摘要
为深入研究小反刍兽疫病毒(peste des petits ruminants virus,PPRV)P蛋白对病毒复制的影响,以小反刍兽疫病毒Nigeria75/1株为研究对象,通过RT-PCR法扩增P基因,将扩增出的P基因克隆到pGEX-6P-1原核表达载体中,成功构建pGEX-6P-1-PPRV-P表达载体,测序鉴定无误后转化至表达宿主菌BL21(DE3)plysS中进行IPTG诱导表达,并通过在线软件预测了P蛋白的磷酸化位点。结果显示,pGEX-6P-1-PPRV-P重组菌在IPTG浓度1.0mmol/L时经37℃诱导4h,目的基因可正确表达,并以可溶性的形式存在。经Western-blot鉴定,目的蛋白可与PPRV Nigeria75/1株病毒小鼠阳性血清发生特异性的免疫印迹反应,证明表达的蛋白具有较好的反应原性。经综合预测分析,可能被磷酸化的位点有46个,其中PKC、PKA、CKII/CKI、cdc2可能作用的位点分别为15个、7个、10个、6个,其他激酶作用位点可能为8个。结果表明,P蛋白的克隆表达及磷酸化位点的成功预测为小反刍兽疫病毒的深入研究奠定了基础。
For studying effects of P protein on viral replication of peste des petits ruminants virus （PPRV） ,P gene was amplified by RT-PCR,and then cloned into pGEX-6P-1 prokaryotic expression plasmid and transformed into BL21（DE3） plysS host cells. The recombinant plasmid was expressed by induction with IPTG and P protein＇s phosphorylation sites were predicted through online software. In result, P protein was expressed in a soluble form at the IPTG concentration of 1.0 mmol/L at 37 ~C for 4 h. Western- blot analysis indicated that the purified protein reacted with anti-PPRV antibody. According to this find ing,the expressed P protein had good reactionogenicity. The P protein might have 46 phosphorylated sites. Cloning and expression of P protein gene and the successfully prediction of phosphorylation sites of P protein will provide the foundation for further study of peste des petits ruminants virus.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2013年第12期1252-1256,共5页
Chinese Veterinary Science
基金
国家自然科学基金资助项目(31172342)
国家公益性行业科研专项(200903037)
关键词
P蛋白
小反刍兽疫病毒
原核表达
磷酸化位点
P protein
peste des petits ruminants virus
prokaryotic expression
phosphorylation site
