抗羊口疮病毒蛋白ORFV086多克隆抗体的制备及其应用

摘要/Abstract

摘要： 本试验旨在克隆表达羊口疮病毒(ORFV)蛋白ORFV086,制备兔抗ORFV086蛋白多克隆抗体并检测ORFV086蛋白的表达。以ORFV基因组DNA为模板,利用PCR方法扩增ORFV086的基因片段,将其克隆入原核表达载体pET-33b(+)。将构建的pET33b-086重组表达质粒转化大肠杆菌BL21(DE3) pLys 感受态细胞,经IPTG诱导表达,SDS-PAGE鉴定融合蛋白的表达,以纯化蛋白作为免疫原,免疫新西兰大耳白兔制备多克隆抗体。应用ELISA和Western blotting方法对所得抗体进行检测。结果显示,重组蛋白pET33b-086主要以包涵体形式存在,分子质量约为100 ku;目的蛋白经切胶纯化回收后作为免疫原制备的多克隆抗体效价达1:128000;纯化的多克隆抗体可用于检测重组及天然ORFV086蛋白,特异性好。本试验制备了ORFV086多克隆抗体,为深入研究ORFV086蛋白在羊口疮病毒感染过程中的作用奠定了基础。

关键词: 羊口疮病毒; ORFV086蛋白; 原核表达; 包涵体; 多克隆抗体

Abstract: This study was aimed to clone and express ORFV086 protein of Orf virus (ORFV) in prokaryocytes and prepare polyclonal antibody anti-ORFV086 which was used to detect the expression of ORFV086 protein. The ORFV086 gene was amplified from genome DNA isolated from ORFV by PCR, then cloned into prokaryotic expression vector pET-33b (+) to construct a recombinant expression vector pET33b-086. The pET33b-086 was transformed into E.coli BL21 (DE3) pLys and induced by IPTG from which the fusion protein was identified by SDS-PAGE. The purified protein was injected into New Zealand rabbits and the polyclonal antibody was prepared, which was identified by the indirect ELISA and Western blotting methods. The results showed that the recombinant protein mainly existed in the inclusion body with the expected molecular weight of about 100 ku. After purified by cutting the gel slices, target protein was used as immunogen to prepare polyclonal antibody. The titer of the polyclonal antibody was 1:128000. Western blotting method revealed that the purified polyclonal antibody had a specific affinity for the reorganizational and natural ORFV086 protein. Thus, the rabbit anti-ORFV086 polyclonal antibody was successfully prepared, providing a tool for further investigation on the role of ORFV086 protein infection.

Key words: Orf virus; ORFV086 protein; prokaryotic expression; inclusion body; polyclonal antibody

中图分类号:

S852.65

王小平, 郝文波, 罗树红, 宁章勇. 抗羊口疮病毒蛋白ORFV086多克隆抗体的制备及其应用[J]. , 2014, 41(11): 7-13.

WANG Xiao-ping, HAO Wen-bo, LUO Shu-hong, NING Zhang-yong. Preparation and Application of the Polyclonal Antibody against Orf Virus ORFV086 Protein[J]. , 2014, 41(11): 7-13.

