猪丹毒丝菌CbpB基因的克隆表达及其间接ELISA抗体检测方法的建立与应用

doi:10.3969/j.issn.1004-1524.2021.05.06

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (5): 816-824.DOI: 10.3969/j.issn.1004-1524.2021.05.06

猪丹毒丝菌CbpB基因的克隆表达及其间接ELISA抗体检测方法的建立与应用

刘君雯¹(), 王迪¹, 朱艳艳¹, 邢刚², 占松鹤³, 刘晓露¹, 魏建忠¹, 孙裴¹, 刘雪兰¹, 李郁¹^,^*()

1.安徽农业大学动物科技学院,安徽合肥 230036
2.马鞍山史记动物健康管理有限公司,安徽马鞍山 238251
3.安徽省动物疫病预防与控制中心,安徽合肥 230091

收稿日期:2020-07-27 出版日期:2021-05-25 发布日期:2021-05-25
通讯作者: 李郁
作者简介:*李郁,E-mail:liyouer@163.com
刘君雯(1997—),女,安徽全椒人,硕士研究生,研究方向为动物传染病学。E-mail:840491505@qq.com
基金资助:
国家星火计划重点项目(2014GA710002);安徽省重点研究与开发计划(面上攻关)项目(201904a06020013);安徽省长三角联合科技攻关项目(1101c0603065);安徽省生猪产业体系基金(皖农科〔2016〕84号)

Establishment and application of an indirect ELISA based on protein by expression and cloning of CbpB gene of Erysipelothrix rhusiopathiae

LIU Junwen¹(), WANG Di¹, ZHU Yanyan¹, XING Gang², ZHAN Songhe³, LIU Xiaolu¹, WEI Jianzhong¹, SUN Pei¹, LIU Xuelan¹, LI Yu¹^,^*()

1. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
2. Maanshan Shiji Animal Health Management Co., Ltd., Maanshan 238251, China
3. Anhui Animal Disease Prevention and Control Center, Hefei 230091, China

Received:2020-07-27 Online:2021-05-25 Published:2021-05-25
Contact: LI Yu

摘要/Abstract

摘要：

利用表达纯化的猪丹毒丝菌(ER)胆碱结合蛋白B(CbpB)建立一种检测ER抗体的间接ELISA方法。克隆扩增ER的CbpB基因并与原核表达载体pGEx-6P-1连接,经PCR、双酶切及测序鉴定,将阳性重组质粒转入E.coil BL21,IPTG诱导表达,SDS-PAGE和Western-blot鉴定产物。以纯化重组CbpB蛋白作为包被抗原,通过一系列反应条件优化,建立检测ER抗体的间接ELISA方法(CbpB-ELISA),并用于临床猪血清样品检测。建立该方法的最适条件:包被抗原浓度为1.0 μg·mL^-1,4 ℃过夜;5%脱脂奶粉于37 ℃封闭2 h;血清稀释度为1:100,37 ℃孵育60 min;酶标二抗稀释度为1∶1 000,37 ℃作用45 min;显色时间为37 ℃作用10 min。该方法可特异性地检测ER抗体,与其他猪病原阳性血清均无交叉反应性;阳性血清稀释至1:12 800时仍可检出;批内及批间变异系数均小于10.00%;与全菌体-ELISA、SpaA-ELISA、商品化ELISA试剂盒及Western-blot比较,该方法总符合率分别为91.67%、93.33%、86.67%和90.00%,其中阳性符合率分别为92.45%、94.55%、88.89%、90.91%,阴性符合率分别为85.71%、80.00%、66.67%、80.00%。对进行和未进行ER免疫的猪血清样品检测,免疫抗体阳性率为86.67%,感染抗体阳性率为30.50%。该方法具有良好的特异性、敏感性和重复性以及临床应用的可靠性,为ER的免疫监测和流行病学调查提供了技术手段。

