绵羊梅迪-维斯纳病毒CA蛋白可溶性表达、多克隆抗体制备及鉴定

doi:10.12160/j.issn.1672-5190.2022.05.001

畜牧与饲料科学 ›› 2022, Vol. 43 ›› Issue (5): 1-6.doi: 10.12160/j.issn.1672-5190.2022.05.001

• 基础研究 • 下一篇

绵羊梅迪-维斯纳病毒CA蛋白可溶性表达、多克隆抗体制备及鉴定

李慧萍^1,^2,³,陈思旭^1,^2,³,张良^1,^2,³,史晓娜^1,^2,³,刘淑英^1,^2,³

1.内蒙古农业大学兽医学院，内蒙古呼和浩特 010018
2.农业农村部动物疾病临床诊疗技术重点实验室，内蒙古呼和浩特 010018
3.内蒙古自治区基础兽医学重点实验室，内蒙古呼和浩特 010018

收稿日期:2022-05-06 出版日期:2022-09-30 发布日期:2022-09-21
通讯作者: 刘淑英（1968—），女，教授，博士，博士生导师，主要研究方向为动物病毒病理学。
作者简介:李慧萍（1985—），女，实验师，硕士，主要研究方向为羊病毒性传染病病原检测。
基金资助:
国家自然科学基金面上项目(32072819);内蒙古科技重大专项计划项目(2021ZD0010);内蒙古应用研究项目(2019GG240);内蒙古草原英才创新团队项目(20151031)

Soluble Expression of CA Protein of Maedi-visna Virus of Sheep Origin and Preparation and Identification of Its Polyclonal Antibody

LI Hui-ping^1,^2,³,CHEN Si-xu^1,^2,³,ZHANG Liang^1,^2,³,SHI Xiao-na^1,^2,³,LIU Shu-ying^1,^2,³

1. College of Veterinary Medicine，Inner Mongolia Agricultural University，Hohhot 010018，China
2. Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease，Ministry of Agriculture and Rural Affairs，Hohhot 010018，China
3. Inner Mongolia Key Laboratory of Basic Veterinary Science，Hohhot 010018，China

Received:2022-05-06 Online:2022-09-30 Published:2022-09-21

摘要/Abstract

摘要：

[目的]制备绵羊梅迪-维斯纳病毒（maedi-visna virus，MVV）衣壳蛋白（capsid protein，CA）多克隆抗体，并鉴定其特异性。[方法]根据MVV内蒙古分离株CA基因序列设计特异性引物，扩增CA基因，构建重组质粒；对 CA重组蛋白进行原核表达及纯化，制备兔源MVV CA重组蛋白多克隆抗体,采用间接ELISA方法测定其抗体效价，利用Western blot和免疫组化方法对其进行特异性鉴定。[结果]成功构建MVV CA重组蛋白原核表达系统，纯化后的目的蛋白大小约27 kDa；间接ELISA方法测定制备的多克隆抗体效价为1∶8 192；Western blot检测感染MVV绵羊的病肺组织，在25 kDa处出现特异性条带；免疫组化结果显示感染MVV绵羊的病肺中巨噬细胞的胞浆内有明显棕黄色阳性信号。[结论]利用获得的可溶性重组MVV CA蛋白制备的多克隆抗体具有较好特异性，可为MVV血清学诊断技术提供检测抗体。

关键词: 绵羊, 梅迪-维斯纳病毒, 衣壳蛋白, 原核表达, 多克隆抗体

Abstract:

[Objective] The aims of this study were to prepare the polyclonal antibody against capsid （CA） protein of a maedi-visna virus （MVV） strain isolated from naturally infected sheep and to assess its specificity. [Method] A set of specific primers was designed according to the CA gene sequence of MVV Inner Mongolia strain, and the CA gene was subsequently amplified by PCR assay for constructing a recombinant plasmid. The MVV CA recombinant protein was prokaryotically expressed and purified. The rabbit-derived polyclonal antibody against MVV CA recombinant protein was prepared, and its titer and specificity were determined and identified by using indirect ELISA assay as well as Western blot and immunohistochemical analyses. [Result] The prokaryotic expression system of MVV CA recombinant protein was successfully constructed, and the target protein was about 27 kDa after purification. The titer of the prepared polyclonal antibody was determined as 1∶8 192 by indirect ELISA assay. Western blot analysis showed that there was a specific band with a size of 25 kDa in lung tissues of sheep infected with MVV. Immunohistochemical analysis demonstrated that there were obvious brownish yellow positive signals in cytoplasm of macrophages in lung tissues of sheep infected with MVV. [Conclusion] Polyclonal antibody prepared with soluble recombinant MVV CA protein had good specificity, which might serve as a candidate antibody in MVV serological diagnosis technology.

