基于重组截短蛋白mp-tGAPDH的微小支原体抗体间接ELISA检测方法建立和应用

doi:10.3969/j.issn.1004-1524.2021.05.05

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (5): 809-815.DOI: 10.3969/j.issn.1004-1524.2021.05.05

基于重组截短蛋白mp-tGAPDH的微小支原体抗体间接ELISA检测方法建立和应用

付媛(), 石团员, 孙洪超

浙江省农业科学院畜牧兽医研究所,浙江杭州 310021

收稿日期:2020-10-10 出版日期:2021-05-25 发布日期:2021-05-25
作者简介:付媛(1980—),女,河北深县人,博士,副研究员,主要从事动物寄生虫分子流行病学研究。E-mail:fuy@mail.zaas.ac.cn
基金资助:
浙江省重点研发计划(2019C02052-4);浙江省公益性项目(2017C32018)

Establishment and application of indirect-ELISA for detection of Mycoplasma parvum based on mp-tGAPDH

FU Yuan(), SHI Tuanyuan, SUN Hongchao

Institute of Animal Husbandry and Veterinary, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2020-10-10 Online:2021-05-25 Published:2021-05-25

摘要/Abstract

摘要：

猪源嗜血支原体(Hemotropic mycoplasma,HM)寄生于猪红细胞表面及骨髓中,引起猪传染性贫血,给我国养猪产业造成危害。近年来病原流行病学调查发现我国流行三种HM,猪支原体(Mycoplasma suis,M. suis 猪附红细胞体),微小支原体(M. parvum)和新种Candidate Mycoplasma hemosuis(CMh)。且M. parvum在是我国浙江和海南猪群中的主要流行嗜血支原体,目前仅M. suis有血清抗体检测方法报道。本研究根据M. parvum ZJ9330株的甘油醛-3-磷酸脱氢酶(GAPDH)基因序列设计引物扩增404 bp的部分基因片段、原核表达纯化15 ku截短蛋白mp-tGAPDH,Western-blot表明纯化蛋白mp-tGAPDH与小鼠抗mp-tGAPDH血清有较好的反应性。以mp-tGAPDH为包被抗原,建立了检测M. parvum抗体的间接ELISA方法。特异性实验表明,建立的ELISA方法不与猪圆环病毒病、猪瘟、伪狂犬病、猪繁殖与呼吸综合征和弓形虫阳性血清交叉反应。重复性实验表明,该ELISA方法批间和批内重复性低于18.4%,表明该法有较好的重复性。用建立的ELISA方法对浙江省上虞地区5个猪场248份临床送检血清进行了检测, M. parvum血清抗体阳性率为36.69% (91/248)。本研究建立的间接ELISA方法为猪微小支原体的检测和流行病学调查提供了一种快速简便的血清学诊断方法。

关键词: 嗜血支原体, 微小支原体, 甘油醛-3-磷酸脱氢酶, ELISA

Abstract:

Hemotropic mycoplasma(HM) resides on the surface of red blood cells and bone marrow of pigs, causing infectious anemia and harm to the pig production. In recent years, three HMs have been found in China, including Mycoplasma suis(M. suis), M. parvum and Candidate Mycoplasma hemosuis(CMh). M. parvum is the predominant hemoplasma strain of the pig populations in Zhejiang and Hainan Province. Currently, there was only M. suis serum antibody detection method was reported. According to the sequence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of M. parvum ZJ9330 strain, a 404 bp partial fragment was amplified and expressed in E. coli Transetta DE3. The 15 ku truncated protein mp-tGAPDH was purified. Western-blot showed that mp-tGAPDH had higher reactivity with mouse anti mp-tGAPDH serum. The mp-tGAPDH applied as coating antigen, an indirect ELISA method for detecting M. parvum antibody in swine serum was established. The indirect ELISA using mp-tGAPDH exhibited good specificity and no cross-reactivity with porcine circovirus, classical swine fever, pseudorabies, porcine reproductive and respiratory syndrome and Toxoplasma gondii positive serum. Intra-and inter-assay variability was less than 18.4%, showing a high degree of repeatability. The antibody positive rate of M. parvum was 36.69% (91/248) in 248 detected serum samples from 5 pig farms in Zhejiang Province by ELISA. The results showed that indirect ELISA using mp-tGAPDH provides a rapid and simple serological diagnostic tool for the detection of antibodies against M. parvum.

Key words: Hemotropic mycoplasma, Mycoplasma parvum, glyceraldehyde-3-phosphate dehydrogenase, ELISA

中图分类号:

S858

付媛, 石团员, 孙洪超. 基于重组截短蛋白mp-tGAPDH的微小支原体抗体间接ELISA检测方法建立和应用[J]. 浙江农业学报, 2021, 33(5): 809-815.

FU Yuan, SHI Tuanyuan, SUN Hongchao. Establishment and application of indirect-ELISA for detection of Mycoplasma parvum based on mp-tGAPDH[J]. Acta Agriculturae Zhejiangensis, 2021, 33(5): 809-815.

图/表 11

图1 mp-tGAPDH基因PCR扩增 M,DNA marker DL1000;1、2,M. parvum阳性血液PCR扩增mp-tGAPDH 基因片段;3、4,阴性对照;

Fig.1 Amplified mp-tGAPDH gene fragment by PCR M, DNA marker DL1000; 1, 2, PCR product of mp-tGAPDH from M. parvum positive blood sample; 3, 4, Negative control.

