摘要
参照包含猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV) VR2332株完整ORF5基因的载体pMD18T-ORF5的核酸序列设计引物,扩增全长的PRRSV ORF5基因片段,同时以SOE-PCR技术扩增缺失信号肽和跨膜区的ORF5核酸片段ORF5NC,分别克隆至Bac-to-Bac杆状病毒表达系统的供体质粒pFastBac-HTA,获得重组转移载体pFastBacHTA-ORF5及截短表达的重组转移载体pFastBacHTA-ORF5NC。将供体质粒分别转化DH10Bac细胞,获得重组穿梭质粒,转染Sf9昆虫细胞,获得重组杆状病毒。SDS-PAGE和Western blot检测结果表明,在Sf9细胞中分别成功表达了全长的GP5重组蛋白和缺失信号肽、跨膜区的截短GP5重组蛋白。2种重组蛋白经纯化后,免疫小鼠制备抗血清,ELISA方法检测免疫GP5全长和截短重组蛋白的小鼠血清中抗体滴度分别为1∶800和1∶1 600,表明重组蛋白具有良好的免疫原性。本试验为进一步分析重组蛋白诱导中和抗体产生的能力以及为PRRSV基因工程亚单位疫苗的研究奠定基础。
To express glycoprotein 5 of porcine reproductive and respiratory syndrome virus in baculovirus expression system and analyze the immunogenicity of the expressed product,the primers were designed according to the nucleotide sequence of vector pMD18 T-ORF5 containing ORF5 gene of PRRSV VR2332 strain.Complete ORF5 gene and incomplete ORF5 gene lack of transmembrane domain and signal peptide were amplified by PCR and the two fragments were respectively cloned into the pFast-HTA vector to obtain two recombinant vector pFastBacHTA-ORF5 and pFastBacHTA-ORF5 NC,and then transformed into DH10-Bac cells that contain baculovirus shuttle plasmid.Recombinant transfer vector pFastBacHTA-ORF5 and truncated expressed recombinant transfer vector pFastBacHTA-ORF5 NC were obtained and then respectively transfected into Sf9 cells to obtain two recombinant baculovirus.SDS-PAGE and Western blot result showed that the full-length GP5 recombinant protein and the deletion signal peptide and the truncated GP5 recombinant protein in the transmembrane region were successfully expressed in Sf9 cells,respectively.Mice were immunized with purified recombinant proteins to prepare antiserum,the titer of antiserum were detected by indirect enzyme linked immunosorbent assay(ELISA) with purified PRRSV as coating antigen,the titer of the full length GP5 antiserum was 1∶800 and 1∶1 600 for truncated GP5 antiserum,indicating that the recombinant protein has good immunogenicity.This study laid a foundation for the further analysis of the ability of recombinant protein to induce neutralizing antibody production and for the study of PRRSV gene engineering subunit vaccine.
作者
许瑞勤
郭嘉
魏凤灵
冯延
马思续
张留君
李想通
李闻
孙杨杨
杨国宇
夏平安
张改平
XU Rui-qin;GUO Jia;WEI Feng-ling;FENG Yan;MA Si-xu;ZHANG Liu-jun;LI Xiang-tong;LI Wen;SUN Yang-yang;YANG Guo-yu;XIA Ping-an;ZHANG Gai-ping(College of Animal Science and. Veterinary Medicine ,Henan Agricultural University ^Zhengzhou 450002,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2019年第4期609-614,621,共7页
Chinese Journal of Veterinary Science
基金
国家自然科学基金重大资助项目(31490600)
国家自然科学基金资助项目(31572520)
