[1]廖荣莉,郑金,熊梦霞,等.基于胞内劳森菌重组flgE 蛋白间接ELISA 检测方法的建立及应用[J].中国预防兽医学报,2021,(02):150-157.[doi::10.3969/j.issn.1008-0589.202012030]
 LIAO Rong-li,ZHENG Jin,XIONG Meng-xia,et al.Establishment and application of an indirect ELISA based onrecombinant protein flgE of Lawsonia intracellularis[J].Chinese journal of preventive veterinary medicine,2021,(02):150-157.[doi::10.3969/j.issn.1008-0589.202012030]
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基于胞内劳森菌重组flgE 蛋白间接ELISA 检测方法的
建立及应用
()
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2021年02
页码:
150-157
栏目:
诊断和检测技术
出版日期:
2021-03-16

文章信息/Info

Title:
Establishment and application of an indirect ELISA based on
recombinant protein flgE of Lawsonia intracellularis
文章编号:
:1008-0589(2021)02-0150-08
作者:
 廖荣莉1郑金2熊梦霞1罗灵芝1赵墩12周阳1唐红剑1葛猛1余兴龙1*
 (1. 湖南农业大学动物医学院,湖南长沙410128;2. 湖南康保特生物科技有限公司,湖南长沙410128)
Author(s):
 LIAO Rong-li1 ZHENG Jin2 XIONG Meng-xia1 LUO Ling-zhi1 ZHAO Dun12 ZHOU Yang1TANG Hong-jian1 GE Meng1 YU Xing-long1*
 (1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China; 2. Hunan Combetter Biotechnology Co., Ltd.,
Changsha 410128, China)
关键词:
:猪增生性肠病胞内劳森菌鞭毛蛋白flgE间接ELISA
Keywords:
: porcine proliferative enteropathy Lawsonia intracellularis flagellar hook-basal body complex protein flgEindirect ELISA
分类号:
S852.61
DOI:
:10.3969/j.issn.1008-0589.202012030
文献标志码:
A
摘要:
:为建立以胞内劳森菌(LI)重组鞭毛钩基体复合蛋白flgE为包被抗原的间接ELISA检测方法,本研究
将flgE 基因克隆至原核表达载体pET-28a(+)中,构建重组表达质粒pET28a-flgE,转化至大肠杆菌BL21(DE3)感
受态细胞,经诱导后获得了可溶性表达的flgE重组蛋白。以纯化的重组flgE蛋白为包被抗原,经优化各反应条件
后建立了检测LI 抗体的间接ELISA 方法。特异性试验结果显示所建立的ELISA 方法与CSFV、PRV、PRRSV、
PCV2、FMDV、Mhp和APP等阳性血清均无交叉反应。该方法能检测到1 600倍稀释的LI阳性血清,批内和批间
变异系数均小于10%。利用该方法检测多个不同类型猪场的猪血清(血清来源:2个种猪场1~5胎次的母猪;3个
商品猪场30日龄~140日龄的猪;仔猪30头,从30日龄跟踪至80日龄,共采集4次血清),结果显示,母猪和育
肥猪的感染率较高可达100%;母源抗体下降后,自50日龄~70日龄起到育肥猪阶段,LI阳性率和抗体水平随着
日龄的增长而不断上升;30头跟踪仔猪在单独密闭的饲养条件下,从开始到全群感染LI所需的时间较短(35 d左
右)。这些结果与目前已报道的LI血清流行病学调查研究一致,表明该方法能较准确反映LI感染猪的抗体水平。
上述结果表明本研究建立的间接ELISA抗体检测方法,具有良好的特异性、敏感性和重复性,可初步用于临床LI
血清流行病学的调查。
Abstract:
To develop an indirect ELISA for detection of antibody to Lawsonia intracellularis(LI) using recombinant flagellar
hook-basal body complex protein flgE as antigen, the flgE gene was cloned into prokaryotic expression pET-28a(+) vector, resulting
in recombinant expression plasmid pET-28a-flgE. The pET-28a-flgE was transformed into E. coil strain BL21(DE3) and the soluble
recombinant protein flgE was obtained after induced. The purified recombinant protein flgE was used as the coating antigen to develop an indirect ELISA assay for detecting LI antibody after optimization of reaction conditions. The specificity tests showed
that the method had no cross reactions with other positive sera, such as CSFV, PRV, PRRSV, PCV2, FMDV, Mhp and APP. It can
detect the LI positive serum with the maximum dilution of 1600. In addition, the coefficients of variations in both intra- and interassay
were less than 10%. This method was used to detect the serum from several different types of piggery (these serum included:
sows of 1 to 5 parities from 2 breeding farms; Pigs of different weights aged from 30 to 140 days from 3 commercial farms; Thirty
piglets were followed from 30 to 80 days of age, and serum was collected for 4 times during this period). The results showed that
the infection rate of sows and finishing pigs was as high as 100%. After the maternal antibody decreased, the positive rate and
antibody level increased with the increase of age during 50-70 days of age to finishing pigs. The time from the initial LI infection
to the whole group infection was relatively short (about 35 days) when 30 tracking piglets were reared in isolation. These results
are consistent with reported studies so far and showed that this method could accurately reflect the antibody level and antibody
positive rate of LI infected pigs. These results indicated that the indirect ELISA antibody detection method established in this study
had strong specificity, high sensitivity and good repeatability. It can be preliminarily used for clinical seroepidemiological survey of
LI.

相似文献/References:

[1]白 云 *,郑英帅 *,左 奕,等. 胞内劳森菌的纳米 PCR 检测方法的建立[J].中国预防兽医学报,2016,(12):972.[doi:10.3969/j.issn.1008-0425.2016.12.11]
 BAI Yun*,ZHENG Ying-Shuai*,ZUO Yi,et al. The nano-PCR assay establishment for detection of lawsonia intracellularis[J].Chinese journal of preventive veterinary medicine,2016,(02):972.[doi:10.3969/j.issn.1008-0425.2016.12.11]

更新日期/Last Update: 2021-03-17