非洲猪瘟病毒P30蛋白原核表达及间接ELISA检测方法建立

doi:10.16431/j.cnki.1671-7236.2023.05.028

摘要/Abstract

摘要： 【目的】建立快速、准确检测非洲猪瘟病毒(African swine fever virus,ASFV)抗体的间接ELISA方法。【方法】根据GenBank收录的ASFV分离株全基因组中的CP204L基因序列,在不影响氨基酸序列的前提下进行密码子优化,克隆到pCold TF冷休克表达载体,构建重组质粒pCold TF-p30。利用大肠杆菌原核表达系统进行IPTG诱导表达并对诱导时间和诱导浓度进行优化。用His标签镍柱对重组蛋白P30进行纯化,通过SDS-PAGE和Western blotting鉴定后,用纯化后的P30重组蛋白作为抗原,建立ASFV抗体间接ELISA检测方法并对反应条件进行优化,检验该方法的特异性、灵敏性和重复性,并用该方法与商品化试剂盒进行对比。【结果】重组质粒成功转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导后以可溶性蛋白形式表达,在约82 ku处有目的蛋白特异性条带。当16 ℃诱导12 h时蛋白表达量最高,不同IPTG浓度诱导无明显差异。Western blotting结果显示,P30重组蛋白可与ASFV阳性血清产生特异性反应,表明其具有良好的反应原性。建立的ASFV抗体间接ELISA检测方法抗原最佳包被浓度为1 μg/mL,使用1% BSA溶液封闭效果最佳,血清最佳稀释度为1∶600,酶标二抗最佳工作浓度为1∶8 000,该方法具有良好的特异性、灵敏性和重复性,与商品化试剂盒总符合率达到96.7%。【结论】本试验成功表达并纯化了ASFV的P30蛋白,建立了ASFV抗体间接ELISA检测方法,为非洲猪瘟的监测、诊断及试剂盒开发提供了参考。

关键词: 非洲猪瘟病毒(ASFV); P30蛋白; 原核表达; 间接ELISA

Abstract: 【Objective】 This study was aimed to establish an indirect ELISA method for rapid and accurate detection of antibodies to African swine fever virus (ASFV).【Method】 The CP204L gene sequence was extracted from the whole genome of ASFV isolate collected by GenBank,without affecting the amino acid sequence,the codon was optimized,and the pCold TF cold shock expression vector was cloned to construct the recombinant plasmid pCold TF-p30.The prokaryotic expression system of Escherichia coli was used to induce IPTG expression,and the induction time and concentration of IPTG were optimized.The recombinant protein P30 was purified with His labeled nickel column.After identification by SDS-PAGE and Western blotting,the purified recombinant protein P30 was used as antigen to establish an indirect ELISA method for detecting ASFV antibodies and optimize the reaction conditions.The specificity,sensitivity and repeatability of the method were tested and compared with commercial kits.【Result】 The recombinant plasmid was successfully transferred into the competent cells of Escherichia coli BL2 (DE3) and expressed in the form of soluble protein after induced by IPTG.There was a specific band of the target protein at about 82 ku.When the induction condition was 16 ℃ and the induction time was 12 h,the protein expression was the highest.There was no significant difference in the induction effect among different IPTG concentrations.Western blotting showed that P30 recombinant protein could react specifically with ASFV positive serum,which proved that it had good reactivity.The optimal concentration of antigen coating of the established ASFV indirect ELISA antibody detection method was 1 μg/mL,using 1% BSA solution had the best sealing effect,the optimal dilution of serum was 1∶600,and the optimal working concentration of enzyme labeled secondary antibody was 1∶8 000.This method had good specificity,sensitivity and repeatability,and the total coincidence rate was 96.7% when compared with the commercial kit.【Conclusion】 P30 protein of ASFV was successfully expressed and purified,and an indirect ELISA method for detecting ASFV antibody was established,which provided a reference for the monitoring,diagnosis and development of the kit of African swine fever.

Key words: African swine fever virus (ASFV); P30 protein; prokaryotic expression; indirect ELISA

中图分类号:

S852.65^＋1

张芳源, 蔺雅婷, 杨大为, 陈虎, 李桂梅, 单虎. 非洲猪瘟病毒P30蛋白原核表达及间接ELISA检测方法建立[J]. 中国畜牧兽医, 2023, 50(5): 2012-2022.

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 and Veterinary Medicine, 2023, 50(5): 2012-2022.

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