用噬菌体抗体库分离NK细胞抑制受体特异性的ScFv片段

Isolation of ScFv fragment specific for human NK cell inhibitory receptor from the phage display library

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 The cytotoxicity of NK cells is regulated by the killer cell inhibitory receptors (KIRs) expressed on NK cell surface with specificities of MHC class I molecules. Some of these receptors have been cloned, and a small number of monoclonal antibodies against these receptors were available. However, more reagents are needed to reliably distinguish the products of various loci and their alleles as well as to understand the precise function of these receptors. This work is to explore the possibility by using a phage display library to identify the soluble single chain Fv (ScFv) fragment with the specificity for the KIR. Methods A KIR(NKAT2) was expressed as fusion protein with the hinge and Fc portions of human IgG1. The phage display library used in this work was a semi synthetic “Nissim”library. The library was subjected to 5 rounds panning using immunotubes coated with NKAT2 IgG1.The soluble ScFv was induced from bacteria by IPTG. The effect of panning was analysed by titering the phage colony form units and fingerprinting. The specificity and immunological characteristics of ScFv were analysed by ELISA, SDS PAGE and Western blotting. Nucleotide sequencing of ScFv was also carried out. Results After 5 rounds panning, the number of NKAT2 binding phages increased resulting in a 200 fold amplification. Fingerprinting was clear, and there was an enrichment for certain digestion patterns. Thirty seven clones out of 40 were found to react strongly with NKAT2. SDS PAGE revealed an approximate 30kD protein which was also detected by Western blotting. The sequences of these clones were pairing of unique complete sequence and their V H segments derived from same V H family DP 35. Conclusions Our results show that highly specific reagents directed against KIR can be obtained from a phage display library by relatively simple selection procedures.