关键词: 猪丹毒丝菌, 胆碱结合蛋白B, 间接ELISA, 抗体检测

Abstract:

Using the expression of purified choline binding protein B (CbpB) of Erysipelothrix rhusiopathiae(ER) to establish an indirect ELISA method for detecting ER antibodies, the CbpB gene of ER was cloned and linked to pGEx-6P-1 prokaryotic expression vector. The positive recombinant plasmid was identified by PCR, double enzyme digestion and sequencing.The positive recombinant plasmid was transferred into E.coil BL21, induced by IPTG, and the product was identified by SDS-PAGE and Western-blot. Using purified recombinant CbpB protein as coating antigen, an indirect ELISA method (CbpB-ELISA) for detection of ER antibody was established through a series of optimization of reaction conditions, and was applied to the detection of clinical pig serum samples. The optimal conditions for this method were as follows: the concentration of encapsulated antigen was 1.0 μg·mL^-1 at 4 ℃ overnight; 5% skim milk powder was sealed at 37 ℃ for 2 hours. The serum dilution was 1∶100 which was incubated at 37 ℃ for 60 min. The dilution of the secondary antibody was 1∶1 000, and it was treated at 37 ℃ for 45 min. The color development time was 10 min at 37 ℃. This method could specifically detect ER antibody, and had no cross reactivity with other pig pathogen positive serum. The method was sensitive to detect antibody even when the positive serum was diluted to 1∶12 800. Both the intra- and inter-coefficients of variation were less than 10.00%. Compared with the methods of whole cell-ELISA, SpaA-ELISA, commercial ELISA kit and Western-blot, the total coincidence rate of these methods were 91.67%, 93.33%, 86.67% and 90.00%, respectively. The positive coincidence rates were 92.45%, 94.55%, 88.89% and 90.91%, respectively. The negative coincidence rates were 85.71%, 80.00%, 66.67% and 80.00%, respectively. The detection of pig serum samples with and without ER immunization showed that the immune antibody positive rate was 86.67%, and the infection antibody positive rate was 30.50%. The indirect ELISA detection method of ER antibody based on recombinant CbpB protein had good specificity, sensitivity, repeatability and reliability of clinical application, which provided a technical support for immune surveillance and epidemiological investigation of ER.

Key words: Erysipelothrix rhusiopathiae, choline binding protein B, indirect ELISA, antibody detection

中图分类号:

S858.28

刘君雯, 王迪, 朱艳艳, 邢刚, 占松鹤, 刘晓露, 魏建忠, 孙裴, 刘雪兰, 李郁. 猪丹毒丝菌CbpB基因的克隆表达及其间接ELISA抗体检测方法的建立与应用[J]. 浙江农业学报, 2021, 33(5): 816-824.

LIU Junwen, WANG Di, ZHU Yanyan, XING Gang, ZHAN Songhe, LIU Xiaolu, WEI Jianzhong, SUN Pei, LIU Xuelan, LI Yu. Establishment and application of an indirect ELISA based on protein by expression and cloning of CbpB gene of Erysipelothrix rhusiopathiae[J]. Acta Agriculturae Zhejiangensis, 2021, 33(5): 816-824.

图/表 9

图1 AEr21CbpB基因PCR扩增结果 M,DNA marker 2 000;1-2,CbpB 基因PCR产物;3,阴性对照。

Fig.1 PCR amplification results of CbpB gene of AEr21 M, DNA marker DL2 000; 1-2, PCR product of CbpB gene; 3, Negative control.

图2 重组质粒pGEX-6P-1-CbpB的双酶切鉴定 M,DNA marker;1-2,BamHⅠ/XhoⅠ双酶切重组质粒pGEX-6P-1-CbpB。

Fig.2 Restriction enzyme digestion identification of recombinant plasmid pGEX-6P-1-CbpB M, DNA marker; 1-2, Digested pGEX-6P-1-CbpB.

