Prokaryotic expression and characterization of two recombinant receptor-binding domain(RBD) proteins of human coronavirus NL63(HcoV-NL63).
- Author:
Hui CHANG
1
;
Yao YI
;
Min ZHAO
;
Wei-Min ZHOU
;
Guo-Xia ZHAO
;
Hui-Juan WANG
;
Sheng-Li BI
;
Ji-Min GAO
;
Bing LIU
;
Wen-Jie TAN
Author Information
1. Department of Genetics, Norman Bethune College of Medicine, Jilin University, Changchun 130021,China.
- Publication Type:Journal Article
- MeSH:
Animals;
Coronavirus Infections;
metabolism;
virology;
Coronavirus NL63, Human;
chemistry;
genetics;
metabolism;
Escherichia coli;
genetics;
metabolism;
Gene Expression;
Humans;
Mice;
Mice, Inbred BALB C;
Protein Engineering;
Protein Structure, Tertiary;
Receptors, Virus;
metabolism;
Viral Envelope Proteins;
chemistry;
genetics;
metabolism
- From:
Chinese Journal of Virology
2013;29(2):106-111
- CountryChina
- Language:Chinese
-
Abstract:
The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.
