Truncated Expression of the S1-CTD Fragment of Porcine Deltacoronavirus and Establishment of an Indirect ELISA for Detecting Its Antibody

doi:10.13560/j.cnki.biotech.bull.1985.2020-1181

Abstract

Abstract:

Porcine deltacoronavirus（PDCoV）is a novel porcine intestinal coronavirus that causes vomiting,diarrhea and dehydration of piglets. The epitope region（S1-CTD）expressing S gene of PDCoV was studied,and an indirect ELISA for the detection of PDCoV antibody was established. The primer was designed according to the S gene sequence,the S gene fragment containing epitope region（S1-CTD）（located at 832-1 848 bp in S gene）was amplified and inserted into pET28a,and the recombinant plasmid pET28a-S1-CTD was constructed. The expressed recombinant S1-CTD protein was about 41 kD by SDS-PAGE and good reactogenicity was proved by Western Blot. The indirect ELISA was developed by using the recombinant S1-CTD protein as coating antigen,and the optimal conditions were as follows：the coating concentration of S1-CTD protein was 1 μg/well,the dilution concentration of serum was 1∶50,the detection positive threshold was OD_450nm≥0.377. The ELISA was very specific,because there was no cross reaction with positive sera of other eight common swine diseases. The method was of high duplicability with the < 10% variation of intra- and inter-batch coefficients. The coincidence rate between ELISA and virus neutralization test（VNT）was 83.3%. A total of 490 clinical swine sera samples collected from Sichuan province from 2012 to 2017 were detected by the ELISA,and the PDCoV-antibody positive rate was 49.18%.

Key words: porcine deltacoronavirus, S1-CTD protein, prokaryotic expression, indirect ELISA

QU Huan, LI Cheng, CHEN Rui, LIAO Yi-jie, CAO San-jie, WEN Yi-ping, YAN Qi-gui, HUANG Xiao-bo. Truncated Expression of the S1-CTD Fragment of Porcine Deltacoronavirus and Establishment of an Indirect ELISA for Detecting Its Antibody[J]. Biotechnology Bulletin, 2021, 37(5): 273-280.

Figures/Tables 8

Fig.1 Construction process of pET28a-S1-CTD

Fig. 2 Identification of recombinant plasmid pET28a-S1CTD by PCR (A) and double digestion (B) A: Identification of recombinant plasmid pET28a-S1CTD by PCR (M: DL2000 DNA marker, 1: Target gene, N: Negative control, P: Positive control). B: Identification of recombinant plasmid pET28a-S1CTD by double digestion (M: DL5000 DNA marke, 1: Undigested recombinant plasmid, 2: Digested recombinant plasmid)

Fig. 3 The SDS-PAGE and Western blot analysis of recombinant S1-CTD protein A: The SDS-PAGE analysis of S1-CTD (M: Protein marker, 1: Post-induced BL21-pET28a, 2: Pre-induced pET28a-S1-CTD, 3: Post-induced pET28a-S1-CTD, 4: Hyperacoustic full bacteria after induction, 5: Hyperacoustic supernatant, 6: Hyperacoustic precipitation). B: Purification of S1-CTD protein (M: Protein marker, 1: Pre-lysing inclusion body protein, 2: Dissolved inclusion body protein, 3: Target protein eluted by 50 mmol/L imidazole, 4: Target protein eluted by 150 mmol/L imidazole, 5: Target protein eluted by 250 mmol/L imidazole). C: The western blot analysis of S1-CTD (M: Protein marker, 1-4: BL21-pET28a, pET28a-S1-CTD, pET-28a-E, pET-28a-N proteins reacted with pig anti-PDCoV positive serum, 5: pET28a-S1-CTD protein reacted with pig anti-PDCoV negative serum)

Fig.4 Determination of the cut-off value

Fig.5 Specificity of the indirect ELISA

Table 1 Repeatability of the indirect ELISA

血清编号 Serum No.	批内变异系数 Intra batch CV %		批间变异系数 Inter batch CV %
血清编号 Serum No.	±SD	变异系数 CV/%	±SD	变异系数 CV/%
1	0.136±0.011	8.07	0.190±0.017	8.85
2	0.110±0.011	9.57	0.217±0.020	9.22
3	0.127±0.004	3.48	0.122±0.012	9.98
4	0.124±0.005	4.35	0.155±0.013	8.29
5	0.113±0.003	2.65	0.909±0.055	6.01
6	1.251±0.043	3.47	1.022±0.079	7.75
7	0.946±0.032	3.43	0.928±0.085	9.12

Table 2 Comparative detection of serum samples by indirect ELISA and VNT

检测方法 Detection method		VNT
检测方法 Detection method		阳性数 Positive number	阴性数 Negative number	合计 Total
	阳性数 Positive number	9	3	12
	阴性数 Negative number	2	16	18
间接ELISA	合计 Total	11	19	30
间接ELISA	阳性符合率 Positive coincidence rate	81.8%
Indirect ELISA	阴性符合率 Negative coincidence rate		84.2%
	总符合率 Total coincidence rate			83.3%

Table 3 Clinical detection of PDCoV samples

来源 Origin	样本数 Sample number	阳性总数 Positive number	阳性率 Positive rate/%
成都 Chengdu	52	34	65.38
南充 Nanchong	57	26	45.61
绵阳 Mianyang	30	12	40
眉山 Meishan	33	3	9.09
遂宁 Suining	16	0	0
德阳 Deyang	61	24	39.34
达州 Dazhou	27	27	100
泸州 Luzhou	9	8	88.89
宜宾 Yibin	1	1	100
阿坝 Aba	23	22	95.65
巴中 Bazhong	90	84	93.33
凉山 Liangshan	4	0	0
资阳 Ziyang	1	0	0
雅安 Ya’an	56	0	0
内江 Neijiang	12	0	0
乐山 Leshan	18	0	0
总数 Total	490	241	49.18

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