地方株马驽巴贝斯虫Bc48截短体单克隆抗体的制备及应用

doi:10.11843/j.issn.0366-6964.2019.09.016

摘要/Abstract

摘要： 本研究旨在建立马驽巴贝斯虫抗体的快速、准确检测方法。以纯化的马驽巴贝斯虫Bc48基因片段的原核表达产物免疫6周龄雌性BALB/c小鼠制备单克隆抗体，并利用重组抗原与单克隆抗体建立间接竞争ELISA（CI-ELISA）方法检测马驽巴贝斯虫抗体。结果显示：制备出3株能稳定分泌单克隆抗体的杂交瘤细胞株，命名为1H2、7F4、11F4，通过对CI-ELISA条件筛选得出，抗原最佳包被浓度为0.19 μg·mL^-1，单克隆抗体11F4的最佳工作浓度为1：3.2×10⁵，通过检测30份马驽巴贝斯虫阴性血清及20份阳性血清，确定该检测方法临界值为45%；特异性试验发现，该CI-ELISA方法不与感染马泰勒虫病的阳性血清发生反应，具有特异性；用所建立的CI-ELISA检测临床血清90份，与标准c-ELISA试剂盒总符合率为92.2%、阳性符合率92.1%、阴性符合率94.1%。试验结果表明，该CI-ELISA方法特异性强，敏感性高，稳定性和重复性好，操作简便。基于单克隆抗体（11F4）与重组蛋白（HIS-Bc48）所建立的CI-ELISA特异性、重复性好，可为新疆马驽巴贝斯虫的检测、监控提供有效手段。

Abstract: This study aimed to establish a rapid and accurate detection method for Babesia caballi. Six-week-old female BALB/c mice were immunized with purified Bc48 recombinant protein to prepare monoclonal antibodies, and a CI-ELISA method was established by Bc48 recombinant antigen and monoclonal antibody. The results showed that three hybridoma cell lines stably secreting monoclonal antibodies were prepared and named as 1H2, 7F4 and 11F4. By screening for CI-ELISA conditions, the optimal coating concentration of the antigen was 0.19 μg·mL^-1, and the optimal working concentration of monoclonal antibody 11F4 was 1:3.2×10⁵. The CI-ELISA was determined to have a critical value of 45% by detecting 30 Babesia caballi negative sera and 20 positive sera. Specificity test showed that the CI-ELISA does not reacted with positive sera from Theileria equi infected horses. Using the established CI-ELISA to detected 90 clinical sera, the total coincidence rate with the standard c-ELISA kit was 92.2%, the positive coincidence rate was 92.1%, and the negative coincidence rate was 94.1%. This results indicated that CI-ELISA method has characteristics of strong specificity, high sensitivity, good stability and repeatability, and simple operation. These results suggested that CI-ELISA established by monoclonal antibody (11F4) and recombinant protein (His-Bc48) was specific and reproducible, which could provide an effective means for the detection and monitoring of Babesia caballi infection in XinJiang, China.

中图分类号:

S852.723

王盼举, 樊新丽, 张梦圆, 宋晶晶, 李敏, 吾力江, 巴音查汗. 地方株马驽巴贝斯虫Bc48截短体单克隆抗体的制备及应用[J]. 畜牧兽医学报, 2019, 50(9): 1882-1887.

WANG Panju, FAN Xinli, ZHANG Mengyuan, SONG Jingjing, LI Min, WU Lijiang, Bayin Chahan. Preparation and Application of Monoclonal Antibody against Truncated Bc48 of Babesia caballi Local Strains[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(9): 1882-1887.

