猪轮状病毒VP6蛋白截短表达及间接ELISA抗体检测方法的建立

doi:10.16431/j.cnki.1671-7236.2025.03.025

Abstract

Abstract: 【Objective】 The purpose of this experiment was to establish a prokaryotic expression system of Porcine rotavirus (PoRV) VP6 truncated protein,and establish an indirect ELISA method to detect PoRV antibody,so as to provide a means for serological monitoring of PoRV.【Method】 A pair of specific primers for truncated VP6 gene was designed to amplify VP6 truncated gene using pMD19-T-VP6 recombinant plasmid as template.The recombinant plasmid pET32a-PoRV-VP6 was constructed using pET-32a(+) prokaryotic expression vector.Using Escherichia coli Rosetta(DE3) competent cells as host bacteria,the recombinant plasmid was induced to express and the expression conditions were optimized.pET32a-PoRV-VP6 recombinant protein was purified by His labeled nickel column and used as coated antigen.Single variable method was used to optimize the working conditions such as antigen coated concentration,serum dilution,secondary antibody dilution and incubation time of primary antibody and secondary antibody.The critical value of negative and positive of indirect ELISA method was determined,and its specificity,sensitivity,repeatability and compliance were tested.384 clinical pig serum samples collected from different pig farms in Guizhou during 2021-2024 were tested.【Result】 The recombinant expression vector pET32a-PoRV-VP6 was successfully constructed,and the prokaryotic truncated expression of VP6 protein was realized.The molecular weight of the protein was about 40 ku,and it had good reactivity.The optimal concentration of VP6 coated antigen was 2 μg/mL,the dilution of positive serum was 1∶100,the incubation time of serum samples and the second antibody was 60 min,the TMB reaction time was 10 min,and the negative and positive critical value (D_{450 nm}) was 0.410.The established indirect ELISA method did not react with Porcine epidemic diarrhea virus (PEDV),Porcine coronavirus (PDCoV),Porcine transmissible gastroenteritis virus (TGEV) and other standard positive sera,and had strong specificity.The variation coefficients of both intra-batch and inter-batch replicate tests were both ＜10%,and the repeatability was good.The overall coincidence rate with ELISA antibody detection kit of PoRV A was 96.80%.The total positive rate of 384 clinical pig serum samples detected by indirect ELISA was 28.91%.【Conclusion】 In this study,truncated expression of PoRV VP6 protein was successfully achieved,and an indirect ELISA method for PoRV antibody detection was established,which was suitable for clinical PoRV antibody detection and vaccine immune effect evaluation.

Key words: Porcine rotavirus (PoRV); VP6 protein; truncated expression; indirect ELISA

CLC Number:

S852.65

PAN Xiangying, ZENG Zhiyong, LIANG Haiying, TANG Deyuan, WANG Bin, YE Ni, TIAN Hongli, BIAN Mengting, LIU Jiajia, HUANG Shu. Truncated Expression of Porcine Rotavirus VP6 Protein and Establishment of an Indirect ELISA Antibody Detection Method[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(3): 1231-1240.

