巴泰病毒G1189aa-239aa肽段的原核表达、纯化及间接ELISA方法的建立

doi:10.16431/j.cnki.1671-7236.2021.10.029

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (10): 3770-3778.doi: 10.16431/j.cnki.1671-7236.2021.10.029

巴泰病毒G1_189aa-239aa肽段的原核表达、纯化及间接ELISA方法的建立

员晓庆¹, 李丽霞¹, 唐甜¹, 陈盛楠¹, 梁晓彤¹, 黄良宗¹, 司兴奎¹, 张浩吉¹, 孙秀涛², 段文学³, 金宁一⁴, 刘昊¹

1. 佛山科学技术学院, 佛山 528225;
2. 红河州动物疫病预防控制中心, 蒙自 661199;
3. 建水动物疫病预防控制中心, 建水 654399;
4. 军事医学研究院军事兽医研究所, 长春 130122

收稿日期:2021-02-24 出版日期:2021-10-20 发布日期:2021-09-30
通讯作者: 刘昊 E-mail:liuhao_lh@hotmail.com
作者简介:员晓庆(1995-),男,河南三门峡人,硕士生,研究方向:人兽共患病防控,E-mail:yuanxiaoqing_fs@126.com;李丽霞(1986-),女,山东青岛人,硕士,助理研究员,研究方向:人兽共患病防控,E-mail:lilixialyt@126.com
基金资助:
国家自然科学基金项目（31802199）；佛山科学技术学院研究生自由探索基金（2020ZYTS44）

Prokaryotic Expression and Purification of rHis-G1_189aa-239aa Peptide of Batai Virus and Establishment of Indirect ELISA Method

YUAN Xiaoqing¹, LI Lixia¹, TANG Tian¹, CHEN Shengnan¹, LIANG Xiaotong¹, HUANG Liangzong¹, SI Xingkui¹, ZHANG Haoji¹, SUN Xiutao², DUAN Wenxue³, JIN Ningyi⁴, LIU Hao¹

1. Foshan University, Foshan 528225, China;
2. Honghe Animal Disease Prevention and Control Center, Mengzi 661199, China;
3. Jianshui Animal Disease Prevention and Control Center, Jianshui 654399, China;
4. Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130122, China

Received:2021-02-24 Online:2021-10-20 Published:2021-09-30

摘要/Abstract

摘要： 本研究旨在获得高纯度巴泰病毒（Batai virus，BATV） G1蛋白强抗原性肽段，并建立一种用于检测BATV羊血清抗体的间接ELISA方法。首先对G1蛋白51个氨基酸编码的189－239位氨基酸强抗原肽段进行密码子优化，基因合成后构建重组表达质粒pET-32a-G1_189aa-239aa，转化大肠杆菌BL21感受态细胞进行诱导表达。优化表达条件获得大量rHis-G1_189aa-239aa肽段，利用镍柱亲和层析方法进行纯化，并用BCA试剂盒进行蛋白浓度的测定。通过Western blotting对rHis-G1_189aa-239aa肽段进行抗原特异性分析，以rHis-G1_189aa-239aa肽段为抗原，羊BATV阳性血清作为抗体，通过方阵滴定优化反应条件，建立BATV间接ELISA检测方法。SDS-PAGE结果显示，rHis-G1_189aa-239aa肽段在大肠杆菌中主要以包涵体形式存在，分子质量为24.5 ku，在0.1 mmol/L的IPTG诱导5 h后rHis-G1_189aa-239aa肽段表达量达到峰值。通过镍柱亲和层析纯化后，测得蛋白浓度为0.296 mg/mL。Western blotting结果显示，rHis-G1_189aa-239aa肽段特异性较好。以rHis-G1_189aa-239aa肽段为抗原建立的间接ELISA方法，通过条件优化确定最佳抗原包被浓度为2.5 μg/mL，血清和二抗稀释度为1∶60和1∶10 000。样品D_{450 nm}值≥0.367为阳性，D_{450 nm}值≤0.319为阴性。该方法与山羊痘病毒和布鲁氏杆菌的阳性血清均无交叉反应，批内和批间的变异系数均<10%，阳性血清最高可稀释至1 600倍，该方法具有特异性强、灵敏性高和重复性好的特点。利用本方法对云南地区的120份羊血清样本进行检测，血清阳性率为21.67%。本研究获得了特异性强和纯度较高的rHis-G1_189aa-239aa肽段，建立了间接ELISA方法，可应用于羊BATV血清流行病学调查，为疫病防控及致病机制研究奠定基础。

