猪嵴病毒结构蛋白VP0与VP1原核表达及间接ELISA方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2021-1550

摘要/Abstract

摘要：

分别建立基于猪嵴病毒（PKV）结构蛋白VP0与VP1的间接ELISA检测方法。对PKV结构蛋白VP0与VP1的基因进行合成并连接至原核表达载体pET-32a后，转入感受态细胞BL21中，用IPTG诱导表达的重组蛋白经Ni柱纯化后，分别以重组蛋白pET-32a-VP0与pET-32a-VP1作为包被抗原，采用棋盘滴定法建立两种间接ELISA检测方法，并进行重复性、敏感性、特异性实验和临床检测。结果显示重组蛋白pET-32a-VP0与pET-32a-VP1抗原的最佳包被浓度分别为2 mg/mL和2.5 mg/mL，反应条件均为37℃、1 h；封闭液最佳条件为5%脱脂乳，37℃、2 h；血清最佳孵育条件为1∶200，37℃、1 h；二抗最佳孵育条件分别为1∶20 000和1∶15 000，37℃、1 h；最佳显色时间为10 min，两种间接ELISA检测方法临界值分别为0.306和0.277。试验结果表明本研究成功建立了能够有效检测PKV血清特异性抗体的间接ELISA方法，且方法具有重复性好、敏感性高、特异性强、稳定性好等特点，对今后PKV的临床诊断及抗体检测试剂盒的开发奠定了重要基础。

关键词: 猪嵴病毒, VP0, VP1, 原核表达, 间接ELISA

Abstract:

In this study，the indirect-ELISA method was established based on the structural proteins VP0 and VP1 of porcine Kobuvirus（PKV）. The genes of structural proteins VP0 and VP1 of PKV were synthesized and ligated into the prokaryotic expression vector pET-32a，and then transferred into competent cells BL21. The expressions of two proteins were induced by IPTG and then were purified by Ni column. The two indirect ELISA assays were established while recombinant protein pET-32a-VP0 and pET-32a-VP as coating antigen through a checkerboard titration method for repeatability，sensitivity，specificity for experiments，and clinical testing. The results showed that the optimal coating conditions of recombinant protein pET-32a-VP0 and pET-32a-VP1 antigen were 2.0 mg/mL and 2.5 mg/mL at 37℃ for 1 h，respectively. The optimal conditions for the blocking solution were 5% skimmed milk，37℃，2 h；the optimal incubation conditions for the serum were 1∶200，37℃，1 h；the optimal incubation conditions for the secondary antibody were 1∶20 000 and 1∶15 000，37℃，1 h；optimal colour development time was 10 min. The critical values of the two indirect-ELISA assays were 0.306 and 0.277 respectively. The results of experiment indicated that the indirect-ELISA methods were successfully established，which are accurate and efficient for detecting serum specific antibody of PKV. The methods are characterized as good reproducibility，sensitivity，specificity and stability. It is very helpful for future clinical diagnosis and the development of antibody detection kits of PKV.

Key words: porcine Kobuvirus, VP0, VP1, prokaryotic expression, indirect-ELISA

沈俊强, 张莉萍, 于瑞明, 王永录, 潘丽, 刘霞, 刘新生. 猪嵴病毒结构蛋白VP0与VP1原核表达及间接ELISA方法的建立[J]. 生物技术通报, 2022, 38(10): 243-253.

SHEN Jun-qiang, ZHANG Li-ping, YU Rui-ming, WANG Yong-lu, PAN Li, LIU Xia, LIU Xin-sheng. Porcine Kobuvirus Structural Proteins VP0 and VP1 Prokaryotic Expression and Establishment of Indirect ELISA Method[J]. Biotechnology Bulletin, 2022, 38(10): 243-253.

图/表 11

参考文献 31

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血清稀释度Serum dilution	PN值 PN value	抗原包被浓度Antigen encapsulation concentration/（mg·mL^-1）
血清稀释度Serum dilution	PN值 PN value	5	2.5	2	1	0.5	0.25	0.1
1∶100	N	0.594	0.418	0.353	0.279	0.148	0.240	0.163
	P	2.477	2.234	2.167	2.234	2.273	2.265	1.991
	P/N	4.170	5.344	6.136	8.001	15.368	9.456	12.185
1∶150	N	0.466	0.244	0.305	0.237	0.178	0.155	0.126
	P	2.307	2.080	2.049	1.956	2.039	1.964	1.883
	P/N	4.953	8.531	6.728	8.267	11.483	12.636	14.910
1∶200	N	0.374	0.230	0.194	0.179	0.169	0.164	0.132
	P	2.212	2.141	2.040	1.896	1.960	1.930	1.699
	P/N	5.907	8.547	10.515	10.614	11.616	11.791	12.852
1∶300	N	0.242	0.205	0.257	0.211	0.132	0.126	0.101
	P	2.144	1.856	1.669	1.609	1.732	1.604	1.212
	P/N	8.868	9.050	6.504	7.644	13.134	12.746	12.019
1∶400	N	0.191	0.133	0.188	0.134	0.120	0.107	0.090
	P	2.023	1.640	1.521	1.401	1.545	1.249	1.075
	P/N	10.574	12.321	8.075	10.497	12.929	11.706	11.976
1∶500	N	0.144	0.105	0.107	0.111	0.099	0.096	0.079
	P	2.111	1.708	1.591	1.367	1.337	1.148	0.791
	P/N	14.642	16.249	14.824	12.318	13.474	11.906	9.982