参考文献

1 于在江,马学恩,周建华. 切胶纯化表达蛋白包涵体的可行性分析[J]. 生物技术,2007,17(3):46～48.
2 白慧玲,裴景堂,杜耀武,等. 兔抗人DR5分子多克隆抗体的制备与初步应用[J]. 免疫学杂志,2008,24(6):634～637.
3 向智龙,卓建华,程振涛,等. 贵州地区羊口疮病毒B2L基因克隆及原核表达载体构建[J]. 中国畜牧兽医,2011,38(7):76～79.
4 何萍,张斯,尹元琴,等. 兔抗Cap43多克隆抗体制备和初步纯化[J]. 中国公共卫生,2007,23(3):289～291.
5 张少华,贾万忠,骆学农,等. 猪囊尾蚴CE18重组蛋白的复性纯化及抗原性鉴定[J]. 生物工程学报,2008,24(8):1490～1495.
6 李向茸,冯若飞,谢晶莹,等. 人血白蛋白的原核表达及应用[J]. 生物技术通报,2013(11):130～135.
7 邹雨妃,王衡,远立国,等. 猪IL-15多克隆抗体的制备及其生物活性的检测[J]. 中国畜牧兽医,2012,39(9):26～29.
8 赵雪超,张学炜,张守峰,等. 狂犬病病毒BD-06株基质蛋白的原核表达、纯化及其多克隆抗体的制备[J]. 中国畜牧兽医,2013,40(4):36～39.
9 徐锋,彭珍,万翠香,等. 高特异性长双歧杆菌多克隆抗体的制备[J]. 中国生物制品学杂志,2010,23(2):182～184.
10 徐静,李树香,刘立成,等. 血管内皮细胞生长因子的表达及其单克隆抗体的制备[J]. 中国生物制品学杂志,2008,21(11):1002～1005.
11 高闪电,常惠芸,独军政,等. 猪FMDV受体整联蛋白β1亚基LBD多克隆抗体的制备和初步应用[J]. 中国人兽共患病学报,2008,24(11):999～1001.
12 康彬,童哲. 一种利于蛋白质回收的快速SDS-聚丙烯酰胺凝胶电泳染色—脱色方法[J]. 生物化学与生物物理进展,2000,27(2):210～211.
13 Billinis C, Mavrogianni V S, Spyrou V, et al. Phylogenetic analysis of strains of Orf virus isolated from two outbreaks of the disease in sheep in Greece[J]. Virol J, 2012, 9: 24.
14 Chi X, Zeng X, Hao W, et al. Heterogeneity among orf virus isolates from goats in Fujian province, southern China[J]. PLoS One, 2013, 8(10): e66958.
15 Delhon G, Tulman E R, Afonso C L, et al. Genomes of the parapoxviruses Orf virus and bovine papular stomatitis virus[J]. J Virol, 2004, 78(1): 168～177.
16 Diel D G, Luo S, Delhon G, et al. A nuclear inhibitor of NF-kappaB encoded by a poxvirus[J]. J Virol, 2011, 85(1): 264～275.
17 Du H, Li W, Hao W, et al. Taqman Real-time PCR assay based on ORFV024 gene for rapid detection of Orf infection[J]. Toxicol Mech Methods, 2013, 23(5): 308～314.
18 Gao F, Yuan H, Ling H, et al. An outbreak of human Orf disease caused by introduced black goats[J]. Zhonghua Liu Xing Bing Xue Za Zhi, 2011, 32(9): 905～907.
19 Heljasvaara R, Rodriguez D, Risco C, et al. The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles[J]. J Virol, 2001, 75(13): 5778～5795.
20 Hosamani M, Scagliarini A, Bhanuprakash V, et al. Orf: An update on current research and future perspectives[J]. Expert Rev Anti Infect Ther, 2009, 7(7): 879～893.
21 Jesus D M, Costa L T, Goncalves D L, et al. Cidofovir inhibits genome encapsidation and affects morphogenesis during the replication of vaccinia virus[J]. J Virol, 2009, 83(22): 11477～11490.
22 Kane E M, Shuman S. Vaccinia virus morphogenesis is blocked by a temperature-sensitive mutation in the I7 gene that encodes a virion component[J]. J Virol, 1993, 67(5): 2689～2698.
23 Li H, Ning Z, Hao W, et al. Identification and characterization of monoclonal antibodies against the ORFV059 protein encoded by Orf virus[J]. Virus Genes, 2012a, 44(3): 429～440.
24 Li W, Ning Z, Hao W, et al. Isolation and phylogenetic analysis of orf virus from the sheep herd outbreak in northeast China[J]. BMC Vet Res, 2012b, 8: 229.
25 Vanslyke J K, Franke C A, Hruby D E. Proteolytic maturation of vaccinia virus core proteins: Identification of a conserved motif at the N termini of the 4b and 25K virion proteins[J]. J Gen Virol, 1991a, 72 (2): 411～416.
26 Vanslyke J K, Whitehead S S, Wilson E M, et al. The multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein: A degenerate conserved cleavage motif within core proteins[J]. Virology, 1991b, 183(2): 467～478.
27 Vikoren T, Lillehaug A, Akerstedt J, et al. A severe outbreak of contagious ecthyma (Orf) in a free-ranging musk ox (Ovibos moschatus) population in Norway[J]. Vet Microbiol, 2008, 127(1～2): 10～20.
28 Zhao K, Song D, He W, et al. Identification and phylogenetic analysis of an Orf virus isolated from an outbreak in sheep in the Jilin province of China[J]. Vet Microbiol, 2010, 142(3～4): 408～415.
29 Zheng M, Liu Q, Jin N, et al. A duplex PCR assay for simultaneous detection and differentiation of Capripoxvirus and Orf virus[J]. Mol Cell Probes, 2007, 21(4): 276～281.