关键词: 巴泰病毒(BATV); G1蛋白; 重组肽段; 原核表达; 蛋白纯化; ELISA

Abstract: The aim of this study was to obtain a highly purified antigenic peptide of Batai virus (BATV) G1 protein and to establish an indirect ELISA method for detecting sheep serum antibodies against BATV. Firstly, the codon of 189-239 amino acids strong antigen peptide encoded by 51 amino acids of G1 protein were optimized. After gene synthesis, a recombinant expression plasmids pET-32a-G1_189aa-239aa was constructed, and transformed into E. coli BL21 competent cells to induce expression. A large number of rHis-G1_189aa-239aa peptides were obtained by optimizing the expression conditions, which were purified by nickel column affinity chromatography, and the protein concentration was determined by BCA kit. Then the antigen specificity of rHis-G1_189aa-239aa peptide was analyzed by Western blotting. Using rHis-G1_189aa-239aa peptide as antigen and positive serum of BATV sheep as antibody, the reaction conditions were optimized by square titration, and the indirect ELISA detection method of BATV was established. SDS-PAGE result showed that the rHis-G1_189aa-239aa peptide existed mainly in the form of inclusion bodies in E. coli, the protein molecular weight was 24.5 ku, and the expression of rHis-G1_189aa-239aa peptide reached the peak after induced by 0.1 mmol/L IPTG for 5 h. After purification by nickel column affinity chromatography, the protein concentration was 0.296 mg/mL. Western blotting results showed that the specificity of rHis-G1_189aa-239aa peptide was good. The indirect ELISA method using rHis-G1_189aa-239aa peptide as antigen was established. Through the optimization of conditions, the optimum antigen coating concentration was 2.5 μg/mL, serum and the dilution of the second antibody were 1:60 and 1:10 000, respectively. When the sample D_{450 nm} value ≥ 0.367 was positive, D_{450 nm} value ≤ 0.319 was negative. This method had no cross reaction with the positive serum of Goatpox virus and Brucella abortus, the coefficient of variation within and between batches was less than 10%, and the positive serum could be diluted to 1 600 times at most. The method had the characteristics of strong specificity, high sensitivity and good repeatability. This method was used to detect 120 sheep serum samples in Yunnan, and the results showed that the serum positive rate was 21.67%. The rHis-G1_189aa-239aapeptide with high specificity and purity was obtained in this study, and the indirect ELISA method was established, which could be applied to the seroepidemiological investigation of sheep BATV and laid a foundation for the study of epidemic prevention and control and pathogenic mechanism.

Key words: Batai virus (BATV); G1 protein; recombinant peptide; prokaryotic expression; protein purification; ELISA

中图分类号:

S852.65

员晓庆, 李丽霞, 唐甜, 陈盛楠, 梁晓彤, 黄良宗, 司兴奎, 张浩吉, 孙秀涛, 段文学, 金宁一, 刘昊. 巴泰病毒G1_189aa-239aa肽段的原核表达、纯化及间接ELISA方法的建立[J]. 中国畜牧兽医, 2021, 48(10): 3770-3778.

YUAN Xiaoqing, LI Lixia, TANG Tian, CHEN Shengnan, LIANG Xiaotong, HUANG Liangzong, SI Xingkui, ZHANG Haoji, SUN Xiutao, DUAN Wenxue, JIN Ningyi, LIU Hao. Prokaryotic Expression and Purification of rHis-G1_189aa-239aa Peptide of Batai Virus and Establishment of Indirect ELISA Method[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3770-3778.

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巴泰病毒G1_189aa-239aa肽段的原核表达、纯化及间接ELISA方法的建立

Prokaryotic Expression and Purification of rHis-G1_189aa-239aa Peptide of Batai Virus and Establishment of Indirect ELISA Method

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巴泰病毒G1189aa-239aa肽段的原核表达、纯化及间接ELISA方法的建立

Prokaryotic Expression and Purification of rHis-G1189aa-239aa Peptide of Batai Virus and Establishment of Indirect ELISA Method

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巴泰病毒G1_189aa-239aa肽段的原核表达、纯化及间接ELISA方法的建立

Prokaryotic Expression and Purification of rHis-G1_189aa-239aa Peptide of Batai Virus and Establishment of Indirect ELISA Method