[1]童 欣,赵 霞,陆康妍,等.人空肠弯曲菌FlaA蛋白重组表达与免疫血清制备以及间接ELISA检测血清抗体的方法建立及初步应用[J].现代检验医学杂志,2026,41(03):50-54+60.[doi:10.3969/j.issn.1671-7414.2026.03.009]
　TONG Xin,ZHAO Xia,LU Kangyan,et al.Recombinant Expression of the Human Campylobacter jejuni FlaA Protein, Preparation of Immuniserum, and Establishment and Preliminary Application of an Indirect ELISA Assay for the Detection of Serum Antibodies[J].Journal of Modern Laboratory Medicine,2026,41(03):50-54+60.[doi:10.3969/j.issn.1671-7414.2026.03.009]
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人空肠弯曲菌FlaA蛋白重组表达与免疫血清制备以及间接ELISA检测血清抗体的方法建立及初步应用()

《现代检验医学杂志》[ISSN:/CN:]

卷:
第41卷
期数:
2026年03期
页码:
50-54+60
栏目:
论著
出版日期:
2026-05-13

文章信息/Info

Title:
Recombinant Expression of the Human Campylobacter jejuni FlaA Protein, Preparation of Immuniserum, and Establishment and Preliminary Application of an Indirect ELISA Assay for the Detection of Serum Antibodies
文章编号:
1671-7414（2026）03-050-06
作者:
童 欣1,2，赵 霞2，陆康妍2，夏 洁2，黄金林1
1.扬州大学江苏省人兽共患病学重点实验室，江苏扬州 225000;2.宜兴市红十字会血站，江苏宜兴 214200
Author(s):
TONG Xin1,2，ZHAO Xia2，LU Kangyan2，XIA Jie2，HUANG Jinlin1
1.Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Jiangsu Yangzhou 225000, China;2.Yixing Blood Center, Jiangsu Yixing 214200, China
关键词:
空肠弯曲菌; FlaA蛋白; 质粒重组; 蛋白表达纯化; 间接酶联免疫吸附试验; 多克隆抗体
分类号:
R378.3；R446.61
DOI:
10.3969/j.issn.1671-7414.2026.03.009
文献标志码:
A
摘要:
目的?表达与纯化空肠弯曲菌（C.jejuni）FlaA蛋白，建立检测人C.jejuni抗体的ELISA诊断试剂盒。方法对C.jejuni的FlaA蛋白进行生物信息学分析，构建pET-28a-flaA重组质粒，通过BL21(DE3)感受态细胞的转化，进行重组蛋白的表达。用免疫雌性新西兰大白兔，制备相应多克隆抗血清，筛选最佳包被浓度，优化实验条件，建立间接ELISA的检测方法，并检测58份腹泻患者血清和697份健康献血者血清样本，评价方法的灵敏度和特异度。结果成功构建pET-28a-flaA重组质粒，通过原核表达系统获得的重组蛋白均有较高表达量，Westernblot鉴定证实重组蛋白与阳性血清反应性强，可作为检测靶抗原，建立并优化间接ELISA检测方案，检测结果基本符合临床结论，特异度和灵敏度分别为75%和99.86%。结论?成功表达并纯化C.jejuniFlaA重组蛋白并建立以FlaA作为包被抗原的间接ELISA方法。
Abstract:
Objective To express and purify the Campylobacter jejuni (C. jejuni) FlaA protein and to develop an ELISA diagnos-tic kit for detecting C. jejuni antibodies in humans. Methods Bioinformatic analysis of the C. jejuni FlaA protein was performed, and the recombinant plasmid pET-28a-flaA was constructed. The recombinant protein was expressed by transforming BL21(DE3) competent cells. Female New Zealand white rabbits were immunized to prepare the corresponding polyclonal antiserum. The op-timal coating concentration was screened, experimental conditions were optimized, and an indirect ELISA assay was established. The assay was then applied to serum samples from 58 patients with diarrhea and 697 healthy blood donors to evaluate the sensi-tivity and specificity of the method. Results The pET-28a-flaA recombinant plasmid was successfully constructed. The recom-binant protein obtained via the prokaryotic expression system exhibited high expression levels. Western blot analysis confirmed that the recombinant protein reacted strongly with positive sera and could serve as a target antigen. An optimized indirect ELISA protocol was established, and the test results were generally consistent with clinical findings, with specificity and sensitivity of 75% and 99.86%, respectively. Conclusions The C. jejuni FlaA recombinant protein is successfully expressed and purified, and an indirect ELISA method using FlaA as the coating antigen is established.

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相似文献/References:

[1]李 博,陈 辉,鞠长燕,等.深圳地区不同来源空肠弯曲菌毒力基因分布及分子分型研究[J].现代检验医学杂志,2016,31(05):107.[doi:10.3969/j.issn.1671-7414.2016.05.029]
　LI Bo,CHEN Hui,JU Chang-yan,et al.Study on the Differences of Virulence Genes and Molecular Typing in Campylobacter Jejuni Isolates from Poultry Products and Diarrhea Patients in Shenzhen[J].Journal of Modern Laboratory Medicine,2016,31(03):107.[doi:10.3969/j.issn.1671-7414.2016.05.029]
[2]高正琴.空肠弯曲菌 TaqMan-MGB双重探针实时荧光定量 PCR快速检测[J].现代检验医学杂志,2019,34(06):1.[doi:10.3969 ／ j.issn.1671-7414.2019.06.001]
　GAO Zheng-qin.Rapid Detection of Campylobacter Jejuni with TaqMan-MGB Dual Probe Real-time Fluorescence Quantitative PCR[J].Journal of Modern Laboratory Medicine,2019,34(03):1.[doi:10.3969 ／ j.issn.1671-7414.2019.06.001]

备注/Memo

备注/Memo:
作者简介：童欣（1996-），女，硕士研究生，主管技师，研究方向：病原微生物的免疫学检验，E-mail：2505195117@qq.com。
通讯作者：黄金林（1969-），男，博士，二级教授，研究方向：人畜共患病原微生物与食品安全，E-mail：jinlin@yzu.edu.com。
更新日期/Last Update: 2026-05-15