图3 CbpB重组蛋白的诱导表达 M,蛋白marker;1-2,未纯化的CbpB蛋白。

Fig.3 Induced expression of recombinant CbpB protein M, Protein marker; 1-2, Unpurified CbpB protein.

图4 CbpB重组蛋白的纯化分析 M,蛋白marker;1-2,纯化后的CbpB蛋白。

Fig.4 Induced expression of recombinant CbpB protein M, Protein marker; 1-2, Purified CbpB protein.

图5 CbpB重组蛋白的Western-blot鉴定 M,蛋白marker;1,诱导后的裂解产物;2,非诱导后的裂解产物。

Fig.5 Western-blot identification of CbpB recombinant protein M, Protein marker; 1, Induced pyrolysis products; 2, Non-induced pyrolysis products.

表1 特异性试验结果

Table 1 Result of the specificity test

血清Serum	HPS	SS	APP	E.coli	PRV	CSFV	PCV2	PRRSV	ER(+)	ER (-)
D₄₅₀	0.173	0.149	0.138	0.120	0.127	0.203	0.099	0.168	1.161	0.136
结果Result	-	-	-	-	-	-	-	-	+	-

表2 重复性试验结果

Table 2 Results of repeatability test

血清编号 Serum No.	批内重复Intra-batch repeatability			批间重复Inter-batch repeatability
血清编号 Serum No.	平均值($\bar{X}$)	标准差(s)	变异系数CV/%	平均值($\bar{X}$)	标准差(s)	变异系数CV/%
1	1.146	0.061	5.322	1.128	0.047	4.167
2	1.176	0.048	4.082	1.170	0.042	3.590
3	0.864	0.051	5.902	1.010	0.080	7.921
4	1.363	0.057	4.182	1.396	0.078	5.587

表3 CbpB-ELISA与全菌体-ELISA、SpaA-ELISA、商品化ELISA试剂盒、Western-blot鉴定结果比较

Table 3 The results of identification of CbpB-ELISA compared with whole cell-ELISA, SpaA-ELISA, commercial ELISA kit, Western-blot

鉴定方法 Methods	阳性符合率 Positive Coincidence rate	阴性符合率 Negative Coincidence rate	总符合率 Total Coincidence rate
全菌体-ELISAwhole cell-ELISA	92.45%(49/53)	85.71%(6/7)	91.67%(55/60)
SpaA-ELISA	94.55%(52/55)	80%(4/5)	93.33%(56/60)
商品化ELISA试剂盒Commercial ELISA kit	88.89%(48/54)	66.67%(4/6)	86.67%(52/60)
Western-blot	90.91%(50/55)	80.00%(4/5)	90.00%(54/60)

表4 安徽10个地市ER感染抗体的检测结果

Table 4 Detection results of ER infection antibody in 10 cities of Anhui

地市 Cities	样品数(份) Number of samples	阳性数(份) Number of positive samples	阴性数(份) Number of negative samples	感染率 Infection rate/%
合肥Hefei	20.0	11.0	9.0	55.0 a
阜阳Fuyang	20.0	8.0	12.0	40.0 b
淮南Huainan	20.0	8.0	12.0	40.0 b
蚌埠Bengbu	20.0	7.0	13.0	35.0 bc
六安Luan	20.0	7.0	13.0	35.0 bc
安庆Anqin	20.0	6.0	14.0	30.0 c
亳州Bozhou	20.0	4.0	16.0	20.0 d
池州Chizhou	20.0	4.0	16.0	20.0 d
芜湖Wuhu	20.0	4.0	16.0	20.0 d
马鞍Maanshan 总计Total	20.0 200.0	2.0 61.0	18.0 139.0	10.0 e 30.5

