－ＩｇＧ１为一ＫＩＲ，与人ＩｇＧ１Ｆｃ以融合蛋白形成表达而获得。噬菌体抗体库为“Ｎｉｓｉｍ”抗体库。抗体库ＮＫＡＴ２－ＩｇＧ１包被的免疫管５次筛选。ｓｃＦｖ由特异的噬菌体感染细菌，诱导而得。筛选效应经噬体菌体的菌落形成单位及ＤＮＡ酶切图谱检测。筛选的克隆进行ＤＮＡ序列分析。ｓｃＦｖ的特异性及免疫学特征由ＥＬＩＳＡ、聚丙烯胺凝胶电泳及免疫印迹分析。细胞结合实验由流式细胞仪测试。结果：５次筛选后，ＮＫＡＴ２特异的噬菌体达到２００倍的富集，ＤＮＡ酶切图谱也显示某种特定消化类型的富集。经ＥＬＩＳＡ分析，结果显示３７个克隆与ＮＫＡＴ２反应呈强阳性。这些克隆与ＮＫＡＴ１－ＩｇＧ１、ＮＫＡＴ３－ＩｇＧ１、ＮＫＡＴ４－ＩｇＧ１、人ＩｇＧ及其他非相关蛋白的反应均为阴性。其中一个克隆（ｓｃＨＵＢ１）经流式细胞仪进一步测试，显示?Abstract Objective: Killer cell inhibitory receptors (KIR) on human natural killer (NK) cells interact with suitable HLA class I molecules expressed on other cells. Many of these receptors have been cloned, and a small number of monoclonal antibodies directed against them is available. However, more reagents are needed to reliably distinguish the product of various loci and their alleles as well as to understand the precise function of these receptors. We have employed a phage display library to identify soluble single chain Fv (scFv)-fragments with specificity for a KIR. Methods: A KIR was expressed as soluble fusion Protein with a human IgG 1 Fc portion (NKAT 2 -IgG 1 ). The phage library used in this work was the “Nissim”library (EMBO J. 13:692, 1994). The library was subjected to 5 cycles of selection with NKAT 2 -IgG 1 coated immunotubes. The scFv-fragments were prepared by infecting bacteria with specific phage and induction. The effect of panning was analyzed by titering the phage colony-forming units and DNA fingerprinting. Nucleotied sequencing of these clones was also carried out. The specificity and immunological characteristics of scFv were anlysed by ELISA, SDS-PAGE and western blotting. Cell binding assays of scFv framents were performed using flow cytometry.Results: After 5 rounds of panning, the number of NKAT 2 binding phages increased, resulting in a 200-fold amplification. Fingerprinting demonstrated an enrichment for certain digestion patterns. 37 of 40 clones analyzed by ELISA were found to react strongly with NKAT 2 ,but not with NKAT 1-IgG 1, NKAT 4 IgG 1 , human IgG and several unrelated proteins. A representative clone (scHUB1) recognized the NKAT 2 molecule expressed on the cell surface of transfected Jurkat cells using flow cytometry. SDS-PAGE of the scFv-reagent revealed a 30-kDa protein which was also detected by western blotting. Comparison with the repertoire of human variable genes showed that scHUB1 belonged to the DP-35 group, but

关 键 词：杀伤细胞 分离 抗体 抑制受体 NKAT2 单链抗体 

分 类 号：R392.11[医药卫生—免疫学]