非洲猪瘟病毒p22蛋白表达、多克隆抗体制备及间接ELISA方法的建立

doi:10.16431/j.cnki.1671-7236.2023.01.027

摘要/Abstract

摘要： 【目的】试验旨在表达与纯化非洲猪瘟病毒（African swine fever virus，ASFV）的结构蛋白p22，将其作为包被抗原建立ASFV抗体的间接ELISA检测方法，用于诊断非洲猪瘟。【方法】将ASFV p22编码基因KP177R的截短体（24―145位氨基酸）克隆至原核表达载体pET-32a （+）中，将重组质粒pET-32a-p22转化大肠杆菌BL21（DE3）感受态细胞，经0.1 mmol/L IPTG诱导表达5 h，利用镍柱亲和纯化p22蛋白，并进行Western blotting鉴定。利用p22蛋白免疫BALB/c小鼠制备抗血清，并以含p22全长基因的真核表达质粒pCAGGS-EGFP-fp22转染HEK293T细胞为抗原基质，利用间接免疫荧光（IFA）鉴定抗血清的反应性。以重组p22蛋白为包被抗原，优化最佳抗原包被浓度、待检血清稀释度、封闭条件、抗原抗体反应时间、酶标二抗工作浓度等参数，建立ASFV抗体间接ELISA检测方法，并对临床猪血清样品进行检测。【结果】ASFV p22截短蛋白在大肠杆菌中高水平表达，蛋白产量为0.85 mg/100 g菌体；p22蛋白具有良好的免疫原性，小鼠血清抗体效价为1：12 800。本研究建立的ELISA方法（p22-ELISA）的最佳抗原包被浓度为0.125 μg/孔，最佳待检血清稀释度为1：800，最佳酶标二抗稀释度为1：10 000；阴阳性临界值为0.384；与CSFV、PRRSV、PCV2、PRV等抗体阳性猪血清无交叉反应，说明特异性好；批内与批间变异系数均低于10%，表明重复性好。利用p22-ELISA方法检测了263份临床猪血清样品，与商品化ASFV抗体ELISA检测试剂盒的符合率为97.7%。【结论】本研究基于原核表达的p22截短体蛋白建立了用于检测ASFV抗体的间接ELISA方法，该方法特异性强、灵敏度高、重复性好，为ASFV感染诊断和流行病学调查提供了技术支撑。

关键词: 非洲猪瘟病毒(ASFV); p22蛋白; 原核表达; 间接ELISA; 抗体

Abstract: 【Objective】 This study was aimed to express and purify the structural protein p22 of African swine fever virus (ASFV), and use it as a coating antigen to establish an indirect ELISA method for detecting ASFV antibodies and diagnosis of African swine fever.【Method】 The truncated KP177R gene encoding ASFV p22 protein (amino acids 24-145) was cloned into prokaryotic expression vector pET-32a(+) to construct the expressing plasmid pET-32a-p22.The recombinant plasmid was transformed into Escherichia coli (E.coli) BL21(DE3) competent cells and then induced by 0.1 mmol/L IPTG for 5 h.The corresponding p22 protein was purified by affinity chromatography on Ni ion and identified by Western blotting.BALB/c mice were inoculated with the purified p22 protein to prepare antiserum.Meanwhile, HEK293T cells were transfected with the eukaryotic expression plasmid containing the p22 full-length gene fragment (pCAGGS-EGFP-fp22) and used as antigenic matrix.Indirect immunofluorescence assay (IFA) was used to identify the reactivity of the antiserum.Based on the optimized parameters including the coating antigen concentration, serum dilution, blocking condition, reaction time of the first antibody, and working concentration of the enzyme-conjugated secondary antibody, an indirect ELISA method was established for ASFV antibody detection, and the clinical porcine serum samples were detected using this indirect ELISA method.【Result】 The truncated ASFV p22 protein was expressed in E.coli, and the protein concentration was 0.85 mg/100 g of bacteria.The prokaryotic p22 protein showed good immunogenicity, and the titer of the corresponding antiserum could reach 1:12 800.The optimized parameters of the ELISA (p22-ELISA)were as follows:The concentration of coating antigen was 0.125 μg/well, the dilution of serum was 1:800, and the dilution of the secondary antibody was 1:10 000.The cut-off value of p22-ELISA was 0.384.There was no cross reaction with CSFV, PRRSV, PCV2 and PRV antibody positive porcine serum, indicating good specificity.The coefficients of variation intra- and inter-batch were lower than 10%, indicating good repeatability.The p22-ELISA was used to detect 263 clinical pig serum samples, and the coincidence rate with thecommercial ASFV ELISA kit was 97.7%.【Conclusion】 This study established an indirect ELISA method for ASFV antibody detection based on the prokaryotic truncated ASFV p22 protein, the method had strong specificity, high sensitivity and good repeatability, and provided technical storage for the diagnosis and epidemiological investigation of ASFV infection.

Key words: African swine fever virus(ASFV); p22 protein; prokaryotic expression; indirect ELISA; antibody

中图分类号:

S852.65⁺1

张凯, 张春平, 戈胜强, 孙春喜, 程杰, 伏春雨, 王刚, 牛星, 彭军. 非洲猪瘟病毒p22蛋白表达、多克隆抗体制备及间接ELISA方法的建立[J]. 中国畜牧兽医, 2023, 50(1): 270-279.

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 and Veterinary Medicine, 2023, 50(1): 270-279.

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