[1]	焦琳琳, 成玉婷, 吴庆国, 吴双, 吴植, 朱善元, 钱莺娟. 鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2395-2402.
[2]	李灿, 李楚楚, 曾维, 张宁, 纪春晓, 陈韬. 猪RegⅢγ蛋白的重组表达及功能研究[J]. 中国畜牧兽医, 2023, 50(6): 2414-2426.
[3]	宋越, 苏胜杰, 张帆, 白帆, 戴伶俐, 王娜, 王大伟, 杨茜雯, 赵世华, 张月梅. 溶血性曼氏杆菌截短型白细胞毒素的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2479-2486.
[4]	欧云文, 潘琴, 汪洋, 代军飞, 任绍科, 张洋, 张杰. 猪细小病毒6型ORF2基因的截短表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2487-2495.
[5]	张芳源, 蔺雅婷, 杨大为, 陈虎, 李桂梅, 单虎. 非洲猪瘟病毒P30蛋白原核表达及间接ELISA检测方法建立[J]. 中国畜牧兽医, 2023, 50(5): 2012-2022.
[6]	王雪平, 张孝俊, 李晓月, 王国栋, 张福良, 宋玉伟, 张明亮, 马磊. 高致病性禽腺病毒血清4型Hexon蛋白的原核表达及多克隆抗体的制备[J]. 中国畜牧兽医, 2023, 50(5): 2053-2060.
[7]	胡乐玉, 胡欣瑶, 李颖, 赵盛淼, 沈凤臻, 王长法, 李亮亮, 王彤彤, 任慧英. 德州驴HO-1基因生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1499-1510.
[8]	王文婕, 茹鹏辉, 郁川, 杜付熙, 陈松彪, 尚珂, 张春杰, 程相朝. 猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1511-1521.
[9]	刘家兴, 韩雪英, 张鑫茹, 邓嘉昕, 姚伦广, 刘阳坤. 携带猪流行性腹泻病毒抗原表位的铁蛋白纳米颗粒制备与免疫原性分析[J]. 中国畜牧兽医, 2023, 50(4): 1567-1574.
[10]	贾新月, 马静, 张艳艳, 马勋, 王正荣, 薄新文. 细粒棘球绦虫EGR-07243和EGR-07244基因生物信息学分析及原核表达[J]. 中国畜牧兽医, 2023, 50(3): 847-858.
[11]	章志涛, 李祥龙, 孔令丽, 罗雨昕, 王冬英. 大片形吸虫丝氨酸/苏氨酸蛋白磷酸酶2A的原核表达及不同时期表达水平分析[J]. 中国畜牧兽医, 2023, 50(3): 1107-1117.
[12]	牛小杰, 徐明国, 肖莉, 刘文豪, 陈创夫, 王勇. 鸡白痢沙门氏菌IPAJ蛋白的表达纯化、多克隆抗体制备及ELISA方法的建立[J]. 中国畜牧兽医, 2023, 50(3): 1218-1228.
[13]	甘露, 郑会珍, 诺明达来, 刘燕, 金敏, 何文文, 温丽翠, 李永畅, 巴音查汗·盖力克. 伊氏锥虫伊犁株扩繁及其PFR基因克隆表达和生物信息学分析[J]. 中国畜牧兽医, 2023, 50(2): 469-478.
[14]	吕双飞, 马勋, 王静, 孔翠莲, 寇丽君, 刘彩霞, 史唯地, 康立超, 任慧杰. 炭疽芽孢杆菌PA-LF1融合蛋白的原核表达及其反应原性研究[J]. 中国畜牧兽医, 2023, 50(2): 674-683.
[15]	但一昕, 向华, 张焕容, 杨璐, 任玉鹏, 徐颂为, 何翃闳, 朱江江. 羊口疮病毒129蛋白互作宿主蛋白的筛选与C1QBP基因的克隆分析[J]. 中国畜牧兽医, 2023, 50(1): 1-14.