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

摘要/Abstract

摘要： 【目的】通过构建猪轮状病毒(Porcine rotavirus，PoRV) VP6截短蛋白(第177—382位氨基酸)的原核表达系统，建立检测PoRV抗体的间接ELISA方法，为PoRV血清学监测提供检测手段。【方法】设计1对针对截短VP6基因的特异性引物，以pMD19-T-VP6重组质粒为模板扩增VP6截短基因。利用pET-32a (+)原核表达载体构建pET32a-PoRV-VP6重组质粒。以大肠杆菌Rosetta (DE3)感受态细胞为宿主菌，对重组质粒进行诱导表达，并优化表达条件。利用His标签镍柱对pET32a-PoRV-VP6重组蛋白进行纯化，以此作为包被抗原，通过单一变量法优化抗原包被浓度、血清稀释度、二抗稀释度及一抗、二抗孵育时间等工作条件。确定间接ELISA方法的阴阳性临界值，检验其特异性、敏感性、重复性及符合性，并对2021—2024年采自贵州地区不同猪场的384份临床猪血清进行检测。【结果】试验成功构建pET32a-PoRV-VP6重组表达载体，实现VP6蛋白的原核截短表达，蛋白分子质量大小约40 ku，具有良好的反应原性。对间接ELISA方法条件进行优化，确定VP6包被抗原最佳浓度为2 μg/mL、阳性血清稀释度为1∶100、血清样品和二抗孵育时间均为60 min、TMB反应时间为10 min、阴阳性临界值(D_{450 nm})为0.410。建立的间接ELISA方法与猪流行性腹泻病毒(PEDV)、猪冠状病毒(PDCoV)、猪传染性胃肠炎病毒(TGEV)等病毒标准阳性血清不发生反应，特异性强；批内和批间重复试验变异系数均＜10%，重复性好；与PoRV A型ELISA抗体检测试剂盒的总体符合率为96.80%。间接ELISA方法检测临床384份猪血清样品结果显示，样品总阳性率为28.91%。【结论】本研究成功实现了PoRV VP6蛋白的截短表达，并建立了PoRV间接ELISA抗体检测方法，适用于临床PoRV抗体检测和疫苗免疫效果评价。

关键词: 猪轮状病毒(PoRV); VP6蛋白; 截短表达; 间接ELISA

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

中图分类号:

S852.65

潘向英, 曾智勇, 梁海英, 汤德元, 王彬, 叶泥, 田红利, 边孟婷, 柳佳佳, 黄书. 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[J]. 中国畜牧兽医, 2025, 52(3): 1231-1240.

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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