猪δ冠状病毒S1-CTD的截短表达及间接ELISA抗体方法的建立

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

摘要/Abstract

摘要：

猪δ冠状病毒（Porcine deltacoronavirus,PDCoV）是一种能引起仔猪呕吐、腹泻和脱水的新的猪肠道冠状病毒,表达了PDCoV的S基因抗原表位区（S1-CTD）并建立了检测抗体的间接ELISA方法。根据S基因序列设计引物,扩增含中和抗原表位的S1-CTD区（位于S基因的832-1 848 bp）,构建原核表达载体pET28a-S1-CTD,表达的重组S1-CTD蛋白经SDS-PAGE鉴定大小约41 kD,Western Blot证明其有良好的反应原性。以重组S1-CTD 蛋白为抗原建立间接ELISA,抗原最佳包被浓度为1μg/孔,待检血清的最佳稀释度为1∶50,阴阳性临界值为OD_450nm≥0.377。该ELISA检测其他8种常见猪病阳性血清无交叉反应,特异性好;批内和批间变异系数均小于10%,重复性好。ELISA与病毒中和试验的总符合率达83.3%。用建立的ELISA检测了2012-2017年四川部分猪场490份临床血清,结果显示血清中PDCoV抗体阳性率为49.18%。

关键词: 猪丁型冠状病毒, S1-CTD蛋白, 原核表达, 间接ELISA

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

瞿欢, 李成, 陈汭, 廖艺杰, 曹三杰, 文翼平, 颜其贵, 黄小波. 猪δ冠状病毒S1-CTD的截短表达及间接ELISA抗体方法的建立[J]. 生物技术通报, 2021, 37(5): 273-280.

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.

图/表 8

图1 pET28a-S1-CTD构建流程图

Fig.1 Construction process of pET28a-S1-CTD

图2 重组质粒pET28a-S1CTD的PCR（A）及双酶切鉴定（B） A：重组质粒pET28a-S1CTD的PCR鉴定（M：DL2000 DNA相对分子质量标准;1：目标基因;N：阴性对照;P：阳性对照）;B：重组质粒pET28a-S1CTD的双酶切鉴定（M：DL5000 DNA相对分子质量标准;1：未酶切的重组质粒;2：双酶切的重组质粒）

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)

图3 重组蛋白S1-CTD的SDS-PAGE与Western blot分析 A：重组蛋白S1-CTD的SDS-PAGE分析（M：蛋白质相对分子质量标准;1：BL21-pET28a诱导后;2：pET28a-S1-CTD诱导前;3：pET28a-S1-CTD诱导后;4：诱导后超声破碎全菌;5：超声破碎后上清液;6：超声破碎后包涵体）;B：S1-CTD蛋白的纯化（M：蛋白质相对分子质量标准;1：溶解前包涵体蛋白;2：溶解后包涵体蛋白;3：50mmol/L咪唑洗脱;4：150 mmol/L咪唑洗脱;5：250mmol/L咪唑洗脱）;C：重组蛋白的Western blot分析（M：蛋白质相对分子质量标准;1-4：BL21-pET28a空载、pET28a-S1-CTD、pET-28a-E、pET-28a-N蛋白分别与猪抗PDCoV阳性血清反应;5：pET28a-S1-CTD 与猪抗PDCoV 阴性血清反应）

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)

图4 临界值的测定

Fig.4 Determination of the cut-off value

图5 间接ELISA的特异性试验

Fig.5 Specificity of the indirect ELISA

表1 间接ELISA的重复性试验

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

表2 间接ELISA与VNT对血清样品的比较性检测

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%

表3 PDCoV临床样品检测结果

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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