人抗菌肽HBD-2的原核表达及其多克隆抗体的制备与鉴定

Production of Polyclonal Antibody Against HBD-2 by Immunization with Recombinant GST-HBD-2 Fusion Protein

制备人β-防御素2(HBD-2)多克隆抗体,以用于HBD-2蛋白水平表达的检测。提取人肠腺上皮细胞株Caco-2总RNA,设计引物,应用RT-PCR从其总RNA中扩增HBD-2成熟肽cDNA片段,以pGEX-1λT为载体,构建原核表达质粒pGEX-1λT-HBD-2。大肠杆菌裂解物经亲合层析纯化后获得分子量约30kDa的融合蛋白,将此融合蛋白免疫新西兰兔,并用正辛酸-硫酸铵分步沉淀法初步纯化抗血清,ELISA法显示抗血清对HBD-2的效价为1∶12800,Western印迹显示抗血清可识别HBD-2。本实验结果证明应用重组融合小肽代替白蛋白或甲状腺-肽偶联免疫可获得其高效价的识别HBD-2的多克隆抗体,为今后从蛋白质水平研究HBD-2基因的诱导表达,包括组织分布和基因表达调控等研究打下了基础。

For the purpose of detecting the HBD-2 expression at protein level, the recombinant prokaryotic expression vector pGEX-1λT-HBD-2 was constructed and the E. coli-based product of GST-HBD-2 fusion protein was prepared. When rabbit was immunized with the fusion protein, the anti-serum against HBD-2 was produced. After caprylic acid and ammonium sulfate precipitation, high titer of specific polyclonal antibody against HBD-2, which was detected by ELISA and Western blot, was obtained. This result suggests that recombinant peptide fusion protein could be used instead of the conjugate of peptide-albumin or peptide-thyroid globulin to produce antibody. The obtained antibodies could be used for revealing the tissue distribution of HBD-2 and the regulation of its gene expression.