中国农业科学 ›› 2021, Vol. 54 ›› Issue (20): 4478-4486.doi: 10.3864/j.issn.0578-1752.2021.20.020
• 畜牧·兽医·资源昆虫 • 上一篇
李敏雪1(),李剑男1,周红1,肖宁1,蔺辉星1,马喆1,范红结1,2(
)
收稿日期:
2020-09-09
接受日期:
2021-04-12
出版日期:
2021-10-16
发布日期:
2021-10-25
通讯作者:
范红结
作者简介:
李敏雪,Tel:13218030982;E-mail: 基金资助:
LI MinXue1(),LI JianNan1,ZHOU Hong1,XIAO Ning1,LIN HuiXing1,MA Zhe1,FAN HongJie1,2(
)
Received:
2020-09-09
Accepted:
2021-04-12
Online:
2021-10-16
Published:
2021-10-25
Contact:
HongJie FAN
摘要:
【目的】胞内劳森菌(Lawsonia intracellularis,LI)是引起猪增生性肠炎(porcine proliferative enteritis, PPE)的肠道病原菌,主要表现为动物福利下降,给世界养猪业造成严重的经济损失。研究通过制备鼠抗LI SodC的单克隆抗体,建立一种针对LI的免疫过氧化物酶细胞单层试验(IPMA)抗原检测方法,检验其在临床上的应用性,从而为LI的病原诊断提供一种科学有效的手段。【方法】选取胞内劳森菌弱毒疫苗为研究对象,通过PCR扩增其sodc片段,将其克隆至原核表达载体pGex-6p-1上,成功构建出pGex-6p-1-sodc重组质粒,诱导表达重组蛋白SodC。Western Blot分析重组蛋白的反应原性,一抗使用鼠抗GST标签的抗体。以该重组蛋白为免疫原,免疫4—6周龄BALB/c小鼠,利用常规细胞融合技术、有限稀释法和间接ELISA技术筛选阳性杂交瘤细胞,并制备腹水。通过间接免疫荧光(IFA)鉴定该单抗的特异性。以该单抗为一抗,摸索并建立了LI IPMA抗原检测方法,并评价该方法的特异性、敏感性和重复性。用优化后的IPMA方法对来自江苏周边地区猪场回肠组织样品进行检测,评价该方法的临床价值。【结果】纯化后SodC蛋白浓度较高,与鼠抗GST标签的抗体发生特异性结合,表明该蛋白反应原性较好。经3次亚克隆后最终共筛选获得2株阳性杂交瘤细胞,分别命名为1D6和1F7。单抗亚型鉴定结果显示:1D6亚型为IgA,1F7亚型为IgG3;ELISA检测1D6单抗效价为1﹕1 024 000;1F7单抗效价为1﹕1 024 000;间接免疫荧光(IFA)结果表明2株单抗均与LI菌株发生特异性反应,与猪霍乱沙门氏菌(Salmonella Cholerasuis,S. Cholerasuis)、猪流行性腹泻病毒(PEDV)和猪传染性胃肠炎病毒(TGEV)等猪常见病原无交叉反应。优化后IPMA反应条件为:一抗稀释倍数为1﹕800,作用45min;二抗稀释倍数为1﹕2 500,作用1h,此时IPMA检测效果最佳。用该方法检测S. Cholerasuis、PEDV、TGEV、伪狂犬病毒(PRV)、猪圆环病毒2(PCV2)均为阴性;最低检测限为103个/mL,说明该方法特异性强、敏感性高。临床样品检测结果显示146份病料中共检测出92份阳性样品,3个不同猪场的阳性样品检出率分别为65.6%、68.2%和53.7%,总体阳性率为63.0%。普通PCR方法检测出82份阳性样品,两种方法的阳性符合率为94.6%。【结论】成功制备了鼠抗LI SodC蛋白的单克隆抗体,建立了针对LI抗原的IPMA检测方法,并对临床样品进行了检测。该方法具有良好的特异性和敏感性,并具有一定的临床价值,为实验室LI的分离鉴定、在感染细胞中的定位以及流行病学调查和相关检疫提供一种有效的技术手段。
李敏雪,李剑男,周红,肖宁,蔺辉星,马喆,范红结. 基于SodC单克隆抗体的胞内劳森菌IPMA抗原检测方法的建立及应用[J]. 中国农业科学, 2021, 54(20): 4478-4486.
LI MinXue,LI JianNan,ZHOU Hong,XIAO Ning,LIN HuiXing,MA Zhe,FAN HongJie. Establishment and Preliminary Application of Lawsonia intracellularis IPMA Antigen Detection Method Based on SodC Monoclonal Antibody[J]. Scientia Agricultura Sinica, 2021, 54(20): 4478-4486.
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