鸭坦布苏病毒RNA依赖RNA聚合酶的真核表达及生物信息学分析  

Eukaryotic Expression and Bioinformatics Analysis of RNA-dependent RNA Polymerase of Duck tembusu virus

作　　者：韩凯凯[1,2,3] 严若峰 刘青涛[1,2,3] 刘宇卓 李银[1,2,3] 赵冬敏[1,2,3] 黄欣梅[1,2,3] 章丽娇 杨婧[2] 付晨 HAN Kaikai;YAN Ruofeng;LIU Qingtao;LIU Yuzhuo;LI Yin;ZHAO Dongmin;HUANG Xinmei;ZHANG Lijiao;YANG Jing;FU Chen(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;School of Life Sciences,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]江苏省农业科学院兽医研究所,江苏南京210014 [3]江苏大学生命科学学院,江苏镇江212013

出　　处：《华北农学报》2021年第6期204-210,共7页Acta Agriculturae Boreali-Sinica

基　　金：江苏省农业科技自主创新资金项目(cx(20)3093);国家自然科学基金(31502101)。

摘　　要：旨在对鸭坦布苏病毒NS5蛋白功能基团RNA依赖RNA聚合酶进行真核表达,同时对其蛋白结构和功能进行生物信息学分析。首先查找GenBank数据库中收录的DTMUV基因序列,以RdRp基因为研究对象,通过软件设计并合成特异性引物,RT-PCR技术扩增RdRp基因,回收PCR产物并与pCMV-N-Flag真核表达载体相连接,构建真核表达质粒pCMV-Flag-RdRp,将测序无误的质粒转染至BHK-21细胞,通过Western Blot和间接免疫荧光技术检测该蛋白在BHK-21细胞内的表达。同时针对RdRp蛋白,利用生物信息学软件进行序列分析、结构解析及功能预测。通过PCR方法成功扩增RdRp基因,构建的真核表达质粒pCMV-Flag-RdRp经双酶切鉴定证明正确,进一步通过间接免疫荧光技术与Western Blot检测发现,重组质粒pCMV-Flag-RdRp在BHK-21细胞内正常表达,生物信息学分析发现,RdRp蛋白编码606个氨基酸,分子式为C_(3091)H_(4810)N_(870)O_(894)S_(41),编码蛋白亲水性平均系数为-0.536,不稳定指数为42.15,含有丰富的磷酸化位点及O-糖基化位点。其二级结构包含46.37%的α-螺旋、12.54%的延伸链以及35.31%的无规则卷曲。上述结果为进一步研究坦布苏病毒致病性打下了基础。The purpose of this study is to express the RdRp of DTMUV in eukaryotic cells and to analyze its structure and function by bioinformatics.Firstly,specific primers were designed and synthesized based on the sequence of the RdRp gene of DTMUV JS804 strain in GenBank,and then the gene was amplified by RT-PCR,subcloned into the pCMV-N-Flag eukaryotic expression vector,transfected into BHK-21 cells.Next,the expression of RdRp was determined by immunofluorescence observation and Western Blot.After that,bioinformatics software was used to analyze the sequence,structure and function of RdRp.RdRp gene was successfully amplified by PCR,and the constructed eukaryotic expression plasmid PCMV-Flag-RdRp was verified to be correct by double enzyme digestion and identification.Further,indirect immunofluorescence and Western Blot detection showed that the recombinant plasmid PCMV-Flag-RdRp was normally expressed in BHK-21 cells.Bioinformatics analysis found that,DTMUV RdRp consisted of 606 amino acids,with a formula of C_(3091)H_(4810)N_(870)O_(894)S_(41),an average hydropathicity of-0.536,and an instability index of 42.15.The RdRp was rich in glycosylation and phosphorylation sites.The ratios ofα-helix,random coil and extending strand in the secondary structure were 46.37%,35.31%,12.54%,respectively.All of these results provided supporting data for the further research on biological functions of DTMUV.

关 键 词：鸭坦布苏病毒 RNA依赖RNA聚合酶 生物信息学 

分 类 号：Q78[生物学—分子生物学] S432.4[农业科学—植物病理学]