图2 mp-tGAPDH在大肠埃希菌中的表达分析 M,Protein marker;Lane 1, 2,pET28-mp-tGAPDH在大肠埃希菌Transetta(DE3)表达目的片段的SDS-PAGE电泳;3,pET28a 在大肠埃希菌Transetta(DE3)表达目的片段的SDS-PAGE分析。

Fig.2 SDS PAGE analysis expression of mp-tGAPDH in different E. coli M, Protein marker; Lane 1, 2, pET28-mp-tGAPDH in Transetta(DE3); 3, pET28a in Transetta(DE3).

图3 重组蛋白mp-tGAPDH Western-blot分析免疫反应性 M,Protein marker;1,纯化mp-tGAPDH的SDS-PAGE电泳分析;2,小鼠抗mp-tGAPDH 血清和mp-tGAPDH纯化蛋白的Western-blot反应,箭头指示反应条带。

Fig.3 Western-blot analysis immunoreactivity of mp-tGAPDH M, Protein marker; 1, SDS-PAGE of purified mp-tGAPDH; 2, mp-tGAPDH Western-blot using mouse anti-mp-tGAPDH serum. Reaction band indicated by arrow.

表1 mp-tGAPDH ELISA最佳封闭液优化结果

Table 1 The optimized result of blocking solution for mp-tGAPDH ELISA

指标 Index	脱脂乳Skim milk			BSA
指标 Index	2.5%	5.0%	7.5%	1%	2%	3%
P	0.774	0.494	0.490	1.321	1.225	1.167
N	0.387	0.270	0.223	0.866	0.741	0.663
P/N	2.002	1.832	2.194	1.526	1.653	1.760

表2 mp-tGAPDH ELISA封闭液封闭时间优化结果

Table 2 The optimized result of blocking solution and blocking time for mp-tGAPDH ELISA

指标 Index	5.0%脱脂乳Skim milk			7.5%脱脂乳Skim milk
指标 Index	0.5 h	1.0 h	1.5 h	0.5 h	1.0 h	1.5 h
P	0.781	0.571	0.637	0.548	0.509	0.657
N	0.387	0.278	0.246	0.236	0.231	0.32
P/N	2.019	2.049	2.586	2.322	2.204	2.057

表3 mp-tGAPDH ELISA血清作用时间优化结果

Table 3 The optimized result of serum reaction time for mp-tGAPDH ELISA

指标 Index	血清不同作用时间Different serum reaction time
指标 Index	0.5 h	1.0 h	1.5 h
P	0.413	0.627	0.532
N	0.196	0.272	0.255
P/N	2.103	2.303	2.086

表4 mp-tGAPDH ELISA二抗稀释倍数优化结果

Table 4 The optimized result of secondary antibody dilution for mp-tGAPDH ELISA

指标 Index	不同二抗浓度Different secondary antibody dilution
指标 Index	1:7500	1:10000	1:12500
P	0.497	0.574	0.609
N	0.196	0.183	0.193
P/N	2.540	3.131	3.160

表5 mp-tGAPDH ELISA二抗作用时间优化结果

Table 5 The optimized result of secondary antibody incubating time for mp-tGAPDH ELISA

指标 Index	不同二抗作用时间 Different secondary antibody incubating time
指标 Index	30 min	45 min	60 min
P	0.480	0.627	0.666
N	0.155	0.208	0.202
P/N	3.099	3.020	3.302

表6 mp-tGAPDH 间接ELISA方法的特异性

Table 6 Specificity of mp-tGAPDH ELISA

阳性血清 Positive serum	D₄₅₀	结果判定Result
M. parvum	1.043	+
PCV2	0.191	-
CSFV	0.238	-
PRV	0.297	-
PRRSV	0.076	-
TG	0.078	-
M. suis	0.114	-
CMh	0.533	+

表7 mp-tGAPDH间接ELISA同批抗原包被板批内变异系数

Table 7 The coefficients of variation for mp-tGAPDH ELISA using the same batch of antigen

抗原批号 Batch of antigen	血清mp-tGAPDH ELISA重复检测的D₄₅₀值 D₄₅₀ values of mp-tGAPDH ELISA at duplicate detection
抗原批号 Batch of antigen	1	2	3	4	5	6	7
0707	0.946	0.675	0.604	0.607	0.224	0.209	0.151
0707	0.863	0.600	0.560	0.557	0.236	0.190	0.142
0707	0.965	0.659	0.633	0.580	0.231	0.185	0.130
$\bar{X}$	0.905	0.638	0.582	0.582	0.230	0.200	0.147
SD	0.059	0.053	0.031	0.035	0.009	0.013	0.006
CV/%	6.5	8.4	5.3	6.1	3.8	6.7	4.1

表8 mp-tGAPDH间接ELISA不同批抗原包被板批间变异系数

Table 8 The coefficients of variation for mp-tGAPDH ELISA test using the different batches of antigen

抗原批号 Batch of antigen	血清mp-tGAPDH ELISA重复检测的D₄₅₀值 D₄₅₀ values of mp-tGAPDH ELISA at duplicate detection
抗原批号 Batch of antigen	1	2	3	4	5	6
0408	0.612	0.511	0.951	0.161	0.125	0.054
0413	0.508	0.391	0.816	0.128	0.096	0.052
0423	0.500	0.428	0.655	0.127	0.101	0.057
$\bar{X}$	0.540	0.443	0.807	0.139	0.107	0.054
SD	0.062	0.061	0.148	0.019	0.015	0.003
CV/%	11.6	13.8	18.4	13.8	14.3	4.7

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基于重组截短蛋白mp-tGAPDH的微小支原体抗体间接ELISA检测方法建立和应用

Establishment and application of indirect-ELISA for detection of Mycoplasma parvum based on mp-tGAPDH

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