鸡新城疫病毒F基因部分片段克隆表达及其抗体制备

目的]为进一步研究新城疫病毒的分子结构和基因疫苗奠定基础。方法]用自行设计的l对引物,通过RT-PCR从接毒的SPF鸡胚的尿囊液中克隆了新城疫病毒F基因部分片段,将该基因片段插入含谷胱甘肽(GST)基因的质粒pGEX-4T-1,构建了重组质粒,对提取的融合蛋白进行亲和层析纯化和琼扩、ELISA及W estern-blot检测。结果]通过克隆和PCR扩增获得新城疫病毒F基因的部分片段,大小为509 bp;对构建的重组质粒pGEX-4T-1-F509经诱导表达,获得分子大小约为44 000 bp的融合蛋白,其中F基因的部分片段大小约18 000 bp;经亲和层析,获得纯化GST-F509融合蛋白,进一步用该蛋白免疫小鼠,制备了鼠源抗鸡新城疫病毒F蛋白抗体。结论]琼脂扩散试验、ELISA及W estern-blot检测表明,该融合蛋白中的F片段具有良好的抗原性。

Cloning and Expression of Partial F Gene Segement in Chicken Newcastle Disease Virus and Its Antibody Preparation

Objective] The research aimed to lay the foundation for further study on the molecular structure and gene vaccine of Newcastle disease virus.Method] Using one pair of self-made primers,partial F gene segment in Newcastle disease virus was cloned from allantoic fluid in SPF chicken embryo after inoculating the virus through RT-PCR.This gene segment was inserted into plasmid pGEX-4T-1 with glutathione(GST) gene to construct the recombinant plasmid.The extracted fusion protein was purified by affinity chromatography and detected by agar diffusion test,ELISA and Western-blot.Result] Partial segment of F gene in Newcastle disease virus was obtained through cloning and PCR amplification,with the length of 509 bp.The induced expression was made on the constructed recombinant plasmid pGEX-4T-1-F509,the fusion protein with the molecular size of 44 000 bp was obtained,among them the partial segment size in F gene was 18 000 bp.Through affinity chromatography,the purified GST-F509 fusion protein was obtained.The fusion protein was further used to immunize mice to prepare mice F protein antibody against Newcastle disease virus in chicken.Conclusion] Agar diffusion test,ELISA and Western-blot detection showed that F segment in this fusion protein had good antigenicity.