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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 of cytochrome P450CYP4G19 gene and induce its expression. The expressed fusion protein was purified and polyclonal antibody against CYP4G19 was prepared. Methods The partial CYP4G19 gene fragment was amplified by RT-PCR. The product was identified by TA cloning and sequencing, subcloned into prokaryotic expression vector pET-28a, induced in E. coli BL21 (DE3), and purified by nickel ion affinity chromatography After immunization, the mice were immunized to obtain polyclonal antibodies, and the titer and specificity of the polyclonal antibodies were detected by ELISA and Western-blot. Results A fragment of 771bp of hydrophilic gene was cloned from Blattella germanica cDNA and expressed in E. coli as P450 recombinant protein in the form of inclusion bodies at about 32000Mr. The purified, renatured recombinant protein was immunized in mice to obtain titers higher than 1: 106 high titer polyclonal antibody. Western-blot showed that this polyclonal antibody specifically binds to 32000Mr of recombinant protein and recognizes native Blattella germanica microsomal P450 protein. Conclusion The prokaryotic expression of CYP4G19 fusion protein has good immunogenicity. The preparation of the polyclonal antibody against Blattella germanica CYP4G19 with high titer and specificity provided an important experimental tool for further research on the expression characteristics and drug resistance function of CYP4G19 protein of Blattella germanica.