References

[1] VANCOTT J L，FRANCO M A，GREENBERG H B，et al.Protective immunity to Rotavirus shedding in the absence of interleukin-6：Th1 cells and immunoglobulin A develop normally[J].Journal of Virology，2000，74(11):5250-5256.
[2] 李妍花，周兵强，康笃利，等.猪轮状病毒胶体金检测试纸条的研制及应用[J].中国畜牧兽医，2024，51(3)：1223-1231. LI Y H,ZHOU B Q,KANG D L,et al.Development and application of colloidal gold test strip against Porcine rotavirus[J].China Animal Husbandry & Veterinary Medicine,2024,51(3):1223-1231.(in Chinese)
[3] CHENG X，WU W，TENG F，et al.Isolation and characterization of Bovine RVA from Northeast China，2017-2020[J].Life (Basel,Switzerland)，2021，11(12):1389.
[4] XUE R，TIAN Y，ZHANG Y，et al.Diversity of group A Rotavirus of Porcine rotavirus in Shandong province China[J].Acta Virologica，2018，62(3):229-234.
[5] LI C，LU H，GENG C，et al.Epidemic and evolutionary characteristics of Swine enteric viruses in South-Central China from 2018 to 2021[J].Viruses，2022，14(7):1420.
[6] AMIMO J O，RAEV S A，CHEPNGENO J，et al.Rotavirus interactions with host intestinal epithelial cells[J].Frontiers in Immunology，2021，12:793841.
[7] DIAN Z，SUN Y，ZHANG G，et al.Rotavirus-related systemic diseases：Clinical manifestation，evidence and pathogenesis[J].Critical Reviews in Microbiology，2021，47(5):580-595.
[8] 刘小兰，刘昌锦，余文洋，等.猪轮状病毒江西株AY01的分离鉴定[J].中国畜牧兽医，2022，49(8)：3151-3162. LIU X L,LIU C J,YU W Y,et al.Isolation and identification of Porcine rotavirus Jiangxi strain AY01[J].China Animal Husbandry & Veterinary Medicine,2022,49(8):3151-3162.(in Chinese)
[9] 龙飞翔，施开创，张珍，等.PEDV、TGEV和PRoV多重RT-PCR检测方法的建立及应用[J].中国畜牧兽医，2016，43(10)：2518-2526. LONG F X,SHI K C,ZHANG Z,et al.Establishment and application of a multiplex RT-PCR assay for detection of PEDV,TGEV and PRoV[J]. China Animal Husbandry & Veterinary Medicine, 2016,43(10):2518-2526.(in Chinese)
[10] MEMON A M，CHEN F，KHAN S B，et al.Development and evaluation of polyclonal antibodies based antigen capture ELISA for detection of Porcine rotavirus[J]. Animal Biotechnology，2023，34(5):1807-1814.
[11] NAGESHA H S，HOLMES I H.Direct serotyping of Porcine rotaviruses using VP7-specific monoclonal antibodies by an enzyme immunoassay[J].Journal of Medical Virology，1991，35(3):206-211.
[12] 崔蕾.猪轮状病毒抗体间接ELISA与分型qRT-PCR检测方法的建立[D].武汉：华中农业大学，2023. CUI L.Development of antibody indirect ELISA and genotyping qRT-PCR detection method for Pocine rotavirus[D].Wuhan：Huazhong Agricultural University,2023.(in Chinese)
[13] ZHU X，FAN B，SONG S，et al.Transcriptomic and antiviral analyses of PoIFN-delta5 against Porcine enteric viruses in porcine intestinal epithelial cells[J].Veterinary Microbiology，2023，280:109718.
[14] NYAGA M M，ESONA M D，JERE K C，et al.Genetic diversity of Rotavirus genome segment 6(encoding VP6) in Pretoria，South Africa[J].Springerplus，2014，3:179.
[15] ZHOU X，WANG Y H，GHOSH S，et al.Genomic characterization of G3P
[6]，G4P
[6] and G4P
[8] Human rotaviruses from Wuhan，China：Evidence for interspecies transmission and reassortment events[J].Infection Genetics and Evolution，2015，33:55-71.
[16] TAO R，CHANG X，ZHOU J，et al.Molecular epidemiological investigation of group A Porcine rotavirus in East China[J].Frontiers in Veterinary Science，2023，10:1138419.
[17] CHOI A H，BASU M，MCNEAL M M，et al.Intranasal administration of an Escherichia coli-expressed codon-optimized Rotavirus VP6 protein induces protection in mice[J].Protein Expression and Purification，2004，38(2):205-216.
[18] TONG D W，SONG J D，WANG J W，et al.Improved expression of Human rotavirus VP6 antigen in the recombinant Adenoviruse by codon optimization[J].Chinese Journal of Experimental and Clinical Virology，2011，25(6):420-422.
[19] SHOJA Z，JALILVAND S，LATIFI T，et al.Rotavirus VP6：Involvement in immunogenicity，adjuvant activity，and use as a vector for heterologous peptides，drug delivery，and production of nano-biomaterials[J].Archives of Virology，2022，167(4):1013-1023.
[20] 陈淑华，库旭钢，闫贵伟，等.猪A群轮状病毒VP6的表达及IgG抗体ELISA检测方法的建立与初步应用[J].动物医学进展，2014，35(7)：1-6. CHEN S H,KU X K,YAN G W,et al.Expression of VP6 and development of an ELISA for detecction of IgG antibodies against Porcine rotavirus group A[J].Progress in Veterinary Medicine,2014,35(7):1-6.(in Chinese)
[21] ZHU J，YANG Q，CAO L，et al.Development of Porcine rotavirus VP6 protein based ELISA for differentiation of this virus and other viruses[J].Virology Journal，2013，10:91.
[22] 王奕斐，洪大林，米雪，等.猪轮状病毒VP4、VP6蛋白原核表达及多克隆抗体的制备[J].中国兽医学报，2022，42(11)：2171-2180. WANG Y W,HONG D L,MI X,et al.Prokaryotic expression of VP4 and VP6 proteins of Porcine rotavirus and preparation of polyclonal antibodies[J].Chinese Journal of Veterinary Science,2022,42(11):2171-2180.(in Chinese)
[23] 田广原,王宇琛,周雅坪,等.绵羊肺炎支原体P60_58aa-928aa蛋白的原核表达及间接ELISA方法的建立[J].中国畜牧兽医,2022,49(5)：1960-1969. TIAN G Y,WANG Y C,ZHOU Y P,et al.Prokaryotic expression and indirect ELISA method establishment of the P60_58aa-928aa protein of Mycoplasma ovipneumoniae[J].China Animal Husbandry & Veterinary Medicine,2022,49(5):1960-1969.(in Chinese)