Key words: sheep, maedi-visna virus, capsid protein, prokaryotic expression, polyclonal antibody

中图分类号:

李慧萍, 陈思旭, 张良, 史晓娜, 刘淑英. 绵羊梅迪-维斯纳病毒CA蛋白可溶性表达、多克隆抗体制备及鉴定[J]. 畜牧与饲料科学, 2022, 43(5): 1-6.

LI Hui-ping, CHEN Si-xu, ZHANG Liang, SHI Xiao-na, LIU Shu-ying. Soluble Expression of CA Protein of Maedi-visna Virus of Sheep Origin and Preparation and Identification of Its Polyclonal Antibody[J]. Animal Husbandry and Feed Science, 2022, 43(5): 1-6.

图/表 7

图1

图2

图3

图4

图5

图6

图7

参考文献 18

[1]	MUZ D, OGUZOGLU T Ç, ROSATI S, et al. First molecular characterization of visna/maedi viruses from naturally infected sheep in Turkey[J]. Archives of Virology, 2013, 158(3):559-570. doi: 10.1007/s00705-012-1518-1 pmid: 23124887
[2]	KALOGIANNI A I, BOSSIS I, EKATERINIADOU L V, et al. Etiology, epizootiology and control of maedi-visna in dairy sheep:A review[J]. Animals, 2020, 10(4):616. doi: 10.3390/ani10040616
[3]	SINGH R, KUMAR P, SINGH R, et al. Pathology and polymerase chain reaction detection of ovine progressive pneumonia (maedi) cases in slaughtered sheep in India[J]. Veterinary World, 2017, 10(11):1401-1406. doi: 10.14202/vetworld.2017.1401-1406 pmid: 29263606
[4]	BORQUEZ C M Y, HERNANDEZ C J F, ARMENTA L B, et al. Ovine progressive pneumonia:Diagnosis and seroprevalence in the south of Sonora, Mexico[J]. Case Reports in Veterinary Medicine, 2021, 2021:6623888.
[5]	邓普辉, 简子健, 孟庆文, 等. 北疆绵羊慢病毒感染的血清学调查[J]. 新疆农业科技, 1996, 19(2):6-9.
[6]	王多智, 史晓娜, 张洁, 等. 内蒙古绵羊梅迪-维斯纳病的病理组织学和巢式PCR诊断[J]. 中国兽医科学, 2019, 49(5):651-658.
[7]	PÉTURSSON G, MATTHÍASDÓTTIR S, SVANSSON V, et al. Mucosal vaccination with an attenuated maedi-visna virus clone[J]. Vaccine, 2005, 23(24):3223-3228. pmid: 15837223
[8]	丁忠庆. 绵羊梅迪-维斯纳病毒核酸疫苗的制备及免疫学研究[D]. 哈尔滨: 东北农业大学, 2006.
[9]	GOMEZ-LUCIA E, BARQUERO N, DOMENECH A. Maedi-visna virus:Current perspectives[J]. Veterinary Medicine, 2018, 9:11-21.
[10]	赵玲. 内蒙古绵羊梅迪-维斯纳病毒全基因组序列的测定与分析[D]. 呼和浩特: 内蒙古农业大学, 2021.
[11]	PREZIUSO S, TACCINI E, ROSSI G, et al. Experimental maedi-visna virus infection in sheep:A morphological,immunohistochemical and PCR study after three years of infection[J]. European Journal of Histochemistry, 2003, 47(4):373-378. doi: 10.4081/849
[12]	李艳华, 李爱华. 肿瘤组织中冷诱导RNA结合蛋白的提取及定量检测[J]. 生物技术世界, 2016, 13(5): 125.
[13]	赵玲, 王振玲, 史晓娜, 等. 内蒙古地区绵羊梅迪-维斯纳病毒全基因组序列的测定与分析[J]. 中国预防兽医学报, 2021, 43(11):1140-1145.
[14]	DE ANDRÉS D, KLEIN D, WATT N J, et al. Diagnostic tests for small ruminant lentiviruses[J]. Veterinary Microbiology, 2005, 107(1/2):49-62. doi: 10.1016/j.vetmic.2005.01.012
[15]	古努尔·吐尔逊, 王登峰, 杨学云, 等. 梅迪-维斯纳病毒gag蛋白的原核表达及其交叉反应原性[J]. 江苏农业学报, 2020, 36(4):978-983.
[16]	张彦红, 肖盛中, 李岩, 等. 梅迪-维斯纳病毒Gag蛋白原核表达与间接ELISA检测方法的建立[J]. 动物医学进展, 2020, 41(5):25-28.
[17]	LUJÁN L, BEGARA I, COLLIE D, et al. Ovine lentivirus (maedi-visna virus) protein expression in sheep alveolar macrophages[J]. Veterinary Pathology, 1994, 31(6):695-703. pmid: 7863586
[18]	GAYO E, POLLEDO L, BALSEIRO A, et al. Inflammatory lesion patterns in target organs of visna/maedi in sheep and their significance in the pathogenesis and diagnosis of the infection[J]. Journal of Comparative Pathology, 2018, 159:49-56. doi: S0021-9975(17)30575-3 pmid: 29599005

