融合表达β趋化因子受体5NH_2端膜外第一襻及其特异抗体F(ab′)_2的制备

Fusion expression of the first extracellular membrane loop chemokine receptor 5 NH 2 terminal and preparation of its specific antibody F (ab′) 2

目的：研究β趋化因子受体５（ＣＣＲ５）ＮＨ２端膜外第一襻结构域的功能及其特异抗体Ｆ（ａｂ′）２的制备。方法：计算机分析趋化因子超基因家族，定位超基因家族中同源顺序最低的结构域ＮＨ２端膜外结构域，ＰＣＲ扩增出该结构域１１４个核苷酸序列。构建融合表达载体ｐＧＥＸ－ＩＮ／ＮＲ５，经测序鉴定正确后，在Ｅ．ｃｏｌｉ中表达，经纯化后免疫新西兰兔。蛋白Ａ亲和层析和胰蛋白酶消化ＩｇＧ，经Ｓｅｐｈａｒｏｓｅ－１２柱层析制备Ｆ（ａｂ′）２。结果：经还原和非还原聚丙烯酰胺凝胶电泳和ＦＡＸ分析证明得到抗ＣＣＲ５ＮＨ２端的特异性抗体。结论：本方法为一简捷快速的特定功能结构域抗体Ｆ（ａｂ′）２制备方法，对研究该基因在体内的表达及生物学功能提供了重要的实验材料，同时也对超基因家族中亚家族特异抗体研制提供了一种研究思路和方法。

Objective:To study the functions of the first extracellular domain of β chemokine receptor 5(CCR5) NH 2 terminal and to prepare its specific antibody F(ab′) 2. Methods: Some β chemokine superfamily members were analyzed by computer and the least homologous domain of the extracellular loops were located. The first extracellular domain 114 nucleotides fragment was defined, sequenced and amplified by PCR,the expression vector pGEX IN/NR 5 of a recombinant GST fusion protein was constructed. After confirming the correctness of the inserted sequence,the transformation and expression of this fusion protein were performed in E. coli . The expression products of the fusion protein were purified and 2 New Zealand rabbits were immunized. An anti CCR5 NH 2 terminal antibody F (ab′) 2 was prepared by protein A affinity chromatography,pepsin digestion and Sepharose 12 column chromatography. Results: Reduced, unreduced SDS PAGE and FAX analysis demonstrated that this F (ab′) 2 had high specificity to combine with CCR5. Conclusion:In this paper,we introduce a simple and quick method to get a specific antibody F (ab′) 2 of certain functional domain. By that,not only can we get an important experiment material for studying gene expression,but also a good idea and technique to study other high similar superfamily members.