禽4型副黏病毒F基因的克隆及原核表达载体构建  

Cloning and Construction of Prokaryotic Expression Vector of F Gene of Avian Paramyxovirus Type 4

作　　者：王雪竹 伍晔晖 陶乔孝慈 刘建华[2] 张慧敏 WANG Xuezhu;WU Yehui;TAO Qiaoxiaoci;LIU Jianhua;ZHANG Huimin(College of Animal Science and Technology,Xinjiang Agricultural Vocational and Technical College,Changji 831199,China;College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Research and Development Center,TECON,Urumqi 830052,China)

机构地区：[1]新疆农业职业技术学院动物科技分院,新疆昌吉831199 [2]新疆农业大学动物医学学院,新疆乌鲁木齐830052 [3]天康生物制药有限公司研发中心,新疆乌鲁木齐831100

出　　处：《兽医导刊》2023年第2期1-5,共5页Veterinary Orientation

摘　　要：扩增禽4型副黏病毒(Avian paramyxovirus type 4,APMV-4)新疆野鸭源分离株APMV-4/Pintail/CH(XJ)/01/2016的F基因,并构建原核表达载体。参考GenBank上公布的APMV-4序列,通过DNAStar设计F基因特异性引物,以APMV-4/Pintail/CH(XJ)/01/2016基因组RNA为模板,采用RT-PCR方法扩增F基因编码区,连接于pMD19-T载体,用BamHⅠ和HindⅢ酶切原核表达载体pET-30a重组质粒,将酶切产物克隆至pET-30a多克隆位点上,转化至大肠杆菌DH5α感受态细胞中,构建F基因原核表达载体,并对阳性重组质粒进行PCR和测序鉴定。结果显示,扩增出的片段大小为1701 bp,经测序鉴定该扩增片段为F基因全长,与GenBank中收录的APMV-4序列核苷酸相似性为99%;连接表达载体结果表明,原核表达载体pET-30a-F成功构建,为APMV-4诊断方法的建立提供了良好的技术基础。In order to clone the F gene of Avian Paramyxovirus type 4(APMV-4)/Pintail/CH(XJ)/01/2016 from wild ducks in Xinjiang and to construct the prokaryotic expression vector.The coding sequence(CDS)of F gene was amplified by RT-PCR with a pair of primers,which was designed according to the published sequence of APMV-4 F gene using DNAStar software.The amplified gene was cloned into the pMD19-T vector,follewed by being digested by BamHI and HindⅢrecycled product was inserted into the multiple cloning sites of the PET-30a.Which was transformed into DH5αand the positive plasmid of the recombinant plasmid,that was identified by restriction enzyme,PCR and sequencing.The results showed that the ful-length of F gene 1701bp was amplified.The F gene were cloned into the vector pET-30a-F recombinant expression vectors were constructed successfully.The construction of recombinant expression vectors could provide a technical basis for the diagnosis of APMV-4.

关 键 词：禽4型副黏病毒 F基因 克隆 原核表达载体 

分 类 号：S852.65[农业科学—基础兽医学]