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

Abstract

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

CLC Number:

S852.65

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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Optimization and Prokaryotic Expression of the σB Protein Gene Codons of the New-type Duck Reovirus

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