Truncated Expression of Porcine Rotavirus VP6 Protein and Establishment of an Indirect ELISA Antibody Detection Method

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	ZHANG Pian, CHEN Jing, ZHANG Xiaoxiao, MAI Xiaopeng, TANG Ke, XIANG Hua, WANG Gang, LUO Shengjun, MA Huihai, YUAN Ziguo, WANG Xiaohu. Expression of GRA1 Protein of Toxoplasma gondii and Establishment of an Indirect ELISA Detection Method [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3308-3320.
[2]	WEI Huangsiwu, ZHANG Xingyi, HUANG Xiaohua, LIU Changjin, WU Wenjie, SHEN Zhengqiao, LUO Feng, DENG Shunzhou. Eukaryotic Expression of Porcine Rotavirus VP6 Protein and Preparation and Application of Its Monoclonal Antibody [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2750-2761.
[3]	OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHAI Jiajia, ZHANG Jie. Truncated Expression of Cap Protein of Porcine Circovirus Type 3 and Establishment of Indirect ELISA Method for Its Antibody [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(7): 3056-3066.
[4]	GAO Le, SI Duoduo, GUO Lei, CHEN Can, WANG Wei, WANG Jianlin, WANG Lingling, LI Jidong. Prokaryotic Expression of GA Module-containing Protein in Mycoplasma synoviae and Establishment of Indirect ELISA Method [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(6): 2621-2632.
[5]	LI Yanhua, ZHOU Bingqiang, KANG Duli, HAO Liying, WANG Zhiyan, ZHAO Shaoruo, HUANG Tian, PANG Xinghao, LIU Junyang, CHU Yuanyuan, ZHANG Yunjing, GAO Xiaojing, DENG Junhua, TIAN Kegong. Development and Application of Colloidal Gold Test Strip Against Porcine Rotavirus [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(3): 1223-1231.
[6]	REN Jianle, LIN Yiting, JI Kang, TAN Shanshan, CHEN Xinxin, JIN Yi, WANG Ying, NIU Sheng, LIANG Libin, LI Junping, ZHAO Yujun, TIAN Wenxia. Preparation of Polyclonal Antibody and Development of an Indirect ELISA Method for Senecavirus A VP2 Protein [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(2): 678-688.
[7]	ZHANG Yichen, SUN Mingjun, DENG Changshun, DING Jiabo, XU Baoliang, CHEN Lei, QUAN Chunlan, ZHAO Kang, FAN Xuezheng, QIN Tong. Development and Evaluation of an Indirect ELISA Based on Recombinant E2 Protein for Detection of Bovine Viral Diarrhea Virus Antibody [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(12): 5459-5469.
[8]	XU Haojun, LIU Ying, MEI Chen, XU Tong, LI Kai, WANG Hongjun. Development of an Indirect ELISA for Antibody Detection Against Ornithobacterium rhinotracheale Based on Outer Membrane Proteins [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(10): 4420-4428.
[9]	WANG Cheng, XIA Yingju, WANG Haidong, LIU Yebing. Prokaryotic Expression of P54 Protein of African Swine Fever Virus and Establishment of an Indirect ELISA Detection Method [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(10): 4440-4449.
[10]	OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHANG Jie. Truncated Expression of ORF2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody [J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(6): 2487-2495.
[11]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(5): 2012-2022.
[12]	NIU Xiaojie, XU Mingguo, XIAO Li, LIU Wenhao, CHEN Chuangfu, WANG Yong. Expression and Purification of IPAJ Protein of Salmonella Pullorum, Preparation of Polyclonal Antibody and Establishment of ELISA Method [J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(3): 1218-1228.
[13]	ZHANG Yan, ZHAN Liyuan, JIANG Fujie, MA Hongxia, KONG Lingcong. Prokaryotic Expression, Polyclonal Antibody Production and Development of an Indirect ELISA Antibody Detection Method of Recombinant Protein P48 of Mycoplasma bovis [J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(11): 4621-4631.
[14]	ZHANG Kai, ZHANG Chunping, GE Shengqiang, SUN Chunxi, CHENG Jie, FU Chunyu, WANG Gang, NIU Xing, PENG Jun. Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method [J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(1): 270-279.
[15]	HAN Rui, YANG Xing, ZHANG Wenbo, CHEN Jialing, ZHOU Xiaoli. Prokaryotic Expression of Autolysin from Streptococcus suis and Establishment of an Indirect ELISA Method for Detection of Its Antibody [J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(9): 3508-3519.