摘要
【目的】克隆、分析羊口疮病毒(ORFV)的ORF019基因,进而表达、纯化和鉴定重组表达产物.【方法】根据GenBank登录的ORFV OV-SA00株(AY386264)E2L基因序列设计1对引物,以提取的ORFV/QH02/2010株基因组DNA为模板,通过PCR获得该毒株的ORF019基因,将该基因连接至原核表达载体pET-32a(+)上,获得重组质粒pET-32a-ORF019,并对其测序,测序结果与副痘病毒属及其脊椎动物痘病毒亚科代表毒株的E2L蛋白进行一致性分析;并转化BL21(DE3)感受态细胞,经IPTG诱导获得重组融合蛋白,用SDS-PAGE和Western-blot对表达的目的蛋白进行分析.【结果】成功获得重组质粒pET-32a-ORF019,读码框正确;生物信息学分析发现,ORF019基因在副痘病毒属各成员间高度保守,也在脊椎动物痘病毒亚科痘病毒的遗传进化过程中保留了相对稳定的氨基酸残基;获得了约100 ku的融合蛋白表达产物,并以包涵体的形式存在于宿主菌.【结论】成功对ORFV019基因进行系统的遗传演化分析,并利用原核表达系统获得了重组表达产物.
【Objective】To clone and analyze the ORF019 gene of sheep aphthous virus(ORFV),and then express,purify and identify recombinant expression products.【Method】A pair of primers were designed based on the E2L gene sequence of ORFV OV-SA00 strain(AY386264)registered in GenBank.The ORF of the ORFV/QH02/2010 strain was used as a template to obtain the ORF019 gene of the strain by PCR.The recombinant plasmid pET-32a-0RF019 was obtained and ligated to the prokaryotic expression vector pET-32a(+),and the sequencing result was consistent with the E2L protein of the parapoxvirus and its vertebrate poxvirus subfamily representative strain.Sexual analysis,and transforming BL21(DE3)competent ce1ls,the recombinant fusion protein was induced by IPTG,and the expressed protein was analyzed by SDS-PAGE and Western-blot.【Result】The recombinant plasmid pET-32a-ORF019 was successfully obtained,and the reading frame was correct.Bioinformatics analysis found that the ORF019 gene is highly conserved among the members of the parapoxvirus,and also the genetic evolution of the vertebrate poxvirus subfamily poxvirus.A relatively stable amino acid residue is retained during the process.A fusion protein expression product of about 100 ku is obtained and is present in the host bacterium as an inclusion body.【Conclusion】The genetic evolution of ORFV019 gone was successfully analyzed,and the recombinant expression product was obtained by,prokaryotic expression system.
作者
沈岩尔
贾怀杰
王晓霞
陈国华
何小兵
房永祥
薛慧文
景志忠
SHEN Yan-er;JIA Huai-jie;WANG Xiao-xia;CHEN Guo-hua;HE Xiao-bing;FANG Yong-xiang;XUE Hui-wen;JING Zhi-zhong(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory of Veterinary Etiological Biology,Key Laboratory of Veterinary,Public Health of Agricultural Ministry,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046.China;Institute of Nutrition and Food Hygiene,School of Public Health,Lanzhou University,Lanzhou,730000,China)
出处
《甘肃农业大学学报》
CAS
CSCD
北大核心
2019年第6期1-7,共7页
Journal of Gansu Agricultural University
基金
“十三五”国家重点研发计划项目(2017YFD0500903,2016YFD0500907)
关键词
羊口疮病毒
ORF019基因
进化分析
原核表达
纯化
sheep aphthous virus
ORF019 gene
evolutionary analysis
prokaryotic expression
purification
