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目的探讨从噬菌体抗体库分离杀伤细胞抑制受体（ＫＩＲｓ）特异性ＳｃＦｖ（ｓｉｎｇｌｅｃｈａｉｎＦｖ）的可行性及其特征。方法所用ＫＩＲ为ＮＫＡＴ２，其可溶性形式为ＮＫＡＴ２－ＩｇＧ１。噬菌体抗体库为Ｎｉｓｓｉｍ等报道的半合成抗体库。抗体库经包被免疫管的ＮＫＡＴ２－ＩｇＧ１５次筛选后，可溶性ＳｃＦｖ由ＩＰＴＧ诱导细菌而获得，筛选效果由测定噬菌体的菌落形成单位及ＤＮＡ酶切图谱分析。ＥＬＩＳＡ、聚丙烯酰胺凝胶电泳及免疫印迹分析ＳｃＦｖ特异性及免疫学特征，并进行ＤＮＡ的序列测定。结果５次筛选后，噬菌体得到２００倍的富集，酶切图谱也显示某种构型噬菌体的富集。４０个克隆的细菌上清经测试，３７个显示与ＮＫＡＴ２有较强的反应。ＳｃＦｖ经聚丙烯酰胺凝胶电泳及免疫印迹显示相对分子质量为３０×１０３，其ＤＮＡ序列完全相同，重链ＣＤＲ３区编码氨基酸为ＥＳＮＬＶＴＣ，重链其它部分与ＤＰ３５一致。结论研究初步结果表明，用噬菌体抗体库可以快速、简便地产生用于ＫＩＲ研究的较高特异性的试剂。
Objective To investigate the feasibility and characterization of phage antibody library for isolation of KIRs-specific scFv (singlechainFv). The KIR used for this method is NKAT2 and its soluble form is NKAT2-IgG1. The phage antibody library is a semi-synthetic antibody library as reported by Nissim et al. The antibody library was immunoblotted with NKAT2-IgG 15 times immunofluorescent tubes. The soluble ScFv was obtained from IPTG-induced bacteria. The screening effect was determined by measuring the phage colony forming units and DNA digestion patterns. ELISA, polyacrylamide gel electrophoresis and Western blot analysis of ScFv specificity and immunological characteristics, and DNA sequence determination. Results Five times of screening, the phage was enriched 200 times, and the digestion pattern also showed the enrichment of certain configuration phage. Forty colonies of bacterial supernatants were tested, and 37 showed strong responses to NKAT2. ScFv by polyacrylamide gel electrophoresis and Western blot showed that the relative molecular mass of 30 × 103, the DNA sequence is identical, the heavy chain CDR3 encoded amino acid ESNLVTC, the rest of the heavy chain and DP35 consistent. Conclusions The preliminary results of the study indicate that phage antibody libraries allow rapid and easy generation of higher specificity reagents for KIR studies.