摘要
3C样蛋白酶(3CLpro)是冠状病毒的主蛋白酶,在病毒复制及致病过程中发挥重要作用。本研究旨在对猪流行性腹泻病毒3C样蛋白酶(PEDV 3CLpro)进行真核表达,并对其结构和功能进行生物信息学分析。根据GenBank上PEDV 3CLpro基因的序列设计并合成特异性引物,通过RT-PCR方法进行3CLpro基因的扩增,亚克隆至pEGFP-N1真核表达载体中,并转染至HEK 293T细胞,通过荧光观察和Western blot鉴定该蛋白的表达。并利用生物信息学软件对3CLpro进行序列分析、结构和功能预测。结果显示:PEDV 3CLpro能够在真核细胞中稳定高效表达,大小约为43 kDa,共编码302个氨基酸,分子式为C1446H2250N400O437S15,亲水性平均系数为0.058,不稳定指数为33.19,含有丰富的糖基化位点及磷酸化位点。其二级结构包含20.53%的α螺旋、38.41%的β折叠以及41.06%的无规则卷曲,对应的2个催化位点分别为41位的组氨酸(His)和144位的半胱氨酸(Cys)。上述结果为进一步研究PEDV 3CLpro在病毒与宿主互作中的生物学功能提供数据支撑。
3 CL protease(3 CLpro)is the main protease of coronavirus and it plays an important role in virus replication and pathogenesis.The purpose of this study was to express the porcine epidemic diarrhea virus(PEDV)3 CLpro in eukaryotic cells and to analyze its structure and function by bioinformatics.Specific primers were designed and synthesized based on the sequence of the 3 CLpro gene in GenBank,and the gene was amplified by RT-PCR,subcloned into the pEGFP-N1 eukaryotic expression vector,and transfected into HEK 293 T.The expression of the protein was determined by fluorescence observation and Western blot.And bioinformatics software was used to analyze the sequence,structure and function of 3 CLpro.The results showed that 3 CLpro could be expressed stably and efficiently in eukaryotic cells,and the size of the protein was about 43 kDa.PEDV 3 CLpro consisted of 302 amino acids,with a formula of C1446H2250N400O437S15,an average hydropathicity of 0.058,and an instability index of 33.19.The protein was rich in glycosylation and phosphorylation sites.And its secondary structure contained 20.53%of helices,38.41%of folds,and 41.06%of irregular curls.The corresponding catalytic sites were His at 41 and Cys at 144,respectively.These results provided supporting data for further research on the biological functions of PEDV 3 CLpro in virus-host interactions.
作者
梁荣
丁芳艺
宋海鑫
张金秋
费荣梅
LIANG Rong;DING Fangyi;SONG Haixin;ZHANG Jinqiu;FEI Rongmei(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonosis,Yangzhou 225009,China)
出处
《畜牧与兽医》
北大核心
2020年第9期80-87,共8页
Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31772701)
江苏省农业科技自主创新资金(CX(17)3027)。
