【作者】 周栋； 刘建柱； 张璐； 成子强； 张利； 刘海涛； 刘永夏； 杨笃宝； 王淑静；

【Author】 ZHOU Dong,LIU Jianzhu~*,ZHANG Lu,CHENG Ziqiang,ZHANG Li,LIU Haitao, LIU Yongxia,YANG Dubao,WANG Shujing (Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention;College of Animal Science and College of Veterinary Medicine,Shandong Agricultural University,Shandong Taian,271018,China)

【机构】 山东农业大学山东省动物生物技术与疫病防治重点实验室；

【摘要】 目的通过重叠区扩增法(PCR-based accurate synthesis,PAS),人工合成猪附红细胞体ORF5基因,并使其在大肠杆菌BL21(DE3)中高效表达。方法根据GenBank注册的AJ504999的ORF5基因序列,选择大肠杆菌偏爱的密码子,设计并合成5对寡核苷酸片段,用PAS方法,人工合成ORF5基因。基因克隆入pMD18-T载体,经测序证实正确后,连接到表达载体PET-32a,获得重组表达质粒PET-32a/ORF5,导入大肠杆菌BL21(DE3),在37℃,IPTG终浓度1mmol/L的条件下诱导表达,经SDS-PAGE分析,Western-blot鉴定,在35kDa附近出现目的片段。结果 ORF5基因能够在大肠杆菌中高效表达;通过生物信息学软件对其3D结构及蛋白功能等方面进行预测,预测结果表明该蛋白具有23个配基结合位点,6个α螺旋,胞浆外蛋白,没有信号肽,不存在糖基化位点。结论 ORF5基因能够在大肠杆菌中高效表达,推测其在猪附红细胞体蛋白的形成,附红细胞体的生物功能等方面有着重要作用。更多还原

【Abstract】 In this study the 0RF5 gene of Mycoplasma suis was obtained by PCR-based accurate synthesis(PAS) and put into E.coli BL21(DE3) for expression.Base on the gene sequence of the ORF5 gene of the AJ504999 which registered in the GenBank,eleven pairs of oligonucleotides were synthesized with E.coli biased codons. The target fragment was amplified with PAS method and cloned into pMD18-T vector.After sequenced,the gene was inserted into expression vector PET-32a.The recombinant plasmid PET-32a/ORF5 was transformed into E. coli BL21(DE3).Under the expression condition 1 mmol/L IPTG at 37℃,the protein was obviously appeared at 35kDa by SDS-PAGE and Western-blot.At the same time,the target protein of the 3D structure and function were predicted with bioinformatics software which showed that there were 23 binding site residues and had not cleavage site.It was extracellular protein and had not glycosylation site.更多还原