摘要
目的应用原核表达系统制备重组新型冠状病毒依赖RNA的RNA聚合酶(RNA-dependent RNA polymerase,RDRP),纯化后免疫新西兰大白兔获得高效价多克隆抗体,并对RDRP进行生物信息学分析。方法鉴定正确的pET-22b RDRP质粒转化表达载体,诱导表达RDRP蛋白,纯化后免疫大白兔,间接ELISA法检测其多克隆抗体效价。运用Expasy PortParam、SWISS MODEL等生物信息学软件对RDRP蛋白的等电点、分子质量、亲疏水性等理化性质、蛋白的空间结构、跨膜结构域、磷酸化位点、蛋白信号肽进行预测;构建系统进化树,进行亲缘性分析。结果pET-22b RDRP重组质粒酶切鉴定正确,经诱导重组蛋白以包涵体的形式表达,8 mol/L尿素梯度复性后得到纯度高的RDRP蛋白。用纯化蛋白免疫大白兔,间接ELISA检测血清抗体效价>1∶256000,Western blot检测抗体具有良好特异性。经生物信息学分析RDRP蛋白为亲水性,非跨膜蛋白,且无信号肽。11种病毒系统进化树显示,SARS-CoV-2 RDRP蛋白与SARS-CoV RDRP亲缘关系较近。结论成功获得重组SARS-CoV-2 RDRP蛋白,用该蛋白免疫大白兔能刺激产生高效价的多克隆抗体。预测与SARS-CoV RDRP亲缘关系较近,为亲水性非跨膜蛋白,为研究SARS-CoV-2 RDRP蛋白的生物学功能及其疫苗的制备奠定了基础。
Objective To use a prokaryotic expression system to prepare recombinant SARS-CoV-2 RNA polymerase(RDRP),to obtain high-titer polyclonal antibodies by immunizing New Zealand white rabbits,and to bioinformatically analyze RDRP.Methods The pET-22 b RDRP was identified as correct and transformed into an expression vector,and then the expression of RDRP was induced.After purification of the RDRP protein,white rabbits were immunized.The titer of polyclonal antibodies was determined using indirect ELISA.Bioinformatic software such as ExPASy ProtParam,and Swiss Model were used to predict the isoelectric point,relative molecular,hydrophilicity and hydrophobicity,spatial structure,transmembrane domains,phosphorylation sites,and protein signal peptides of RDRP and to construct an evolutionary tree for genetic analysis.Results Recombinant pET-22 b RDRP was identified as correct according to enzyme digestion.Expression of the recombinant protein was induced in the form of inclusion bodies.After protein refolding using an 8 mol/L urea gradient,the target protein was obtained with a high level of purity.After rabbits were immunized with the purified protein,the titer of polyclonal antibodies was more than 1:256000 according to indirect ELISA.Western blotting indicated that the antibodies had good specificity.According to bioinformatic analysis,the RDRP protein was a hydrophilic non-transmembrane protein with no signal peptides.A phylogenetic tree of 11 viruses indicated that SARS-CoV-2 and SARS-CoV RDRP had a high level of affinity.Conclusion Recombinant RDRP was successfully prepared,and it yields high-titer antibodies.Results predicted that it is closely related to SARS-CoV RDRP,which is a hydrophilic non-transmembrane protein.These findings have laid the foundation for studying the biological function of the SARS-CoV-2 RDRP protein and the preparation of vaccines.
作者
陈柯
闫硕
宋桃花
高岩
杜昆朋
郑皖怿
伊正君
付玉荣
CHEN Ke;YAN Shuo;SONG Tao-hua;GAO Yan;DU Kun-peng;ZHENG Wan-yi;YI Zheng-jun;FU Yu-rong(School of Basic Medicine,Weifang Medical University,Shandong 261053,China;School of Medicine Laboratory,Weifang Medical University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2022年第1期9-13,共5页
Journal of Pathogen Biology
基金
山东省自然科学基金重大基础研究项目(No.ZR2018ZC1054)。
