溶葡萄球菌素的原核表达及多克隆抗体的制备

Prokaryotic expression of lysostaphin and preparation of its polyclonal antibody

【目的】制备溶葡萄球菌素(lys)的多克隆抗体,为检测其在转基因细胞、胚胎及转基因动物中的表达奠定基础。【方法】以含有lys基因成熟肽序列的PEPB质粒为模板,经PCR扩增,构建pET28a-Ly原核表达载体,并以其转化大肠杆菌BL21(DE3)菌株,经IPTG诱导表达并纯化蛋白后,常规免疫家兔,制备多克隆抗体;用ELISA方法检测抗体效价,Western blot检测抗体的特异性。【结果】构建的原核表达载体pET28a-Ly经IPTG诱导6h后即可高效表达lys蛋白,蛋白经纯化后,其质量浓度可达到3.05mg/mL;抗体经ELISA检测,效价为1∶27 000,West-ern blot检测结果表明,抗体的特异性较好。【结论】成功构建了lys原核表达载体,所制备的lys多克隆抗体具有较高的效价和良好的特异性。

【Objective】 A polyclonal antibody of lysostaphin（lys） protein was prepared to lay a foundation so that the expression of the lys gene could be detected in transgenic cells,embryos and transgenic animals.【Method】 The mature peptide of lys gene was amplified by PCR from the PEPB plasmid with lys gene sequence inside and then cloned into a prokaryotic expression vector pET-28a to construct a recombinant plasmid pET28a-Ly.The pET28a-Ly plasmid was transformed into E.coli BL21（DE3） cells to produce the recombinant lys protein with His tag by inducing 1 mmol/L IPTG for 6 hours.The recombinant lys protein was purified by using a His-tag fusion protein purification kit and injected into a few rabbits to get the specific polyclonal antibody.The specificity and antibody titer of the polyclonal antibody was confirmed by western blot and ELISA.【Result】 The recombinant lys protein was efficiently expressed in E.coli BL21（DE3） after induction with IPTG.3.05 mg/mL of the protein concentration was obtained after purification.Antibody titer of 1∶27 000 was detected by ELISA.The results of Western blot showed that the antibody specificity was better.【Conclusion】 The prokaryotic expression vector pET28a-Ly was constructed successfully.The prepared polyclonal antibody of lys protein has a high titer and good specificity.