牛布鲁氏菌毒力基因Virb8间接ELISA检测方法的建立与应用

doi:10.3864/j.issn.0578-1752.2013.16.017

中国农业科学 ›› 2013, Vol. 46 ›› Issue (16): 3460-3469.doi: 10.3864/j.issn.0578-1752.2013.16.017

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

牛布鲁氏菌毒力基因Virb8间接ELISA检测方法的建立与应用

张剑^1,2, 马卫明², 张亮¹, 何洪彬¹, 丁家波³, 杨宏军¹

1.山东省农业科学院奶牛研究中心，济南 250100
2.山东农业大学动物科技学院，山东泰安 271018
3.中国兽医药品监察所，北京 100081

收稿日期:2013-01-04 出版日期:2013-08-15 发布日期:2013-05-31
通讯作者: 通信作者杨宏军，E-mail：longfei1997@sina.com。丁家波，E-mail：dingjiabo@ivdc.gov.cn
作者简介:张剑，Tel：15339958085；E-mail：zhangjian.1886@163.com
基金资助:
公益性行业（农业）科研专项（200903027）；国家“863”计划项目（2012AA101300）

Development of iELISA for Detection and Application of Brucella abortus Virb8

ZHANG Jian^1,2, MA Wei-ming², ZHANG Liang¹, HE Hong-bin¹, DING Jia-bo³, YANG Hong-jun¹

1.Cow Research Center, Shandong Academy of Agricultural Science, Ji,nan 250100
2.College of Veterinary Medicine, Shangdong Agricultural University, Tai’an 271018, Shandong
3.China Institute of Veterinary Drug Control, Beijing 100081

Received:2013-01-04 Online:2013-08-15 Published:2013-05-31

摘要/Abstract

摘要： 【目的】布鲁氏菌virb8蛋白是布鲁氏菌的重要毒力因子，本研究拟构建含布鲁氏菌Virb8基因的原核表达载体，利用纯化蛋白作为包被抗原，建立检测牛布鲁氏菌血清抗体的间接ELISA方法（iELISA）。【方法】根据GenBank中的牛型布鲁氏杆菌Virb8序列，设计一对引物，以布鲁氏菌疫苗株S2全基因组DNA为模板扩增Virb8的基因，将目的基因克隆入PEASY-T3，经测序正确后，与载体pET-32a(+)连接，构建重组表达质粒pET-32a-Virb8，转化E.coli BL21(DE3)，诱导表达融合蛋白pET-32a-Virb8，经SDS-PAGE及Western-blot分析鉴定后，采用Ni-NTA Spin Kit纯化目的蛋白，利用所建立的iELISA方法对235个临床奶牛血清样本进行检测，并与虎红平板凝集实验进行比较。【结果】成功构建了含Virb8的表达载体，经多次对表达条件的优化，大量表达了目的蛋白，建立的iELISA方法具有稳定、灵敏的特点，与虎红平板凝集试验的符合率可达97.02%。【结论】所建立的iELISA方法是一种有效的牛布鲁氏菌病血清学诊断的方法，为进一步研究其免疫原性和抗感染保护作用奠定了基础。

关键词: 布鲁氏菌 , Virb8 , 间接ELISA

Abstract: 【Objective】 Brucella abortus Virb8 is an important Brucella virulence factor. The aim of this study was to construct a prokaryotic expression vector for Brucella Virb8. Then, an indirect enzyme-linked immunosorbent assay (iELISA) for detecting antibodies to Brucella abortus was established using the purified recombinant protein. 【Method】 Brucella S2 genome sequence was extracted and used as a templet in polymerase chain reaction to amplify Virb8 gene. The PCR product was cloned into the pEASY-T3 vector and subsequently sequenced. Then, the gene was cloned into the expression vector pET-32a to construct the recombinant plasmid pET-32a-Virb8. The fused protein pET-32a-Virb8 was expressed and purified by Ni-NTA Spin Kit. Serum samples (n=235) were simultaneously tested by the iELISA and the rose bengal plate test (RBPT). 【Result】 In this study, Brucella Virb8 was successfully cloned and highly purified protein was obtained. The established iELSIA was stable and sensitive. The coincidence of iELISA with RBPT was 97.02%. 【Conclusion】 The iELISA could be used as a method for serological diagnosis of brucellosis and a foundation for advance study on immunogenicity and protection of brucellosis.

Key words: Brucella abortus , Virb8 , iELISA

张剑, 马卫明, 张亮, 何洪彬, 丁家波, 杨宏军. 牛布鲁氏菌毒力基因Virb8间接ELISA检测方法的建立与应用[J]. 中国农业科学, 2013, 46(16): 3460-3469.

ZHANG Jian, MA Wei-ming, ZHANG Liang, HE Hong-bin, DING Jia-bo, YANG Hong-jun. Development of iELISA for Detection and Application of Brucella abortus Virb8[J]. Scientia Agricultura Sinica, 2013, 46(16): 3460-3469.

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牛布鲁氏菌毒力基因Virb8间接ELISA检测方法的建立与应用

Development of iELISA for Detection and Application of Brucella abortus Virb8

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