拯救新城疫病毒LaSota株辅助质粒的构建

Construction of the Helper Plasmid for Rescue LaSota Strain of Newcastle Disease Virus

本实验室保存的LaSota株新城疫病毒经SPF鸡胚增殖,收集尿囊液并纯化病毒,提取病毒基因组RNA。参考GeneBank收录的LaSota株新城疫病毒基因组序列,设计6对特异性引物,分别扩增病毒的NP、P和L1～L4六个基因片段,并将目的片段回收纯化,克隆至pMD18-T载体,转化大肠杆菌DH5α,小提质粒并酶切鉴定,鉴定正确的样品进行序列测定,并比对测序结果。结果证明测序结果与GeneBank上公布的LaSota株新城疫病毒序列完全一致,没有任何氨基酸和核苷酸的变化,将NP、P、L分别连接至pVAX载体,其中L1～L4为顺次连接。鉴定结果表明NP、P和L基因已正确克隆至pVAX表达载体,证明3个辅助质粒构建成功,分别命名为pVAX-NP、pVAX-P和pVAX-L。

Newcastle disease virus（NDV）LaSota strain was seperated and conserved in my laboratory. Virus was multiplied by SPF embryonated eggs. Allantoic fluid was harvest,purified and then extracted the RNA genome. Six pairs of specific primers were designed to amplify NP,P,L1 ～ L4 gene fragments with the method of RT-PCR, Based on the complete genome sequence of NDV LaSota strain logged in GeneBank. Simultaneously,the target gene fragments were recycled and purified and insert into pMD18-T vector to sequencing. Sequence comparison proved that the gene sequence is same with standard sequence of NDV LaSota stain which included in the GeneBank. No amino acid and nucleotide changes were found in the positive clones,NP,P,L was connected to the pVAX vector. Enzyme cut result showed that NP,P,L were cloned into the pVAX vector,and proved that the three helper plasmid was constructed successfully,named pVAX-NP,pVAX-P and pVAX-L respectively。