Prokaryotic Expression, Purification And Primary Application Of Recombinant Receptor-binding Domain(RBD) Protein Of Human Coronavirus NL63(HCoV-NL63)

Human Coronavirus NL63(HCoV-NL63), as a member of group α serologically,is enveloped, single positive-stranded RNA virus.The RNA genome of HCoV-NL63is27,553bp.The Spike(S) protein of coronavirus forms a layer of long petal-shapedsurface spikes that give the virions a crown-like appearance when visualized byelctron microscope.The S protein mediates cell attachment,membrane fusion,and theproduction of neutralizing antibodies that don’t rely on the complement.And the Sprotein also determines the virus virulence and tissue tropism.Similar to othercoronaviruses,the S protein of HCoV-NL63can be cleaved into two subunits byproteases,one is S1(21~717aa) in the N terminal which includes the receptor bindingdomain(RBD),the other is S2(718~1356aa) in the C terminal consists of thetransmembrane domain(TMD).The S1domain,especially the RBD is responsible forvirus binding to host cell receptors while the S2is the major determinant formembrane fusion.Previous studies have shown that the RBD of NL63was mapped toamino acid residues474~616(RS),but a larger RBD mapped232~684(RL) has abetter binding affinity to the receptor human angiotensin-convertingenzyme2(hACE2).In this paper, we prepared2purified recombinant HCoV-NL63RBD proteinsexpressed in E.coli and identified the proteins by Western blotting. Then we used therecombinat proteins to detect specific antibodies in the serum of ordinary people.Wealso used the recombinant proteins to evaluate the specific antibody titers in the serumof genetically engineered vaccine immuned mice.The results might provide a basis for further exploring the biologicalrole,infection epidemic regularity and vaccine development of HCoV-NL63. First of all, according to the sequence of HCoV-NL63spike protein(Genebank:AY567487),we optimized and synthesized the RL protein(232~684aa) andinserted it into the pET30,the resulting plasmid named pET30-RBD(L) HCoV-NL63.According to the sequence of RS and RL,two pairs of primers were designed andsynthesized with NcoI and XhoI restriction endonuclease site each at the5’ and3’end.We amplified the RL and RS(476-616aa) coding gene via PCR using differentprimers. The RL or RS coding gene was cloned into the pM48expression vectorfused with TrxA tag.The expression plasmid pM48-RS and pM48-RL wereidentified by enzyme digestion and DNA sequence analysis.The recombinant pM48-RS and pM48-RL vector was transformed into E.coliBL21(DE3)plysS. Afer induction optimization, the recombinant RBD proteins weremaximally expressed at37℃with0.8mM IPTG induction for4h and the RBD (RLand RS) of HCoV-NL63were expressed majorly as inclusion body.Collect theinsoluble pellets after ultrasonic broken.After centrifuging the disruption of bacteria cells, the insoluble pellets wasdissolved in the6M guanidine hydrochloride and then the proteins were purified byaffinity chromatography.The purity of the sample is up to95%. According to theresults of WB,the fusion proteins reacted positively with anti-sera from miceimmunized with the recombinant vaccinia virus(Tiantan strain) in which HCoV-NL63RL or RS protein was expressed.The recombinant proteins were used to evaluate the specific antibody titers in theserum of genetically engineered vaccine immuned mice.The results showed that thespecific antibody level in the immune group of plasmid plus vaccinia virus werehigher than the plasmid alone immune group which was consistent with thecellular immune responses.Then，the recombinant proteins were used to detect the specific antibodies amonggeneral population via WBLA.A total of30specimens were tested,results of whichshowed that the infection of human coronavirus NL63was widespread in healthypopulation in our country. The positive rate was the same(60%) by using RL-based WBLA,RS-based WBLA or IFA assays. RL-based WBLA and RS-based WBLAconsistent rate was79.15%; RL-based WBLA、RS-based WBLA and IFA consistentrate were both58.33%.And for proteins as envelope antigens, to establish an indirectELISA detection method has carried on the preliminary exploration.