ZHOU Sheng,GAO Ge,SUN Dang,et al.The Gene Clone,Prokaryotic Expression and AntigenEpitopes Prediction of the Cysteine Domain ofNonstructural Protein 2 of PRRSV[J].HEILONGJIANG AGRICULTURAL SCIENCES,2012,(03):25-28.
PRRSV非结构蛋白2-半胱氨酸结构域的原核表达及抗原表位预测
- Title:
- The Gene Clone,Prokaryotic Expression and AntigenEpitopes Prediction of the Cysteine Domain ofNonstructural Protein 2 of PRRSV
- 文章编号:
- 1002-2767（2012）03-0025-04
- Keywords:
- PRRSV; Nsp2pro; gene clone; prokaryotic expression; antigen epitopes prediction
- 分类号:
- Q78
- 文献标志码:
- A
- 摘要:
- 为了获得PRRSV非结构蛋白2-半胱氨酸结构域的高纯度原核表达蛋白以及了解该蛋白的抗原表位,试验以从PRRSV-VR2332毒株上提取的病毒全基因组RNA为模板，通过RT-PCR扩增非结构蛋白2-半胱氨酸结构域（Nsp2pro）基因，连接构建原核表达载体SUMO-Nsp2pro，转入宿主菌Rosetta2，经IPTG诱导，获得高浓度的可溶蛋白Nsp2pro，经亲和层析His-Bind后得到高纯度的目的蛋白。同时，运用生物信息学方法对Nsp2pro级结构及抗原表位进行预测。结果显示，Nsp2pro的α螺旋及β折叠区域较多，转角区域较少，结构较为复杂，位于蛋白分子表面且亲水的区段很可能是B细胞表位的优势区段。获得较高纯度的蛋白，将为后续进一步研究Nsp2pro基因编码的蛋白在病毒复制过程中的作用奠定基础。
- Abstract:
- In order to acquire highly purified protein of cysteine domain of nonstructural protein 2 of PRRSV and acquaintance its antigen epiopes,the experiment were conducted with the genome extracted from PRRSV-VR2332 and got the gene of the cysteine domain of nonstructural protein 2 of PRRSV(Nsp2pro)by RT-PCR.Then,a recombinant plasmid named SUMO-Nsp2pro had been constructed,and then transformed SUMO-Nsp2pro into Rosetta2.Therefore,a large amount of Nsp2pro was obtained by IPTG.In the end,Nsp2pro was purified by His-Bind affinity chromatography,meanwhile predicted the second structure and antigen epiopes through means of bio-information,the results showed that Nsp2pro owned a great many Alpha regions and Beta regions while few of turn regions.The antigen epitopes were located in hydrolicity regions possibly.The obtained high purified protein would provide foundations for the further researches on Nsp2pro gene.
参考文献/References:
[1]秦爱建,韦平. 重要动物病毒分子生物学 [M]. 北京 : 北京科学出版 ,2008. [2]Jun Han MSR,Kay S Faberg.The porcine reproductive and respiratory syndrome virus Nsp2 cysteine protease domain possesses both trans- and cis-cleavage activities[J].Journal of Virology,2009,83（18）:9449-9463. [3]严景华,瞿李侯.Expression and purification of porcine reproductive and respiratory syndrome virus Nsp2 protein and analysis of cleavage activity[J].微生物学报,2009,49（11）:1502-1508. [4]de lima M PAK,Flores E F,Osorio F A.Mapping of B-cell lineat epitopes on Nsp2 and structural proteins of a North American strain sf porcine reproductive and respiratory syndrome virus[J].Virology,2006,353:310-421. [5]Ying Fang JC-H,Elizabeth Brown,Liu Haixia,et al.Development of genetic markers in the nonstructural protein 2 region of a US type 1 porcine reproductive and respiratory syndrome virus:implications for future recombinant marker vaccine development[J].Journal of General Virology,2008,89:3086-3096. [6]毛君婷,周碧君,史开志.Prrsv特种野猪分离株Nsp2基因序列分析和疫苗免疫效果[J].畜牧与兽医,2009(10):88-90. [7]Oleksiewicz MB BA,Toft P,Normann P,et al.Epitope mapping porcine reproductive and respiratory syndrome virus by phage display:the Nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes[J].J.Virol,2001,75（7）:3277-3290. [8]郭杨,陈世界,林华.高致病PRRSV Nsp2变异株Power SYBR Green real time RT-PCR检测方法的建立[J].中国兽医科学,2009,39(5):410-415. [9]Eric J，Snijder HvT.Norbert Roos,Ketil W Pedersen.Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex[J].Journal of General Virology,2001,82:985-994. [10]邱德新,陈焕春.猪繁殖与呼吸综合征(PRRS)弱毒活疫苗的安全性和体液免疫研究[J].华中农业大学学报,2004,23（4）:437-441.
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备注/Memo
基金项目：南京农业大学大学生创新资助项目（1004A04）