新型冠状病毒N蛋白原核可溶性表达与血清学评价  

Prokaryotic Soluble Expression and Serological Evaluation of SARS-CoV-2 N Protein

作　　者：侯江厚 孙卫国 黄国红 詹晓燕[1] 杨奕梅 HOU Jiang-Hou;SUN Wei-Guo;HUANG Guo-Hong;ZHAN Xiao-Yan;YANG Yi-Mei(Kunming City Matermal and Child Health Hospital,Kunming 650013,China;Eighth Medical Center of General Hospital of PLA,Beijing 100091,China)

机构地区：[1]昆明市妇幼保健院,云南昆明650013 [2]解放军总医院第八医学中心,北京100091

出　　处：《生物技术通讯》2020年第4期427-431,共5页Letters in Biotechnology

摘　　要：目的:利用原核系统可溶性表达新冠病毒(SARS-CoV-2)N蛋白,评价其在血清学诊断上的可行性。方法:将SARS-CoV-2 N蛋白对应的核酸表达序列克隆到载体pET-DsbC上,经原核表达和亲和纯化获得可溶性DsbC-N融合蛋白,通过ELISA试验检测30份确诊新冠肺炎患者、50份健康人血清,评价重组DsbC-N蛋白在新冠肺炎患者血清学诊断中的应用价值。结果:DsbC-N融合蛋白在原核表达系统中以可溶性形式表达,经亲和层析纯化后相对分子质量为68×10^3,纯度为92%。ELISA结果显示纯化后的DsbC-N蛋白与新冠肺炎患者血清有较强的反应,血清学诊断敏感性为96%,特异性为98%。结论:原核可溶性表达的DsbC-N蛋白经纯化后在新冠肺炎诊断方面具有很高的应用价值,可以作为诊断抗原用于检测患者血清中的抗体。Objective:To prokaryotic express soluble N protein of SARS-CoV-2,and to evaluate its serological diagnosis value.Methods:The nucleicacid sequence of corresponding N protein gene was obtained by PCR and cloned into pET-DsbC vector.After soluble expression and affinity purification,the feasibility was evaluated of fusion protein DsbC-N for clinical serological diagnosis by ELISA with 30 COVID-19 patients and 50 healthy people sera.Results:The DsbC-N fusion protein was expressed in soluble form and efficiently in prokaryotic system with the relative molecular weight was about 68×10^3 and the purity was about 92% after purification by affinity chromatography,which had a significantly higher level of reactivity to sera from COVID-19 patients.The sensitivity and specificity were about 96% and 98% with serological diagnosis.Conclusion:The DsbC-N protein expression in solubity and high purity had an important value in diagnosis of SARS-CoV-2,and might be selected as one of diagnostic antigens to detect antibodies in the serum of COVID-19 patients.

关 键 词：新型冠状病毒 N蛋白 可溶性表达 酶联免疫吸附试验 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]