参考文献 18

[1]	李吉平. 猪丹毒流行特点及防控措施[J]. 国外畜牧学(猪与禽), 2018,38(11):59-60.
	LI J P. Epidemic characteristics and prevention measures of Swine erysipelas[J]. Animal Science Abroad (Pigs and Poultry), 2018,38(11):59-60. (in Chinese)
[2]	肖国生, 曹三杰, 文心田. Dot-PAA-ELISA快速检测猪丹毒抗体方法的建立与应用[J]. 中国预防兽医学报, 2005,27(1):59-62.
	XIAO G S, CAO S J, WEN X T. Development and application of Dot-PAA-ELISA rapidly detecting antibodies against swine erysipelas[J]. Chinese Journal of Preventive Veterinary Medicine, 2005,27(1):59-62.(in Chinese with English abstract)
[3]	李富祥, 朱凤琼, 王艳芬, 等. 猪丹毒杆菌抗体间接ELISA检测方法的建立[J]. 中国预防兽医学报, 2018,40(4):311-315.
	LI F X, ZHU F Q, WANG Y F, et al. Development of an indirect ELISA for detection of antibodies against Erysipelothrix rhusiopathiae[J]. Chinese Journal of Preventive Veterinary Medicine, 2018,40(4):311-315.(in Chinese with English abstract)
[4]	姚焱彬, 李倩文, 杨志鹏, 等. 检测猪丹毒杆菌抗体重组SpaA ELISA的建立[J]. 微生物学通报, 2017,44(3):739-748.
	YAO Y B, LI Q W, YANG Z P, et al. Development of recombinant Spa A-based ELISA for detection of antibodies against swine erysipelas[J]. Microbiology China, 2017,44(3):739-748.(in Chinese with English abstract)
[5]	SHI F, HARADA T, OGAWA Y, et al. Capsular polysaccharide of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, and its modification with phosphorylcholine[J]. Infection and Immunity, 2012,80(11):3993-4003. DOI URL
[6]	FISCHETTI V A. Surface proteins on gram-positive bacteria[M]//Gram-Positive Pathogens. Washington, DC, USA: ASM Press, 2019: 19-31.
[7]	SATO H, MIYAZAKI H, SAKAKURA H, et al. Isolation and purification of a protective protein antigen of Erysipelothrix rhusiopathiae[J]. Journal of Veterinary Medicine, Series B, 1999,46(2):73-84.
[8]	陈章, 刘晓露, 姚焱彬, 等. 猪丹毒丝菌灭活疫苗制备及其对小鼠免疫效力的评价[J]. 西北农业学报, 2019,28(6):877-887.
	CHEN Z, LIU X L, YAO Y B, et al. Preparation of inactivated vaccine against Erysipelothrix rhusiopathiae and evaluation of immune efficacy in mice[J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2019,28(6):877-887.(in Chinese with English abstract)
[9]	姚焱彬, 陆萍, 杨志鹏, 等. 猪丹毒丝菌SpaA基因原核表达及表达蛋白质的免疫原性分析[J]. 畜牧兽医学报, 2017,48(3):492-500.
	YAO Y B, LU P, YANG Z P, et al. Prokaryotic expression and immunogenicity analysis of SpaA gene in Erysipelothrix rhusiopathiae[J]. Chinese Journal of Animal and Veterinary Sciences, 2017,48(3):492-500.(in Chinese with English abstract)
[10]	华耀, 王玮, 李郁, 等. 猪流行性腹泻病毒S蛋白主要抗原表位区的原核表达及间接ELISA检测方法的建立[J]. 微生物学通报, 2016,43(2):434-443.
	HUA Y, WANG W, LI Y, et al. Prokaryotic expression of the structural protein S of porcine epidemic diarrhea virus and establishment of an indirect ELISA for an detection of antibody against porcine epidemic diarrhea[J]. Microbiology China, 2016,43(2):434-443.(in Chinese with English abstract)
[11]	朱晓明, 邹垚, 陈攀, 等. 副猪嗜血杆菌不同血清型稳定表达蛋白的筛选及间接ELISA检测方法的建立[J]. 中国兽医科学, 2015,45(1):68-74.
	ZHU X M, ZOU Y, CHEN P, et al. Screening of stably-expressed proteins in different serotypes of Haemophilus parasuis and establishment of an indirect ELISA[J]. Chinese Veterinary Science, 2015,45(1):68-74.(in Chinese with English abstract)
[12]	SHIMOJI Y, OGAWA Y, OSAKI M, et al. Adhesive surface proteins of Erysipelothrix rhusiopathiae bind to polystyrene, fibronectin, and type I and IV collagens[J]. Journal of Bacteriology, 2003,185(9):2739-2748. DOI URL
[13]	李一经. 兽医微生物学[M]. 北京: 高等教育出版社, 2011.
[14]	UCHIYAMA M, SHIMAZAKI Y, ISSHIKI Y, et al. Pathogenic characterization of Erysipelothrix rhusiopathiae Met-203 type SpaA strains from chronic and subacute swine erysipelas in Japan[J]. Journal of Veterinary Medical Science, 2017,79(1):18-21. DOI URL
[15]	BERGMANN S, HAMMERSCHMIDT S. Versatility of pneumococcal surface proteins[J]. Microbiology, 2006,152(2):295-303. DOI URL
[16]	HOLT H R, INTHAVONG P, BLASZAK K, et al. Production diseases in smallholder pig systems in rural Lao PDR[J]. Preventive Veterinary Medicine, 2019,162:110-116. DOI URL
[17]	SHIMOJI Y, OSAKI M, OGAWA Y, et al. Wild boars: a potential source of Erysipelothrix rhusiopathiae infection in Japan[J]. Microbiology and Immunology, 2019,63(11):465-468. DOI URL
[18]	周绪斌, 丹尼, 李聪, 等. 猪丹毒:古老的传染病是否从中国规模化猪场消失了?[J]. 今日养猪业, 2009(5):24-26.
	ZHOU X B, DAN N, LI C, et al. Swine erysipelas-whether ancient infectious diseases have disappeared from large-scale pig farms in China?[J]. Pigs Today, 2009(5):24-26. (in Chinese)

