摘要
目的研制安全有效的预防猪繁殖与呼吸综合征病毒(PRRSV)重组鸡痘病毒活载体疫苗。方法将PRRSV长春株的GP5和GP3基因插入到含有猪IL-18基因的鸡痘病毒转移载体pUTAL复合启动子下游,构建重组鸡痘病毒转移载体pUTAL-IL-18-GP5-GP3。将该重组质粒与鸡痘病毒(FPV)282-E4株共转染鸡胚成纤维细胞(CEF),进行同源重组。通过3次BrdU加压筛选,经PCR、RT-PCR和IFA方法鉴定重组病毒。结果从重组鸡痘病毒基因组和总RNA中扩增出340bp的目的基因片段,感染重组病毒的CEF细胞与特异性荧光抗体发生阳性反应,表明目的基因在重组鸡痘病毒中得到表达。结论已成功构建1株重组鸡痘病毒,并可正确表达PRRSVORF5/ORF3基因。
Objective To develop a safe and effective recombinant fowlpox（FPV） as a vaccine against pig reproduction and respiration syndrome virus （PRRSV）. Methods Insert the GPS/GP3 gene of Changchun strain of PRRSV downstream to the composite promoter of fowlpox virus transferring vector pUTAL-IL-18. Co-transfeet chick embryo fibroblast（CEF） with the constructed recombinant plasmid pUTAL-IL-18-GPS-GP3 and FPV 282-FA strain for homologous recombination. After 3 cycles of pressure screening in presence of 5＇-bromo-deoxyuridine（BrdU） ,the recombinant virus was identified by PCR,RT-PCR and IFA. Results The target gene at a length of 340 bp was amplified from the genome and total RNA of recombinant fowlpox virus. The CEF transfeeted with recombinant fowlpox virus reacted with PRRSV specific fluorescent antibody. It indicated that the target gene was successfully expressed in recombinant fowlpox virus. Conclusion A recombinant fowlpox virus for expression of GPS/GP3 gene of PRRSV was successfully constructed.
出处
《中国生物制品学杂志》
CAS
CSCD
2006年第6期583-585,共3页
Chinese Journal of Biologicals
关键词
猪繁殖与呼吸综合征病毒
GP5
GP3
鸡痘病毒
Pig reproduction and respiration syndrome virus（PRRSV）
GP5
GP3
Fowlpox virus
