新城疫病毒(NDV)F基因的克隆及其真核表达重组质粒的构建

Cloning of the Newcastle Disease Virus (NDV) F Gene and Construction of an Eukaryotic Expression Recombinant Plasmid Containing NDV F Gene

应用反转录聚合酶链反应(RT-PCR)技术和一对自行设计的引物,从新城疫病毒(NDV)F48E9株扩增出长度为1664bp的DNA片段。与已报道的NDV-融合蛋白(F)基因比较,两者大小一致。经限制性内切酶反应,获得两个预期大小的片段。将克隆的F基因定向插入真核表达质粒pcDNA3中,经酶切和测序鉴定,证明构建的重组质粒含有NDV-F基因。

A DNA fragment of the Newcastle disease virus (NDV) fusion (F) protein with a length of 1 664 bp was obtained from a strain F48E9 by reverse transcription polymerase chain reaction (RTPCR) with a pair of designed primers. In comparison with the NDV F gene registered in GenBank, this fragment shows no difference in the length. After a restriction endonuclease cleavage, two expected smaller fragments were observed. Then the RTPCR product was inserted into an eukaryotic expression plasmid pcDNA3. The results of endonuclease cleave and nucleotide sequence indicated that the recombinant plasmid contains NDV F gene DNA sequence.