[1]钟灵毓,王元红,白彩霞,等.羊口疮病毒127基因的克隆表达及亚细胞定位分析[J].江苏农业学报,2019,(03):646-652.[doi:doi:10.3969/j.issn.1000-4440.2019.03.020]
　ZHONG Ling-yu,WANG Yuan-hong,BAI Cai-xia,et al.Prokaryotic expression and subcellular localization of Orf virus 127 gene[J].,2019,(03):646-652.[doi:doi:10.3969/j.issn.1000-4440.2019.03.020]
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羊口疮病毒127基因的克隆表达及亚细胞定位分析()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2019年03期
页码:
646-652
栏目:
畜牧兽医·水产养殖
出版日期:
2019-06-30

文章信息/Info

Title:
Prokaryotic expression and subcellular localization of Orf virus 127 gene
作者:
钟灵毓1; 王元红1; 白彩霞1; 杨侃侃1; 俞赵荣1; 鲁智敏1; 张学琪1; 刘自敏1; 蒋书东1; 李永东2; 王勇1
(1.安徽农业大学动物科技学院，安徽合肥230036；2.宁波市疾病预防控制中心，浙江宁波315010)
Author(s):
ZHONG Ling-yu1; WANG Yuan-hong1; BAI Cai-xia1; YANG Kan-kan1; YU Zhao-rong1; LU Zhi-min1; ZHANG Xue-qi1; LIU Zi-min1; JIANG Shu-dong1; LI Yong-dong2; WANG Yong1
(1.College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China;2.Ningbo Municipal Center for Disease Control and Prevention, Ningbo 315010，China）
关键词:
：羊口疮病毒; ORFV127基因; 原核表达; 亚细胞定位
Keywords:
orf virus; ORFV127 gene; prokaryotic expression; subcellular localization
分类号:
S855.3
DOI:
doi:10.3969/j.issn.1000-4440.2019.03.020
文献标志码:
A
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558 bp，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49 000。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。
Abstract:
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.

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备注/Memo

备注/Memo:
收稿日期：2018-07-26 基金项目：国家自然科学基金项目（31602063）；安徽省自然科学基金项目（1508085QC60） 作者简介：钟灵毓（1996-），女，江西新余人，本科，主要从事动物传染病研究。（E-mail）1071338281@qq.com。王元红为共同第一作者。 通讯作者：王勇，（E-mail）wangyong119@ahau.edu.cn。李永东，（E-mail）liyd@nbcdc.org.cn
更新日期/Last Update: 2019-06-30