新型冠状病毒N蛋白的原核表达及其多克隆抗体的制备

Prokaryotic expression of a SARS-CoV-2 nucleocapsid protein and preparation of rabbit anti-N polyclonal antibodies

目的利用原核表达系统获得新型冠状病毒(SARS-CoV-2)N蛋白,将目的蛋白纯化后制备兔抗N蛋白多克隆抗体。方法对PET28a-2019-EnCoV-N重组质粒进行酶切和测序鉴定后转化E.coli BL21(DE3),筛选阳性重组菌,优化SARS-CoV-2 N重组蛋白表达条件,并对目的蛋白进行纯化,通过SDS-PAGE及Western blot对N蛋白进行鉴定。用纯化的N蛋白免疫新西兰大白兔获得多克隆抗体,利用间接ELISA、Western blot检测多克隆抗体的效价和特异性。结果酶切和测序表明,PET28a-2019-EnCoV-N重组质粒构建成功,经诱导在E.coli BL21(DE3)中成功表达约50 ku的SARS-CoV-2 N蛋白,该蛋白主要以可溶形式存在。用纯化的N蛋白免疫新西兰大白兔,获得高水平抗体血清,抗体效价达1∶128000以上,Western blot检测多克隆抗体与N蛋白具有良好的反应性。结论获得了免疫原性较强的N蛋白及具有特异性的兔抗N蛋白多克隆抗体,为研究N蛋白在新型冠状病毒感染中的作用及新型冠状病毒肺炎的诊断奠定了基础。

Objective To establish a prokaryotic expression system to express a nucleocapsid protein of SARS-CoV-2 and to prepare rabbit anti-N polyclonal antibodies.Methods The positive recombinant plasmid PET28 a-2019-EnCoV-N was transformed into E.coli BL21(DE3)after restriction enzyme digestion and sequencing.Expression of the recombinant N protein was induced and the target protein was purified.The protein was identified using SDS-PAGE and Western blotting.Polyclonal antibodies were prepared by immunizing New Zealand white rabbits with purified N protein and titers were determined using indirect ELISA and Western blotting.Results Restriction enzyme digestion and sequencing indicated that the recombinant plasmid PET28 a-2019-EnCoV-N was successfully constructed.After its expression was induced with IPTG,the target protein was expressed in E.coli BL21(DE3).The protein was about 50 ku and mainly existed in supernatant.New Zealand white rabbits were immunized with purified N protein to obtain high levels of antibodies,and the antibody titer was more than 1:128,000.Western blotting indicated that the polyclonal antibodies specifically recognized the N protein.Conclusion The N protein and rabbit anti-N polyclonal antibodies were obtained by immunizing New Zealand white rabbits with a SARS-CoV-2 nucleocapsid protein.These findings have laid the foundation for the further study of the role of nucleocapsid proteins and the diagnosis of COVID-19.