丝状支原体山羊亚种LPPA蛋白原核表达及间接ELISA方法的建立

doi:10.16431/j.cnki.1671-7236.2022.01.034

摘要/Abstract

摘要： [目的] 建立基于丝状支原体山羊亚种（Mmc）膜脂蛋白LPPA的间接ELISA方法。[方法] 扩增Mmc LPPA基因并将其克隆至pCold-Ⅰ载体，构建重组质粒pCold-Ⅰ-LPPA，测序鉴定正确后转化大肠杆菌DH5α感受态细胞，经IPTG诱导表达后纯化得到Mmc LPPA重组蛋白，采用SDS-PAGE验证该重组蛋白是否表达，并采用Western blotting和传统经典的琼脂扩散血清学试验分析其与Mmc阳性血清的反应原性。以该重组蛋白为包被抗原，建立Mmc重组LPPA蛋白的间接ELISA抗体检测方法，对该方法进行反应条件优化后开展特异性、重复性试验，并将其初步应用于184份山羊血清样本。[结果] 通过PCR扩增得到Mmc LPPA基因，成功构建了重组质粒pCold-Ⅰ-LPPA，经诱导表达后得到Mmc LPPA重组蛋白，SDS-PAGE结果显示，获得了大小约23 ku的LPPA重组融合蛋白；琼脂扩散及Western blotting试验证明该蛋白反应原性良好。ELISA方法反应条件优化结果显示，LPPA抗原蛋白的包被浓度为2.0 μg/100 μL，血清稀释度为1：300，3% BSA封闭1 h为最佳反应条件，临界值为2.738。本试验建立的间接ELISA方法批内和批间变异系数均<10%，证明该方法具有良好的重复性；对绵羊肺炎支原体（Mo）、蓝舌病病毒（BTV）、小反刍兽疫病毒（PPRV）、羊口蹄疫病毒（FMDV）、弓形虫及山羊痘病毒（GPV）标准阳性血清的检测均为阴性，表明该方法特异性较强；184份临床血清临床应用结果显示，该方法与正向间接血凝诊断试剂盒检测结果阳性符合率为93.33%，阴性符合率为75.23%，两者相对符合率为82.61%。[结论] 本研究成功建立了Mmc LPPA蛋白间接ELISA抗体检测方法，为临床Mmc血清抗体水平的监测及Mmc血清流行病学调查奠定了基础。

关键词: 丝状支原体山羊亚种(Mmc); LPPA蛋白; 间接ELISA; 原核表达

Abstract: [Objective] The purpose of this study was to establish an indirect ELISA method based on Mycoplasma mycoides subsp. capri (Mmc) membrane lipoprotein LPPA. [Method] In this study, the Mmc LPPA gene was amplified and cloned into pCold-Ⅰ vector. The recombinant plasmid pCold-Ⅰ-LPPA was constructed. After sequencing identification, it was transformed into E. coli DH5α competent cells. The recombinant protein of Mmc LPPA was purified after induced expression by IPTG. The expression of the recombinant protein was verified by SDS-PAGE. Its reactivity with Mmc positive serum was analyzed by Western blotting test and traditional classical agar diffusion serological test. Using the recombinant protein as the coating antigen, an indirect ELISA antibody detection method for Mmc recombinant LPPA protein was established. After the reaction conditions were optimized, the specificity and repeatability tests were carried out, and it was preliminarily applied to 184 goat serum samples. [Result] The Mmc LPPA gene was amplified by PCR, and the recombinant plasmid pCold-Ⅰ-LPPA was successfully constructed. After induced expression, the recombinant protein Mmc LPPA was obtained. SDS-PAGE result showed that the recombinant fusion protein of LPPA with the size of about 23 ku was obtained. Agar diffusion and Western blotting showed that the protein had good reactivity. The optimized reaction conditions of ELISA showed that the coating concentration of LPPA antigen protein of 2.0 μg/100 μL, serum dilution of 1:300 and blocking with 3% BSA for 1 h were the optimum reaction conditions, and the critical value was 2.738. Intra assay and inter assay coefficients of variation of the indirect ELISA method established in this test were both less than 10%, which proved that the method had good repeatability. The standard positive sera of Mo, BTV, PPRV, FMDV, Toxoplasma gondii and GPV were negative, indicating that the method had strong specificity. The clinical application results of 184 clinical sera showed that the positive coincidence rate between the method and the positive indirect hemagglutination diagnostic kit was 93.33%, the negative coincidence rate was 75.23%, and the relative coincidence rate was 82.61%. [Conclusion] This study successfully established an indirect ELISA method for the detection of antibodies for Mmc LPPA protein, which laid a foundation for the monitoring of serum antibody level of clinical Mmc and the investigation of serum epidemiology of Mmc.

Key words: Mycoplasma mycoides subsp. capri (Mmc); LPPA protein; indirect ELISA; prokaryotic expression

中图分类号:

S852.62

陈静, 吴燕, 李梅, 岳筠, 朱二鹏, 文明, 张双翔, 程振涛. 丝状支原体山羊亚种LPPA蛋白原核表达及间接ELISA方法的建立[J]. 中国畜牧兽医, 2022, 49(1): 318-327.

CHEN Jing, WU Yan, LI Mei, YUE Jun, ZHU Erpeng, WEN Ming, ZHANG Shuangxiang, CHENG Zhentao. Prokaryotic Expression of LPPA Protein of Mycoplasma mycoides subsp. capri and Establishment of Indirect ELISA Method[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 318-327.

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