摘要
为了对羊口疮病毒(ORFV)AH-F10株127基因进行原核表达及亚细胞定位,本试验采用PCR方法扩增出ORFV127基因,成功构建原核表达重组质粒pGEX-6p-1-ORFV127和真核重组质粒pEGFP-N1-ORFV127。将原核表达重组质粒转化到大肠埃希氏菌中表达,并进行纯化和鉴定。以纯化的蛋白质免疫BALB/c雌鼠制备多克隆抗体,利用Western-blot技术鉴定其反应原性。利用脂质体介导法将重组质粒pEGFP-N1-ORFV127转染至Vero细胞,通过倒置荧光显微镜观察其在细胞中的表达及亚细胞定位。结果表明,获得的ORFV127基因序列全长为558bp,ORFV127蛋白在大肠杆菌中获得高效表达,主要以包涵体蛋白质的形式表达,大小约49000。Western-blot结果显示,免疫小鼠获得的抗ORFV127蛋白的多克隆抗体可特异性识别ORFV127蛋白,倒置荧光显微镜观察发现,ORFV127蛋白主要定位于细胞质。本试验结果为后续研究ORFV127蛋白的功能提供了宝贵的生物材料。
This study was designed to achieve prokaryotic expression and subcellular localization of the Orf virus(ORFV)127 gene of AH-F10 strain.The gene was amplified by PCR and cloned into pGEX-6p-1 and pEGFP-N1.The recombinant plasmids were named as pGEX-6p-1-ORFV127 and pEGFP-N1-ORFV127,respectively.Prokaryotic expression recombinant plasmid was transformed into Escherichia coli for expression,purification and identification.Then,the BALB/c mice were immunized with the purified ORFV127 protein to prepare polyclonal antibody and the reaction was detected by Western-blot.The recombinant plasmid pEGFP-N1-ORFV127 was transfected into Vero cells by liposome mediated method.The expression of ORFV127 protein in Vero cells was observed with inverted fluorescence microscopy.The results showed that the full length of ORFV127 gene sequence was 558 bp.The expressed ORFV127 protein was sized about 49 000,and with the form of inclusion body in E.coli.Moreover,Western-blot results showed that ORFV127 protein reacted specifically with the prepared polyclonal antibody and had good reactionogenicity.The results of subcellular localization showed that the ORFV127 protein was mainly located in the cytoplasm.These results provide biomaterials for further study on the function of ORFV127 protein.
作者
钟灵毓
王元红
白彩霞
杨侃侃
俞赵荣
鲁智敏
张学琪
刘自敏
蒋书东
李永东
王勇
ZHONG Ling-yu;WANG Yuan-hong;BAI Cai-xia;YANG Kan-kan;YU Zhao-rong;LU Zhi-min;ZHANG Xue-qi;LIU Zi-min;JIANG Shu-dong;LI Yong-dong;WANG Yong(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China;Ningbo Municipal Center for Disease Control and Prevention,Ningbo 315010,China)
出处
《江苏农业学报》
CSCD
北大核心
2019年第3期646-652,共7页
Jiangsu Journal of Agricultural Sciences
基金
国家自然科学基金项目(31602063)
安徽省自然科学基金项目(1508085QC60)