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参考文献

[1] RÜEGG S R, TORGERSON P, DEPLAZES P, et al. Age-dependent dynamics of Theileria equi and Babesia caballi infections in Southwest Mongolia based on IFAT and/or PCR prevalence data from domestic horses and ticks[J]. Parasitology, 2007, 134(7):939-947.
[2] MACHADO R Z, TOLEDO C Z P, TEIXEIRA M C A, et al. Molecular and serological detection of Theileria equi and Babesia caballi in donkeys (Equus asinus) in Brazil[J]. Vet Parasitol, 2012, 186(3-4):461-465.
[3] AVARZED A, DE WAAL D T, IGARASHI I, et al. Prevalence of equine piroplasmosis in central Mongolia[J]. Onderstepoort J Vet Res, 1997, 64(2):141-145.
[4] FRIEDHOFF K T, TENTER A M, MÜLLER I. Haemoparasites of equines:impact on international trade of horses[J]. Rev Sci Tech, 1990, 9(4):118-1194.
[5] PRESTON P M. Theileriosis[M]//SERVICE M W. Encyclopedia of Arthopod-Transmitted Infections of Man and Domestic Animals. Wallingford:CABI, 2001:487-502.
[6] UILENBERG G. Babesiosis[M]//SERVICE M W. Encyclopedia of Arthropod-Transmitted Infections of Man and Domestic Animals. Wallinford:CABI, 2001:53-60.
[7] 朱玉涛, 恰布旦·阿仔拜,宋瑞其,等. 阿勒泰富蕴县放牧马驽巴贝斯虫和马泰勒虫抗体的检测初报[J]. 畜牧与兽医, 2016, 48(1):111-113.
ZHU Y T, QIABUDAN·A Z B, SONG R Q, et al. Preliminary report on detection of Babesia caballi and Theileria equi antibodies in herding horses in Fuyun county of Altay[J]. Animal Husbandry & Veterinary Medicine, 2016, 48(1):111-113. (in Chinese)
[8] 李永畅, 恩克博力德,哈西巴特,等. 南北疆部分地区马驽巴贝斯虫病血清学调查[J]. 中国兽医杂志, 2016, 52(2):6-8.
LI Y C, ENKEBOLIDE, HAXIBATE, et al. The Serological survey of Babesia caballi in part Southern and Northern of Xinjiang[J]. Chinese Journal of Veterinary Medicine, 2016, 52(2):6-8. (in Chinese)
[9] 瓦热斯·吐尔松,李永畅,朱玉涛,等. 新疆昌吉部分地区马梨形虫病检测初报[J]. 中国动物传染病学报, 2016, 24(1):75-79.
WARIS T, LI Y C, ZHU Y T, et al. A preliminary survey of equine piroplasmosis in part area of Changji, Xinjiang[J]. Chinese Journal of Animal Infectious Diseases, 2016, 24(1):75-79. (in Chinese)
[10] FRERICHS W M, HOLBROOK A A, JOHNSON A J.Euqine piroplasmosis:Complment-fixation titers of horses infected with Babesia caballi[J].Am J Vet Res,1969,30(5):697-702.
[11] BASHIRUDDIN J B, CAMMÀ C, REBÊLO E. Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16S rRNA gene[J]. Vet Parasitol, 1999, 84(1-2):75-83.
[12] KNOWLES D P Jr, PERRYMAN L E, GOFF W L, et al. A monoclonal antibody defines a geographically conserved surface protein epitope of Babesia equi merozoites[J]. Infect Immun, 1991, 59(7):2412-2417.
[13] IKADAI H, TAMAKI Y, XUAN X N, et al. Inhibitory effect of monoclonal antibodies on the growth of Babesia caballi[J]. Int J Parasitol, 1999, 29(11):1785-1791.
[14] IKADAI H, XUAN X N, IGARASHI I, et al. Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay[J]. J Clin Microbiol, 1999, 37(11):3475-3480.
[15] BRÜNING A, PHIPPS P, POSNETT E, et al. Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis[J]. Vet Parasitol, 1997, 68(1-2):11-26.
[16] BÖSE R, PEYMANN B. Diagnosis of Babesia caballi infections in horses by enzyme-linked immunosorbent assay (ELISA) and western blot[J]. Int J Parasitol, 1994, 24(3):347-346.
[17] SCHWINT O N, UETI M W, PALMER G H, et al. Imidocarb dipropionate clears persistent Babesia caballi infection with elimination of transmission potential[J]. Antimicrob Agents Chemother, 2009, 53(10):4327-4332.
[18] 薛书江, 于龙政,曹世诺,等. 驽巴贝虫BC-48基因的克隆及其在大肠杆菌中的表达[J]. 中国兽医科学, 2007, 37(3):214-217.
XUE S J, YU L Z, CAO S N, et al. Cloning of BC-48 gene of Babesia caballi Jilin strain and its expression in Escherichia coli[J]. Veterinary Science in China, 2007, 37(3):214-217. (in Chinese)
[19] 王盼举, 艾日登才次克,许正茂,等. 马驽巴贝斯虫新疆伊犁株Bc48基因克隆、表达及其多克隆抗体的制备[J]. 中国畜牧兽医, 2018, 45(6):1677-1682.
WANG P J, AIRIDENG C, XU Z M, et al. Cloning and expression of Bc48 gene of Babesia caballi Xinjiang Yili strain and preparation of its polyclonal antibody[J]. China Animal Husbandry & Veterinary Medicine, 2018, 45(6):1677-1682. (in Chinese)
[20] WANG C H, GU C, GUO D H, et al. Preparation of monoclonal antibodies against bovine Haptoglobin[J]. Monoclon Antib Immunodiagn Immunother, 2014, 33(5):350-353.
[21] 冷小茜, 叶欢,杜浩,等. 中华鲟Vtg间接竞争ELISA检测方法的建立和应用[J]. 水生生物学报, 2018, 42(4):779-785.
LENG X Q, YE H, DU H, et al. Establishment and application of an ELISA technique for detecting Chinese sturgeon vitellogenin[J]. Acta Hydrobiologica Sinica, 2018, 42(4):779-785. (in Chinese)
[22] 胡庆云. 猪伪狂犬病毒gE蛋白单克隆抗体的制备及竞争ELISA方法的建立[D]. 扬州:扬州大学, 2018.
HU Q Y. Preparation of monoclonal antibody against gE protein of pseudorabies virus and establishment of a competitive ELISA method[D]. Yangzhou:Yangzhou University, 2018. (in Chinese)
[23] 宋瑞其, 王盼群,王振宝,等. 马泰勒虫新疆株EMA-1基因的原核表达及间接ELISA检测方法的建立[J]. 畜牧兽医学报, 2017, 48(9):1737-1743.
SONG R Q, WANG P Q, WANG Z B, et al. Prokaryotic expression of the EMA-1 Gene of Xinjiang strain of Theileria equi and establishment of indirect ELISA detection method[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(9):1737-1743. (in Chinese)
[24] KAPPMEYER L S, PERRYMAN L E, HINES S A, et al. Detection of equine antibodies to Babesia caballi by recombinant B. caballi rhoptry-associated protein 1 in a competitive-inhibition enzymelinked immunosorbent assay[J]. J Clin Microbiol, 1999, 37(7):2285-2290.
[25] KATZ J, DEWALD R, NICHOLSON J. Procedurally similar competitive immunoassay systems for the serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum, and Burkholderia mallei infection in horses[J]. J Vet Diagn Invest, 2000, 12(1):46-50.
[26] 朱建波, 杨振兴,肖雷,等. 流行性出血病多克隆抗体C-ELISA检测方法的建立[J]. 畜牧兽医学报, 2018, 49(7):1440-1450.
ZHU J B, YANG Z X, XIAO L, et al. Establishment of a rapid competitive ELISA for detecting antibodies against epizootic haemorrhagic disease virus[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(7):1440-1450. (in Chinese)