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 and Veterinary Medicine, 2025, 52(3): 1231-1240.

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

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