【作者】 杨建新； 郭育培； 张学东； 王泽； 党源； 张晓东； 丁壮；

【Author】 YANG Jian-xin,GUO Yu-pei,ZHANG Xue-dong,WANG-ze,DANG-yuan,ZHANG Xiao-dong,DING-zhuang(College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062)

【机构】 吉林大学畜牧兽医学院；

【摘要】 为了研究RKIP对新城疫病毒(NDV)复制的影响,从鸡胚成纤维细胞中提取总RNA,采用RT-PCR方法扩增RKIP基因,将其克隆入真核表达载体pEGFP-N1中,经PCR、双酶切和测序鉴定,采用FuGENEHD法将阳性质粒转染Vero细胞,经G418筛选,有限稀释法获取单克隆细胞株,Western blot检测RKIP的表达。利用Real-time PCR测定病毒感染细胞后培养上清的病毒量。结果表明,成功构建鸡RKIP真核表达质粒;转染Vero细胞后经克隆化培养获得稳定表达鸡RKIP的细胞株Vero-RKIP;感染初期,NDV在Vero-RKIP上表现出了更强的复制能力。更多还原

【Abstract】 To research the influence of Raf kinase inhibitor protein(RKIP)on Newcastle Disease Virusreplication,the total RNAs were isolated from chick embryo fibroblast(CEF).RKIP DNA segment was amplified using reverse transcriptase polymerase chain reaction(RT-PCR)and cloned into pEGFP-N1 vector.The positive plasmid identified by PCR、enzyme digestion and DNA sequencing was transfected into Vero by FuGENE HD.The stable transfectants were screening by G418.Cell subclones were isolated by utmost dilution.The expression of RKIP was detected by Western blot.After the NDV infected cells,the viral load of culture supernatant was analyzed by Real-time PCR.These results showed that a recombinant eukaryotic expression plasmid was successfully constructed.A Vero-RKIP cell strain stably expressing RKIP was subsequently obtained.In the early stage of infection,NDV showed significant higher replication ability on Vero-RKIP.更多还原