title: [Prokaryotic expression and characterization of two recombinant receptor-binding domain(RBD) proteins of human coronavirus NL63(HcoV-NL63)].
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.
authors.affiliation: Department of Genetics, Norman Bethune College of Medicine, Jilin University, Changchun 130021,China.
authors.forename: Hui
authors.initials: H
authors.lastname: Chang
authors.forename: Yao
authors.initials: Y
authors.lastname: Yi
authors.forename: Min
authors.initials: M
authors.lastname: Zhao
authors.forename: Wei-Min
authors.initials: WM
authors.lastname: Zhou
authors.forename: Guo-Xia
authors.initials: GX
authors.lastname: Zhao
authors.forename: Hui-Juan
authors.initials: HJ
authors.lastname: Wang
authors.forename: Sheng-Li
authors.initials: SL
authors.lastname: Bi
authors.forename: Ji-Min
authors.initials: JM
authors.lastname: Gao
authors.forename: Bing
authors.initials: B
authors.lastname: Liu
authors.forename: Wen-Jie
authors.initials: WJ
authors.lastname: Tan
mesh_terms: D000818:Animals; D018352:Coronavirus Infections / Q000378:metabolism / Q000821:virology*; D058957:Coronavirus NL63, Human / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011991:Receptors, Virus / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D011991:Receptors, Virus; D014759:Viral Envelope Proteins