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

doi:10.3969/j.issn.1004-1524.2021.05.06

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (5): 816-824.DOI: 10.3969/j.issn.1004-1524.2021.05.06

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S858.28

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.

Figures/Tables 9

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.

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

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

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

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

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	-	-	-	-	-	-	-	-	+	-

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

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

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)

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

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

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