Porcine Kobuvirus Structural Proteins VP0 and VP1 Prokaryotic Expression and Establishment of Indirect ELISA Method

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

Abstract

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

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.

Figures/Tables 11

References 31

[1]	Reuter G, Boldizsár A, Kiss I, et al. Candidate new species of kobuvirus in porcine hosts[J]. Emerg Infect Dis, 2008, 14(12):1968-1970. doi: 10.3201/eid1412.080797 pmid: 19046542
[2]	Yu JM, Jin M, Zhang Q, et al. Candidate porcine kobuvirus, China[J]. Emerg Infect Dis, 2009, 15(5):823-825. doi: 10.3201/eid1505.081518 URL
[3]	Khamrin P, Maneekarn N, Hidaka S, et al. Molecular detection of kobuvirus sequences in stool samples collected from healthy pigs in Japan[J]. Infect Genet Evol, 2010, 10(7):950-954. doi: 10.1016/j.meegid.2010.06.001 pmid: 20547246
[4]	Park SJ, Kim HK, Moon HJ, et al. Molecular detection of porcine kobuviruses in pigs in Korea and their association with diarrhea[J]. Arch Virol, 2010, 155(11):1803-1811. doi: 10.1007/s00705-010-0774-1 URL
[5]	Sisay Z, Wang QH, Oka T, et al. Prevalence and molecular characterization of porcine enteric caliciviruses and first detection of porcine kobuviruses in US swine[J]. Arch Virol, 2013, 158(7):1583-1588. doi: 10.1007/s00705-013-1619-5 pmid: 23456421
[6]	Ribeiro J, Arruda Leme R, Alfieri AF, et al. High frequency of Aichivirus C(porcine kobuvirus)infection in piglets from different geographic regions of Brazil[J]. Trop Animal Heal Prod, 2013, 45(8):1757-1762. doi: 10.1007/s11250-013-0428-x URL
[7]	孟丽, 陶洁, 李本强, 等. 猪7种腹泻相关病毒的临床检测及猪嵴病毒的遗传进化分析[J]. 生物工程学报, 2017, 33(8):1292-1303.
	Meng L, Tao J, Li BQ, et al. Clinical detection of seven porcine diarrhea-associated viruses and evolution analysis of porcine kobuvirus[J]. Chin J Biotechnol, 2017, 33(8):1292-1303.
[8]	Yang F, Liu XW, Zhou YC, et al. Histopathology of porcine Kobuvirus in Chinese piglets[J]. Virol Sin, 2015, 30(5):396-399. doi: 10.1007/s12250-015-3608-1 pmid: 26475611
[9]	Theuns S, Vanmechelen B, Bernaert Q, et al. Nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus[J]. Sci Rep, 2018, 8(1):9830. doi: 10.1038/s41598-018-28180-9 pmid: 29959349
[10]	张莎. 猪嵴病毒流行病学调查及CH/HZ/2011株全基因序列分析[D]. 哈尔滨: 东北农业大学, 2013.
	Zhang S. Epidemiological investigation of porcine kobuvirus and genome sequence analysis of CH/HZ/2011Strain[D]. Harbin: Northeast Agricultural University, 2013.
[11]	胡军勇, 汤细彪, 胡睿铭, 等. 猪库布病毒RT-PCR检测方法的建立及湖北省流行病学初步调查[J]. 华中农业大学学报, 2012, 31(4):485-489.
	Hu JY, Tang XB, Hu RM, et al. Porcine kobuvirus RT-PCR detection assay establishment and application in primary epidemiology investigation in Hubei Province[J]. J Huazhong Agric Univ, 2012, 31(4):485-489.
[12]	王玮, 刘海源, 李鹏飞, 等. 2014年-2016年山东省猪嵴病毒流行病学调查[J]. 中国预防兽医学报, 2018, 40(8):670-674.
	Wang W, Liu HY, Li PF, et al. Epidemiological investigation of porcine Kobuvirus in Shandong Province from 2014 to 2016[J]. Chin J Prev Vet Med, 2018, 40(8):670-674.
[13]	沈素芳, 汤赛冬, 孙泉云. 猪Kobu病毒感染概况[J]. 动物医学进展, 2012, 33(1):115-116.
	Shen SF, Tang SD, Sun QY. Introduction to porcine Kobuvirus infection[J]. Prog Vet Med, 2012, 33(1):115-116.
[14]	Yamashita T, Kobayashi S, Sakac K, et al. Isolation of cytopathic small round viruses with BS-C-l cells from patients with gastroenteritis[J]. J Infect Dis, 1991, 164(5):954-957. pmid: 1658159
[15]	Yamashita T, Ito M, Kabashima Y, et al. Isolation and characterization of a new species of Kobuvirus associated with cattle[J]. J Gen Virol, 2003, 84(Pt 11):3069-3077. doi: 10.1099/vir.0.19266-0 pmid: 14573811
