德国小蠊细胞色素P450 CYP4G19基因的原核表达及多克隆抗体制备

Prokaryotic Expression of German cockroach Cytochrome P450 CYP4G19 Gene and Preparation of Polyclonal Antibody

目的构建细胞色素P450CYP4G19基因部分片段的原核表达载体并诱导其表达,纯化表达的融合蛋白并制备CYP4G19多克隆抗体。方法应用RT-PCR扩增CYP4G19基因部分片段,产物经T-A克隆、测序鉴定,亚克隆入原核表达载体pET-28a,在大肠杆菌BL21(DE3)中诱导表达,镍离子亲和层析法纯化重组蛋白后,免疫小鼠,获得多克隆抗体,ELISA及Western-blot检测多抗的效价及特异性。结果从德国小蠊cDNA中克隆出一段771bp的亲水性基因片段,在大肠杆菌中诱导表达出约32000Mr、以包涵体形式存在的P450重组蛋白。将纯化、复性的重组蛋白免疫小鼠,得到了滴度高于1∶106的高效价多克隆抗体。Western-blot显示此多抗能与32000Mr的重组蛋白特异结合,并能识别天然的德国小蠊微粒体P450蛋白。结论利用原核表达的CYP4G19融合蛋白具有良好的免疫原性。制备出效价高、特异性强的抗德国小蠊CYP4G19多克隆抗体,为下一步关于德国小蠊CYP4G19蛋白表达特性及其抗药性功能的深入研究提供了重要的实验工具。

Objective To construct prokaryotic expression vector carrying a partial German cockroach cytochrome P450 CYP4G19 gene, and to express the recombinant protein in E. coil. Using the purified fusion protein for the production of polyclonal antibody. Method Part of the CYP4G19 gene was amplified by RT-PCR. PCR products were ligated to the cloning vector pGEM T-easy. The positive clones were sequenced and then subcloned into prokaryotic expression vector pET-28a. The constructed pET-28a-CYP4G19 vetor was transformed to E.coil BL21 （DE3） and the recombinant P450 protein （rP450） was expressed, rP450 was then purified using Ni^＋ affinity column chromatography. Balb/c mice were then immunized using the purified rP450 and the titer of the polyclonal antibody was determined by ELISA. The specificity of the antibody was confirmed by Western-blot. Result A hydrophilic fragment of 771bp was cloned from the German cockroach cDNA. The recombinant protein with a relative molecular weight of 32000 Mr was expressed as inclusion bodies. The protein was then purified and renatured for immunization. The titer of the polyclonal antibody was greater than 1：10^6. Western-blot analysis indicated that the antibody specifically reacted with rP450 and recognized the native German cockroach microsome P450. Conclusion The expressed CYP4G19 fusion protein was antigenic. The CYP4G19 polyelonal antibody may be used for further characterization of the German cockroach CYP4G19 protein, especially for the further studies of the development of pesticide resistance.