猪丹毒丝菌CbpB基因的克隆表达及其间接ELISA抗体检测方法的建立与应用

Establishment and application of an indirect ELISA based on protein by expression and cloning of CbpB gene of Erysipelothrix rhusiopathiae

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 18

相关文章 6

编辑推荐

Metrics

本文评价

[1]	陈章, 刘晓露, 姚焱彬, 吴琼娟, 孙裴, 魏建忠, 李东风, 李郁. 猪丹毒丝菌灭活疫苗免疫佐剂的筛选[J]. 浙江农业学报, 2019, 31(11): 1803-1811.
[2]	吴琼娟, 杨志鹏, 姚焱彬, 陆萍, 魏建忠, 孙裴, 李郁. 猪红斑丹毒丝菌制苗用菌株的筛选[J]. 浙江农业学报, 2018, 30(9): 1467-1475.
[3]	闫慧丽，席俊*，高学梅，贺梦雪，陈哲，陆启玉. 抗Gly m Bd 28K单克隆抗体的制备及其免疫学特性[J]. 浙江农业学报, 2016, 28(5): 748-.
[4]	谢星星;李祥瑞;;李银;;赵冬敏;黄欣梅;韩凯凯;刘宇卓;游园. 检测坦布苏病毒病抗体NS1\\|ELISA方法的建立与初步应用[J]. , 2014, 26(1): 0-140.
[5]	杨少艳;于可响;王华;史玉颖;马秀丽;李建亮;崔言顺;*. 鸭坦布苏病毒E基因的克隆表达及初步应用[J]. , 2012, 24(5): 0-786.
[6]	郑红英;鲁宇文;沈嘉乐;林林;陈炯;陈剑平*. 侵染水仙的虎眼万年青花叶病毒外壳蛋白的原核表达、抗血清制备及应用[J]. , 2009, 21(3): 0-201.