[1]王熙凤,孟庆玲*,乔 军,等.肝片吸虫MeCatL-B融合基因的构建、表达及其间接ELISA检测方法的建立[J].中国预防兽医学报,2019,(03):273-278.[doi:0.3969/j.issn.1008-0589.201807032]
 WANG Xi-feng,MENG Qing-ling*,QIAO Jun,et al.Construction and expression of MeCatL-B fusion gene of Fasciola hepatica and development of indirect ELISA assay for F.hepatica detection[J].Chinese journal of preventive veterinary medicine,2019,(03):273-278.[doi:0.3969/j.issn.1008-0589.201807032]
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肝片吸虫MeCatL-B融合基因的构建、表达及其间接ELISA检测方法的建立()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年03
页码:
273-278
栏目:
诊断和检测技术
出版日期:
2019-04-25

文章信息/Info

Title:
Construction and expression of MeCatL-B fusion gene of Fasciola hepatica and development of indirect ELISA assay for F.hepatica detection
文章编号:
1008-0589(2019)03-0273-06
作者:
 

王熙凤1孟庆玲1*乔 军1张 凯1张国武1贡莎莎1黄运福1才学鹏2

 (1. 石河子大学 动物科技学院,新疆 石河子 832003;2. 中国兽医药品监察所,北京 100081)
Author(s):
 

WANG Xi-feng1 MENG Qing-ling1* QIAO Jun1 ZHANG Kai1 ZHANG Guo-wu1 GONG Sha-sha1 HUANG Yun-fu1 CAI Xue-peng2

 (1. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China;
2. China Institute of Veterinary Drug Control, Beijing 100081, China)
关键词:
 肝片吸虫MeCatL-B融合基因原核表达间接ELISA
Keywords:
Fasciola hepatica  MeCatL-B fusion gene  prokaryotic expression  indirect ELISA
分类号:
S852.7
DOI:
0.3969/j.issn.1008-0589.201807032
文献标志码:
A
摘要:
为分析组织蛋白酶的抗原性及评价其作为肝片吸虫(Fh) ELISA诊断抗原的潜力,本研究采用RT-PCR技术扩增Fh CatL1D和CatB4基因的优势抗原表位集中区段,利用重叠延伸PCR技术融合CatL1D和CatB4基因片段获得约810 bp的 Fh CatL-B融合基因(MeCatL-B),构建原核重组表达载体pET-MeCatL-B,并将其转化入BL21 (DE3)感受态细胞中诱导表达,经SDS-PAGE和western blot检测,结果显示表达的重组蛋白MeCatL-B (rMeCatL-B)约46 ku,可以被Fh阳性血清特异性识别。以纯化的rMeCatL-B为包被抗原,经优化各反应条件后建立检测Fh抗体的间接ELISA方法,该检测方法的特异性强、敏感性高、重复性好。利用该方法对97份绵羊临床血清样品进行检测,并与商品化Fh ELISA抗体试剂盒检测结果对比,结果显示二者符合率达95.88 %。本研究为进一步研发Fh血清学诊断试剂盒奠定了前期基础。
  
Abstract:
To reveal the antigenicity of cathepsins and evaluate its potential value as diagnostic antigen for the detection of Fasciola hepatica via ELISA, the dominant epitope-rich regions of F.hepatica CatL1D and CatB4 genes were amplified by RT-PCR, and fused them by overlap extension PCR to obtain the 810bp CatL-B fusion gene (MeCatL-B). The prokaryotic expression recombinant plasmie pET-MeCatL-B was constructed and then transformed into BL21(DE3) competent cells for expression. The results of SDS-PAGE and western blot showed that the expressed recombinant MeCatL-B (rMeCatL-B) was about 46ku, which reacted with the positive sheep serum against F.hepatica. The purified reMeCatL-B was used as the coating antigen to develop an indirect ELISA assay for detecting F.hepatica antibody after optimization of reaction conditions. The specificity, sensitivity and reproducibility of the indirect ELISA were very high. In addition, a total of 97 serum samples were detected by the indirect ELISA, and the coincidence was 95.88 % between the established indirect ELISA and the commercial F.hepatica antibody ELISA kit, which laid the foundation for the development of F.hepatica serological diagnostic kit.

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(本文编辑:李 爽)

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更新日期/Last Update: 2019-04-26