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

doi:10.3864/j.issn.0578-1752.2021.20.020

摘要/Abstract

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。

关键词: 胞内劳森菌, SodC蛋白, 单克隆抗体, 免疫过氧化物酶细胞单层试验

Abstract:

【Objective】 Lawsonia intracellularis (L. intracellularis) is an enteric pathogenic bacteria that causes porcine proliferative enteropathy (PPE), which mainly shows the decline of animal welfare and causes serious economic losses to the world swine industries. The objective of this study was to prepare monoclonal antibodies against SodC of L. intracellularis, and to establish an immunoperoxidase monolayer assay (IPMA) method for detecting L. intracellularis base the monoclonal antibody, while test its application in clinical practice, so as to provide a scientific and effective means for the diagnosis of L. intracellularis. 【Method】In this study, the commercial live attenuated L. intracellularis vaccine was selected as target strain. The sodc gene was amplified by PCR and cloned into the prokaryotic expression vector pGex-6p-1. The recombinant plasmid pGex-6p-1-sodc was confirmed to be constructed successfully and induced expression of recombinant SodC protein. The reactivity of the recombinant protein was analyzed by Western Blot. The primary antibody was a mouse anti-GST labeled antibody. BALB/c mice aged 4-6 weeks were immunized with purified SodC protein, and hybridoma cells were screened by conventional cell fusion, limited dilution and indirect ELISA, then ascites were prepared. It was confirmed that two monoclonal antibodies had good specificity through indirect immunofluorescence (IFA). Using the monoclonal antibody as the primary antibody, a method of IPMA for detecting L. intracellularis was developed, and the specificity, sensitivity and repeatability of the method were evaluated. The optimized IPMA method was used to detect ileal tissue samples from pig farms in Jiangsu Province and to evaluate the clinical value of the method. 【Result】After purification, the concentration of SodC protein was higher, and it specifically bound to the antibody against GST tag, indicating that the protein had good regenicity. After three times of subcloning, two strains positive hybrid tumor cells were screened, named 1D6 and 1F7, respectively. The titers of two monoclonal antibodies were both reached 1﹕204 000 by ELISA. The subclass identification results of antibodies showed the subclass of 1D6 was IgA,and subclass of 1F7 was IgG3. The result IFA showed that 1D6 and 1F7 had specific reaction with L. intracellularis, but did not cross-react with S. Cholerasuis, PEDV and TGEV. The ascites of the two monoclonal antibodies were both 1﹕1 024 000 by ELISA; IFA confirmed that the two monoclonal antibodies had good specificity. The optimized IPMA reaction conditions showed that when the dilution ratio of the primary antibody was 1:800 for 45 min, and the dilution ratio of the secondary antibody was 1﹕2 500 for 1h, the established IPMA exhibited the best performance. The specificity and sensitivity tests showed that S. Cholerasuis, PEDV, TGEV, PCV2 and PRV were all negative, and the minimum detection limit was 10³L.intracellularis. The optimized IPMA method was used to detect the ileum tissue samples from pig farms in the surrounding areas of Jiangsu Province. A total of 92 positive samples were detected from 146 samples of ileal tissues. The positive rates of 3 different pig farms were 65.6%, 68.1% and 53.7%, respectively, and the overall positive rate was 63.0%. 82 positive samples were detected by PCR method. and the positive coincidence rate of the two methods was 94.6%. These results indicated that this method had clinical value. 【Conclusion】The monoclonal antibodies against the recombinant SodC protein were successfully prepared, and the IPMA method for L. intracellularis was established with good specificity and sensitivity, and the clinical samples were tested. In summary, these results further proved that the IPMA had certain clinical value, and provided an effective technical means for the isolation and identification of L. intracellularis in the laboratory, localization in infected cells, epidemiological investigation and quarantine.

Key words: Lawsonia intracellularis, SodC protein, monoclonal antibody, an immunoperoxidase monolayer assay (IPMA)

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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