绵羊梅迪-维斯纳病毒CA蛋白可溶性表达、多克隆抗体制备及鉴定

Soluble Expression of CA Protein of Maedi-visna Virus of Sheep Origin and Preparation and Identification of Its Polyclonal Antibody

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 18

相关文章 15

编辑推荐

Metrics

本文评价

[1]	岱红艳, 白雪, 张宁, 张家新. 绵羊卵泡中抗氧化酶表达的研究[J]. 畜牧与饲料科学, 2023, 44(6): 66-75.
[2]	白绥明, 辛雅明, 齐盟, 胡亮, 杨银凤. 枯草芽孢杆菌培养上清液对绵羊瘤胃上皮细胞β-防御素-1表达的影响[J]. 畜牧与饲料科学, 2023, 44(5): 8-14.
[3]	刘凯强, 俞进, 段真真, 金敏, 李佳, 巴音查汗·盖力克. 新疆阿克苏地区蜱和羊感染无浆体的分子流行病学调查[J]. 畜牧与饲料科学, 2023, 44(3): 122-128.
[4]	戴伶俐, 王娜, 白帆, 张帆, 宋越, 张月梅, 达来宝力格, 李晓艳, 王根云, 赵世华. 9株绵羊肺炎支原体内蒙古分离株P113基因克隆及蛋白序列分析[J]. 畜牧与饲料科学, 2022, 43(6): 1-5.
[5]	张加宁, 陈名利, 邓金华, 李凯, 彭霞, 信璐瑶, 齐萌. 内蒙古某牲畜交易市场绵羊肠道蠕虫感染情况调查[J]. 畜牧与饲料科学, 2022, 43(5): 112-116.
[6]	宝·阿茹娜, 巴图赛罕, 木其儿, 杨苏日娜, 根锁. 内蒙古自治区绵羊生产成本影响因素的计量分析[J]. 畜牧与饲料科学, 2022, 43(3): 83-87.
[7]	戴伶俐, 王娜, 张帆, 宋越, 白帆, 刘威, 杨斌, 赵世华, 张月梅. 绵羊肺炎支原体分子致病机理研究进展[J]. 畜牧与饲料科学, 2022, 43(2): 124-128.
[8]	戴伶俐, 王娜, 刘威, 白帆, 张帆, 张月梅, 宋越, 杨斌, 陈伟, 赵世华, 达来宝力格. 绵羊肺炎支原体内蒙古分离株DnaJ基因克隆表达及其免疫原性研究[J]. 畜牧与饲料科学, 2022, 43(1): 1-7.
[9]	史静茹, 郭丽丽, 刘在霞, 刘永斌, 张家新, 张文广. 绵羊不同妊娠时期卵巢转录组差异表达比较分析[J]. 畜牧与饲料科学, 2021, 42(4): 37-41.
[10]	玉梅, 李长青, 王利, 郭天龙, 王超, 金海, 张海鹰, 田丰. 内蒙古四子王旗草原土壤—牧草—放牧绵羊生态系统微量元素的季节变化研究与盈亏分析[J]. 畜牧与饲料科学, 2021, 42(4): 74-82.
[11]	赵霏霏, 杨丹, 辛雅明, 杨银凤. 永生化绵羊瘤胃成纤维细胞系的建立[J]. 畜牧与饲料科学, 2021, 42(3): 1-6.
[12]	程家海, 宋前进, 郭昊, 侯丽娜, 杨超, 王凤雪, 关平原, 温永俊. 布鲁菌Rev.1株eryA基因的原核表达及间接ELISA方法的建立[J]. 畜牧与饲料科学, 2020, 41(6): 7-12.
[13]	王力伟, 赵沛义, 王朝, 房永雨, 刘红葵, 郭慧琴, 王蕴华, 何江峰. 梭梭HabHLH74转录因子基因的克隆及在大肠杆菌中的表达[J]. 畜牧与饲料科学, 2020, 41(5): 13-18.
[14]	张帆, 杨斌, 钱琳娜, 陈伟, 宋越, 王娜, 戴伶俐, 罗晓平, 刘威, 高娃, 达来宝力格, 柴春霞, 周蕾, 宋爱军, 卢岩, 赵世华, 张月梅. 微量元素缺乏导致绵羊脱毛症的诊断和治疗[J]. 畜牧与饲料科学, 2020, 41(3): 102-105.
[15]	范文斌, 祁云霞, 刘永斌. 绵羊miRNA-1185-5p的基因组定位、靶基因预测和功能分析[J]. 畜牧与饲料科学, 2020, 41(2): 20-23.