新型鸭呼肠孤病毒σB蛋白基因密码子的优化及原核表达

doi:10.16431/j.cnki.1671-7236.2018.10.006

摘要/Abstract

摘要：

试验旨在优化新型鸭呼肠孤病毒（new-type duck reovirus，NDRV）XX株σB蛋白的原核表达系统，并评价sσB蛋白的免疫原性。根据已测得的NDRV-XX的σB蛋白基因序列，在不改变氨基酸序列的前提下，按照大肠杆菌密码子偏好性对σB蛋白全基因进行优化、合成并连接至pET-32a（+）质粒中，构建原核表达重组质粒pET-32a（+）-sσB，转化大肠杆菌BL21（DE3）菌株，经IPTG诱导表达并优化表达条件。SDS-PAGE结果显示，最佳诱导表达时间、温度及IPTG浓度分别为3 h、32℃和0.25 mmol/L；可溶性分析结果表明，重组蛋白主要以包涵体形式存在于菌体中。表达菌经超声破碎、变性、复性和Ni²⁺柱亲和层析后，得到纯度高于90%的sσB可溶性蛋白，sσB重组蛋白在大肠杆菌BL21（DE3）菌株上的表达量较σB蛋白提升了14.6%，前者占细菌总蛋白量的32.3%。Western blotting结果显示，sσB重组蛋白具备NDRV抗原免疫反应原性。本试验成功构建并优化了NDRV-XX株σB蛋白的原核表达系统，提高了σB蛋白的表达量，并获得具有良好NDRV抗原免疫反应原性的σB重组蛋白，为后续NDRV σB蛋白功能及其应用的深入研究、基因工程疫苗的研发奠定了基础。

关键词: 新型鸭呼肠孤病毒(NDRV); σ B蛋白; 密码子优化; 原核表达; 纯化

Abstract:

The aim of the trial was to optimize the prokaryotic expression system of the σB protein of the new-type duck reovirus (NDRV) XX strain and evaluate its immunogenicity.Based on the measured σB protein sequence of NDRV-XX,the whole gene of σB protein was optimized,synthesized and ligated into pET-32a(+) plasmid according to E.coli codon bias without changing the amino acid sequence.The prokaryotic expression recombinant plasmid pET-32a(+)-sσB was constructed and transformed into E.coli BL21(DE3),induced by IPTG,and its expression conditions were optimized.The SDS-PAGE results showed that the optimal induction time,temperature and IPTG concentration were 3 h,32℃ and 0.25 mmol/L,respectively.Soluble analysis showed that the recombinant protein existed mainly in the form of inclusion bodies.The sσB soluble protein with purity higher than 90% was obtained after ultrasonication,denaturation,renaturation,and Ni²⁺ column affinity chromatography.The expression of sσB recombinant protein on E.coli BL21(DE3) was increased by 14.6% compared with σB protein.It accounted for 32.3% of the total bacterial protein.Western blotting results showed that sσB recombinant protein possessed NDRV antigen immunogenicity.The prokaryotic expression system of σB protein in NDRV-XX strain was successfully constructed and optimized,the expression of σB protein was increased,and recombinant σB protein with good NDRV antigen immunoreactivity was obtained.The results of this study laid the foundation for subsequent in-depth study of NDRV σB protein function and its application and the development of genetic engineering vaccine.

Key words: new-type duck reovirus (NDRV); σB protein; codon optimization; prokaryotic expression; purification

中图分类号:

S852.65

李文锋, 梅敏敏, 黄兴国, 黄雯晶, 李晓文, Saeed El-Ashram, 黄淑坚, 李智丽. 新型鸭呼肠孤病毒σB蛋白基因密码子的优化及原核表达[J]. 《中国畜牧兽医》, 2018, 45(10): 2700-2706.

LI Wenfeng, MEI Minmin, HUANG Xingguo, HUANG Wenjing, LI Xiaowen, Saeed EL-ASHRAM, HUANG Shujian, LI Zhili. Optimization and Prokaryotic Expression of the σB Protein Gene Codons of the New-type Duck Reovirus[J]. , 2018, 45(10): 2700-2706.

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