马驽巴贝斯虫Bc48蛋白的纯化及其应用
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摘要
为了解新疆南北疆部分疫区马匹携带驽巴贝斯虫(Babesia caballi)抗体情况,运用实验室已构建保存的地方虫株重组Bc48为靶抗原,将重组原核表达质粒pGEX4T-1-Bc48转化至BL21表达系统中,诱导表达出36 KDa的重组蛋白。经KCl法切胶纯化重组蛋白后作为rELISA包被抗原,对阿克苏、伊犁、和硕等7个区域的随机采集样品(n=256)进行马驽巴贝斯虫抗体检测。结果显示,Bc48重组抗原能够精确识别马驽巴贝斯虫标准阳性和阴性血清;所检测的临床样品中,驽巴贝斯虫抗体阳性率为19.5%,其中,南疆2个地区(和硕、阿克苏)马驽巴贝斯虫病阳性率分别为30%、9%;北疆5个地区(昭苏种马场、伊犁卡拉苏、洪纳海乡、阿尕什敖包乡和富蕴县区域)马驽巴贝斯虫抗体阳性率分别为17%、12.5%、17%、5%和35%。本次调查结果为新疆驽巴贝斯虫防控措施及疫苗和驱虫药的使用提供必要的理论依据。
In order to understand the prevalence of Babesia caballi infection in parts of southern and northern Xinjiang, an ELISA method was developed to detect serological response using recombinant protein as the coating antigen. The recombinant pGEX4 T-1-Bc48 plasmid was transformed into BL21 expression system and a protein at molecular weight of 36 kDa was expressed and purified through KCL method. Total 256 serum samples were collected from 7 areas such as Aksu, Yili and Heshuo and tested using ELISA method. As a result, overall positive rate of of B.caballi antibodies was 19.5%. The detailed positive rates in different areas were 30% in Heshuo and 9%in Aksu of southern Xinjiang, and 17% in Zhaoxu, 12.5% in Karasu, 17% in Hong Nahai Township, 5% in A Gaishao Baocao and 35% in Fuyun of northern Xinjiang.
In order to understand the prevalence of Babesia caballi infection in parts of southern and northern Xinjiang, an ELISA method was developed to detect serological response using recombinant protein as the coating antigen. The recombinant pGEX4 T-1-Bc48 plasmid was transformed into BL21 expression system and a protein at molecular weight of 36 kDa was expressed and purified through KCL method. Total 256 serum samples were collected from 7 areas such as Aksu, Yili and Heshuo and tested using ELISA method. As a result, overall positive rate of of B.caballi antibodies was 19.5%. The detailed positive rates in different areas were 30% in Heshuo and 9%in Aksu of southern Xinjiang, and 17% in Zhaoxu, 12.5% in Karasu, 17% in Hong Nahai Township, 5% in A Gaishao Baocao and 35% in Fuyun of northern Xinjiang.
引文
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[2]Uilenberg G.Babesia-a historical overview[J].Vet Parasitol,2006,138(1-2):3-10.
[3]de Waal D T.Equine piroplasmosis:a review[J].Br Vet J,1992,148(1):6-14.
[4]Ueti M W,Palmer G H,Scoles G A,et al.Persistently infected horses are reservoirs for intrastadial tickborne transmission of the apicomplexan parasite Babesia equi[J].Infect Immun,2008,76(8):3525-3529.
[5]Rüegg S R,Heinzmann D,Barbour A D,et al.Estimation of the transmission dynamics of Theileria equi and Babesia caballi in horses[J].Parasitology,2008,135(5):555-565.
[6]Health W.Manual of diagnostics tests and vaccines for terrestrial animals[J].Bulletin-Office international desépizooties,2008:1092-1106.
[7]Ikadai H,Osorio C R,Xuan X,et al.Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein[J].Int J Parasitol,2000,30(5):633-635.
[8]高慎阳,查恩辉,王珅,等.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26.
[9]李永畅.新疆马驽巴贝斯虫地方虫株靶基因Bc48克隆、表达和rELISA的建立及其应用研究[D].乌鲁木齐:新疆农业大学,2016.
[10]Rapoport A,Aharonson-Raz K,Berlin D,et al.Molecular characterization of the Babesia caballi rap-1 gene and epidemiological survey in horses in Israel[J].Infect Genet Evol,2014,23:115-120.
[11]张杨.马驽巴贝斯虫二温式、荧光PCR检测方法的建立及其初步应用[D].乌鲁木齐:新疆农业大学,2015.
[12]敖敦格日勒,格日勒图,朱玉涛,等.内蒙古和新疆两地草原革蜱源性马驽巴贝斯虫病病原DNA检测[J].中国兽医杂志,2015,51(5):21-23.
[13]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.
[14]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.
[15]BartoloméDel Pino L E,Nardini R,Veneziano V,et al.Babesia caballi and Theileria equi infections in horses in Central-southern Italy:Sero-molecular survey and associated risk factors[J].Ticks Tick-Borne Dis,2016,7(3):462-469.
[16]Sumbria D,Das Singla L,Sharma A.Theileria equi and Babesia caballi infection of equids in Punjab,India:a serological and molecular survey[J].Trop Anim Health Prod,2016,48(1):45-52.