非洲猪瘟病毒B602L蛋白的原核表达、纯化及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2024.09.026

摘要/Abstract

摘要： 【目的】非洲猪瘟病毒(African swine fever virus，ASFV)是对生猪养殖业危害极大的一种病原体，B602L蛋白作为其重要的非结构蛋白，是衣壳蛋白p72折叠为正确的三级结构的伴侣分子之一，在病毒组装成二十面体结构的过程中发挥着关键作用。特异性抗体对于抗非洲猪瘟免疫应答机制及血清学检测方法研究具有重要意义，本研究旨在获取ASFV B602L蛋白，并制备抗ASFV B602L蛋白的多克隆抗体，为ASFV B602L蛋白功能研究及相关诊断试剂的研发提供材料。【方法】以ASFV China/2018/AnhuiXCGQ株的B602L基因为研究对象，应用生物信息学软件对B602L蛋白的理化性质、信号肽、跨膜结构、抗原性和蛋白结构进行分析后，利用同源重组的方法将其克隆至原核表达载体pET-28a(+)中，转化大肠杆菌BL21(DE3)感受态细胞后经诱导表达获得B602L重组蛋白，利用镍柱亲和层析进行蛋白纯化，通过SDS-PAGE和Western blotting对B602L蛋白纯化效果进行验证。将纯化后的B602L蛋白免疫BALB/c小鼠制备多克隆抗体，通过间接ELISA方法测定多克隆抗体效价，通过Western blotting和Dot blotting检测多克隆抗体特异性。【结果】生物信息学分析表明，B602L蛋白属于稳定性亲水蛋白，无跨膜区和信号肽。二级结构主要含有α-螺旋(54.91%)、延伸链(9.81%)和无规则卷曲(32.08%)。成功构建了ASFV B602L重组质粒，SDS-PAGE和Western blotting结果显示，重组菌pET-28a-B602L-BL21在16 ℃、0.5 mmol/L IPTG诱导下高效表达，获得的重组蛋白主要表达在BL21菌株上清中，大小约为70 ku，且能与ASFV 阳性血清特异性结合。间接ELISA检测结果显示，制备的抗B602L鼠源多克隆抗体效价达1∶40 9600，Western blotting和Dot blotting试验结果表明，抗B602L鼠源多克隆抗体能够特异性识别B602L蛋白。【结论】原核表达的B602L重组蛋白具有良好的免疫原性，能够与ASFV阳性血清特异性结合，制备的鼠抗ASFV B602L蛋白多克隆抗体具备较好的生物学活性。本试验结果可为进一步研究ASFV B602L蛋白的生物学功能和非洲猪瘟诊断方法的建立提供生物材料和理论支撑。

关键词: 非洲猪瘟病毒(ASFV); B602L蛋白; 原核表达; 多克隆抗体

Abstract: 【Objective】 African swine fever virus (ASFV) is a pathogen that is very harmful to the swine industry, and the B602L protein, as an important non-structural protein, is one of the chaperone molecules for the folding of the coat protein p72 into the correct tertiary structure, which plays a key role in the assembly of the virus into an icosahedral structure.Specific antibodies are of great importance for the study of the mechanism of the anti-African swine fever immune response and serological detection methods.This study was aimed to obtain ASFV B602L protein and prepare polyclonal antibodies against ASFV B602L protein, and provide materials for the functional research of ASFV B602L protein and the development of related diagnostic reagents. 【Method】 The present study was conducted on the B602L gene of ASFV China/2018/AnhuiXCGQ strain.After the prediction of the physicochemical properties and the analysis of the antigenicity of B602L protein, the B602L gene was cloned into the prokaryotic expression vector pET-28a(+) by homologous recombination and expressed in Escherichia coli BL21(DE3) competent cells.The protein was purified by nickel column affinity chromatography, the purification effect and specificity were verified by SDS-PAGE and Western blotting.And the purified B602L protein was immunized against BALB/c mice to prepare polyclonal antibodies.The titer of polyclonal antibodies was determined by indirect ELISA method, and the specificity of polyclonal antibodies was detected by Western blotting and Dot blotting. 【Result】 Bioinformatics analysis showed that B602L protein was an unstable hydrophilic protein without transmembrane region and signal peptide.The secondary structure mainly consisted of alpha helix (54.91%), extended chain (9.81%) and random coil (32.08%).ASFV B602L recombinant plasmid was successfully constructed.SDS-PAGE and Western blotting results showed that the recombinant strain pET-28a-B602L-BL21 was highly expressed under the condition of 16 ℃ and 0.5 mmol/L, and the obtained recombinant protein was mainly expressed in the supernatant of strain BL21.It was about 70 ku in size and binded specifically to ASFV-positive serum.Indirect ELISA showed that the prepared murine polyclonal antibody against B602L had a titer of 1∶409 600.Western blotting and Dot blotting results showed that the murine polyclonal antibody against B602L could specifically recognize B602L protein. 【Conclusion】 The prokaryoticly expressed B602L recombinant protein had good immunogenicity, which could specifically bind to ASFV-positive serum, and the prepared polyclonal antibody against mouse anti-ASFV B602L protein had good biological activity.The results could provide experimental materials and theoretical support for further research on the biological function of ASFV B602L protein and the establishment of diagnostic methods for African swine fever.

Key words: African swine fever virus (ASFV); B602L protein; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65⁺1

郭晨云, 宋金星, 王梦翔, 孙俊如, 闫若潜, 谢彩华, 吴亚楠, 张改平. 非洲猪瘟病毒B602L蛋白的原核表达、纯化及多克隆抗体制备[J]. 中国畜牧兽医, 2024, 51(9): 3980-3989.

GUO Chenyun, SONG Jinxing, WANG Mengxiang, SUN Junru, YAN Ruoqian, XIE Caihua, WU Yanan, ZHANG Gaiping. Prokaryotic Expression,Purification and Polyclonal Antibodies Preparation of African Swine Fever Virus B602L Protein[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 3980-3989.

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