猫冠状病毒S蛋白生物信息学分析与原核表达  

Bioinformatics analysis and prokaryotic expression of feline Coronavirus S protein

作　　者：章高强 董波[1,3] 张晓东 陈雪菲 林炜明 ZHANG Gaoqiang;DONG Bo;ZHANG Xiaodong;CHEN Xuefei;LIN Weiming(College of Life Science,Longyan University,Longyan 364012,China;College of Animal Sciences,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Fujian Provincial Key Laboratory of Livestock Infectious Disease Prevention and Biotechnology/Fujian Provincial Universities Key Laboratory of Preventive Veterinary Medicine and Biotechnology,Longyan 364012,China)

机构地区：[1]龙岩学院生命科学学院,福建龙岩364012 [2]福建农林大学动物科学学院,福州350002 [3]福建省家畜传染病防治与生物技术重点实验室/预防兽医学与生物技术福建省高等学校重点实验室,福建龙岩364012

出　　处：《黑龙江畜牧兽医》2023年第3期119-124,129,137,138,共9页Heilongjiang Animal Science And veterinary Medicine

基　　金：福建省自然科学基金项目(2022J011155)。

摘　　要：为了研发基于S蛋白的FCoV诊断试剂盒和疫苗,试验采用生物信息学分析方法分析表达FCoV S蛋白,确定含有抗原表位的蛋白可表达区域,并在大肠杆菌中原核表达重组蛋白,筛选重组蛋白表达条件,鉴定重组蛋白的免疫原性。结果表明:Ⅰ型FCoV S蛋白二级结构中α-螺旋、延伸链、β-转角和无规则卷曲分别占25.82%、28.14%、4.23%和41.80%;有4个比较明显的亲水性区域与1个明显的疏水性区域;该蛋白质具有穿膜螺旋结构与信号肽,并存在3个可能性很高的N-糖基化位点与S蛋白表面存在46个氨基酸较为暴露的片段。以S蛋白1127~1400 aa片段作为体外表达目的片段,命名为S1127-1400aa;重组蛋白最佳诱导温度为37℃、IPTG最佳诱导浓度为0.1 mmol/L、最佳诱导时间为5 h;重组蛋白以包涵体形式表达;成功纯化重组蛋白S1127-1400aa;与FCoV阳性血清有良好的免疫原性。说明试验成功表达并纯化的FCoV S蛋白片段,该蛋白质具有良好的免疫原性。In order to develop FCoV diagnostic kits and vaccines based on S protein,the experiment used bioinformatic analysis to analyze the expression of FCoV S protein,to determine the expressible region of protein containing antigenic epitopes,to express recombinant protein in prokaryote in Escherichia coli,to screen the expression conditions of recombinant protein,and to identify the immunogenicity of recombinant protein.The results showed that theα-helix,extension chain,β-angle and irregular coil accounted for 25.82%,28.14%,4.23%and 41.8%respectively in the secondary structure of typeⅠFCoV S protein.There were 4 obvious hydrophilic regions and 1 obvious hydrophobic region.The protein had a transmembrane helix structure and signal peptide,and there were 3 N-glycosylation sites with a high probability and relatively exposed 46-amino-acid fragments on the surface of S protein.The S protein 1127-1400 aa fragment was used as the fragment of in vitro expression target and named S1127-1400aa.The optimal induction temperature of recombinant protein was 37℃;the optimal induction concentration of IPTG was 0.1 mmol/L,and the optimal induction time was 5 h.Recombinant proteins were inclusion body expression;successful purification of S1127-1400aaprotein was carried out,which had good immunogenicity with FCoV-positive serum.The results suggested that the FCoV S protein fragment was successfully expressed and purified in this experiment,and the protein had good immunogenicity.

关 键 词：S蛋白 生物信息学分析 原核表达 表达条件 免疫原性 鉴定 

分 类 号：S855.3[农业科学—临床兽医学] S829.3[农业科学—兽医学]