血清稀释度Serum dilution	PN值 PN value	抗原包被浓度Antigen encapsulation concentration/（mg·mL^-1）
血清稀释度Serum dilution	PN值 PN value	5	2.5	2	1	0.5	0.25	0.1
1∶100	N	0.594	0.418	0.353	0.279	0.148	0.240	0.163
	P	2.477	2.234	2.167	2.234	2.273	2.265	1.991
	P/N	4.170	5.344	6.136	8.001	15.368	9.456	12.185
1∶150	N	0.466	0.244	0.305	0.237	0.178	0.155	0.126
	P	2.307	2.080	2.049	1.956	2.039	1.964	1.883
	P/N	4.953	8.531	6.728	8.267	11.483	12.636	14.910
1∶200	N	0.374	0.230	0.194	0.179	0.169	0.164	0.132
	P	2.212	2.141	2.040	1.896	1.960	1.930	1.699
	P/N	5.907	8.547	10.515	10.614	11.616	11.791	12.852
1∶300	N	0.242	0.205	0.257	0.211	0.132	0.126	0.101
	P	2.144	1.856	1.669	1.609	1.732	1.604	1.212
	P/N	8.868	9.050	6.504	7.644	13.134	12.746	12.019
1∶400	N	0.191	0.133	0.188	0.134	0.120	0.107	0.090
	P	2.023	1.640	1.521	1.401	1.545	1.249	1.075
	P/N	10.574	12.321	8.075	10.497	12.929	11.706	11.976
1∶500	N	0.144	0.105	0.107	0.111	0.099	0.096	0.079
	P	2.111	1.708	1.591	1.367	1.337	1.148	0.791
	P/N	14.642	16.249	14.824	12.318	13.474	11.906	9.982

血清稀释度Serum dilution	PN值 PN value	抗原包被浓度Antigen encapsulation concentration/（mg·mL^-1）
血清稀释度Serum dilution	PN值 PN value	5	2.5	2	1	0.5	0.25	0.1
1∶100	N	0.348	0.299	0.288	0.220	0.142	0.146	0.106
	P	2.309	2.238	2.234	2.047	2.109	1.904	1.798
	P/N	6.634	7.497	7.751	9.291	14.897	13.012	16.946
1∶150	N	0.258	0.354	0.209	0.160	0.134	0.136	0.118
	P	2.246	2.153	2.050	1.849	1.937	1.516	1.543
	P/N	8.706	6.082	9.799	11.527	14.414	11.169	13.105
1∶200	N	0.230	0.199	0.188	0.164	0.141	0.164	0.104
	P	2.221	2.087	1.986	1.719	1.716	1.426	1.328
	P/N	9.649	10.503	10.562	10.479	12.216	8.672	12.769
1∶300	N	0.159	0.174	0.136	0.112	0.136	0.197	0.256
	P	2.134	2.127	2.169	1.655	1.465	1.154	1.089
	P/N	13.461	12.239	15.898	14.832	10.807	5.864	4.260
1∶400	N	0.137	0.151	0.139	0.098	0.094	0.090	0.090
	P	1.970	1.890	1.632	1.312	1.304	1.155	1.021
	P/N	14.380	12.517	11.741	13.388	13.872	12.833	11.344
1∶500	N	0.120	0.165	0.131	0.092	0.081	0.085	0.074
	P	1.971	1.846	1.588	1.225	1.197	1.107	1.014
	P/N	0.419	4.099	6.638	14.336	20.273	21.851	26.822

血清稀释度Serum dilution	PN值 PN value	抗原包被浓度Antigen encapsulation concentration/（mg·mL^-1）
血清稀释度Serum dilution	PN值 PN value	5	2.5	2	1	0.5	0.25	0.1
1∶100	N	0.348	0.299	0.288	0.220	0.142	0.146	0.106
	P	2.309	2.238	2.234	2.047	2.109	1.904	1.798
	P/N	6.634	7.497	7.751	9.291	14.897	13.012	16.946
1∶150	N	0.258	0.354	0.209	0.160	0.134	0.136	0.118
	P	2.246	2.153	2.050	1.849	1.937	1.516	1.543
	P/N	8.706	6.082	9.799	11.527	14.414	11.169	13.105
1∶200	N	0.230	0.199	0.188	0.164	0.141	0.164	0.104
	P	2.221	2.087	1.986	1.719	1.716	1.426	1.328
	P/N	9.649	10.503	10.562	10.479	12.216	8.672	12.769
1∶300	N	0.159	0.174	0.136	0.112	0.136	0.197	0.256
	P	2.134	2.127	2.169	1.655	1.465	1.154	1.089
	P/N	13.461	12.239	15.898	14.832	10.807	5.864	4.260
1∶400	N	0.137	0.151	0.139	0.098	0.094	0.090	0.090
	P	1.970	1.890	1.632	1.312	1.304	1.155	1.021
	P/N	14.380	12.517	11.741	13.388	13.872	12.833	11.344
1∶500	N	0.120	0.165	0.131	0.092	0.081	0.085	0.074
	P	1.971	1.846	1.588	1.225	1.197	1.107	1.014
	P/N	0.419	4.099	6.638	14.336	20.273	21.851	26.822

优化条件 Optimization condition	反应条件优化Optimization of reaction conditions
优化条件 Optimization condition	抗原包被 Antigen-coated	封闭液 Sealing fluid	一抗孵育 Primary antibody incubation	二抗孵育 Secondary antibody incubation	底物显色 Substrate chromogenesis
最佳稀释度 Optimal dilution	2 mg/mL 2.5 mg/mL	5%脱脂乳 5% Skimmed milk powder	1∶200	1∶20 000 1∶15 000
反应条件 Reaction condition	37℃，1 h	37℃，2 h	37℃，1 h	37℃，1 h	避光10 min Avoid light 10 min