[16]	Reuter G, Boldizsár A, Pankovics P. Complete nucleotide and amino acid sequences and genetic organization of porcine Kobuvirus, a member of a new species in the genus Kobuvirus, family Picornaviridae[J]. Arch Virol, 2009, 154(1):101-108. doi: 10.1007/s00705-008-0288-2 pmid: 19096904
[17]	Sasaki J, Kusuhara Y, Maeno Y, et al. Construction of an infectious cDNA clone of Aichi virus(a new member of the family Picornaviridae)and mutational analysis of a stem-loop structure at the 5' end of the genome[J]. J Virol, 2001, 75(17):8021-8030. pmid: 11483747
[18]	Wang EL, Yang B, Liu W, et al. Complete sequencing and phylogenetic analysis of porcine kobuvirus in domestic pigs in Northwest China[J]. Arch Virol, 2014, 159(9):2533-2535. doi: 10.1007/s00705-014-2087-2 pmid: 24777826
[19]	Sasaki J, Nagashima S, Taniguchi K. Aichi virus leader protein is involved in viral RNA replication and encapsidation[J]. J Virol, 2003, 77(20):10799-10807. pmid: 14512530
[20]	Rossmann MG, Arnold E, Erickson JW, et al. Structure of a human common cold virus and functional relationship to other picornaviruses[J]. Nature, 1985, 317(6033):145-153. doi: 10.1038/317145a0 URL
[21]	Liu PJ, Li P, Lyu WT, et al. Epidemiological study and variation analysis of the porcine kobuvirus 3D gene in Sichuan Province, China[J]. Virol Sin, 2015, 30(6):460-463. doi: 10.1007/s12250-015-3632-1 pmid: 26637336
[22]	Okitsu S, Khamrin P, Thongprachum A, et al. Sequence analysis of porcine Kobuvirus VP1 region detected in pigs in Japan and Thailand[J]. Virus Genes, 2012, 44(2):253-257. doi: 10.1007/s11262-011-0692-7 pmid: 22109708
[23]	谢荣辉, 刘霞, 赵灵燕, 等. 猪嵴病毒荧光定量RT-PCR检测方法的建立和应用[J]. 中国兽医科学, 2018, 48(4):438-442.
	Xie RH, Liu X, Zhao LY, et al. Establishment and application of the real-time reverse transcription polymerase chain reaction for detection of porcine kobuvirus[J]. Chin Vet Sci, 2018, 48(4):438-442.
[24]	李淞, 朱玲, 周远成, 等. 猪嵴病毒RT-PCR检测方法的建立及应用[J]. 中国兽医学报, 2013, 33(8):1150-1153.
	Li S, Zhu L, Zhou YC, et al. Development and application of RT-PCR assay for detection of porcine kobuvirus[J]. Chin J Vet Sci, 2013, 33(8):1150-1153.
[25]	韩磊, 彭志豪, 刘莹, 等. 猪嵴病毒VP0基因的原核表达及间接ELISA检测方法的建立[J]. 中国动物传染病学报, 2021.1-7.
	Han L, Peng ZH, Liu Y, et al. Prokaryotic expression of porcine Kobuvirus VP0 gene and establishment of indirect elisa detection method[J]. Chin J Animal Infect Dis, 2021. 1-7.
[26]	田野. 猪嵴病毒VP1基因克隆与表达及抗体检测ELISA方法的建立[D]. 扬州: 扬州大学, 2013.
	Tian Y. Gene clone and prokaryotic expression of structural protein VP1 of porcine Kobuvirus and development of ELISA for detection of antibody to VP1[D]. Yangzhou: Yangzhou University, 2013.
[27]	祝俊鹏. 猪嵴病毒CH441株VP3基因的原核表达及间接ELISA方法的建立[D]. 兰州: 甘肃农业大学, 2015.
	Zhu JP. Prokaryotic expression of the VP3 gene of porcine kobuvirus CH441 and development of indirect ELISA[D]. Lanzhou: Gansu Agricultural University, 2015.
[28]	朱庆贺, 苗艳, 李文辉, 等. 猪嵴病毒重组VP3蛋白间接ELISA检测方法的建立[J]. 中国兽医科学, 2017, 47(9):1150-1153.
	Zhu QH, Miao Y, Li WH, et al. Establishment of an indirect ELISA based on VP3 protein for the detection of the antibodies against porcine kobuvirus[J]. Chin Vet Sci, 2017, 47(9):1150-1153.
[29]	Reuter G, Kecskeméti S, Pankovics P. Evolution of porcine kobuvirus infection, Hungary[J]. Emerg Infect Dis, 2010, 16(4):696-698. doi: 10.3201/eid1604.090937 pmid: 20350391
[30]	梁丹洁. 广西猪嵴病毒的感染状况调查与基因序列分析[D]. 南宁: 广西大学, 2014.
	Liang DJ. The epidemic situation research and genome sequence analysis of porcine kobuvirus in Guangxi Province[D]. Nanning: Guangxi University, 2014.
[31]	毛亚萍, 卞大伟. SARS-CoV-2毒株S蛋白突变及其影响的生物信息学分析[J]. 病毒学报, 2020, 36(6):1020-1027.
	Mao YP, Bian DW. Bioinformatics analysis of mutations in the spike protein of SARS-CoV-2 and their effects[J]. Chin J Virol, 2020, 36(6):1020-1027.

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