Study On Truncated Hantavirus Nucleocapsid Proteins And Their Application For Serotyping

Hantavirus mainly causes Hemorrhagic Fever with Renal Syndrome (HFRS), which is an acute infectious diseases characterized by fever, hemorrhage, renal impairment and thrombocytopenia, and hantavirus pulmonary syndrome (HPS). Four distinct hantaviruses are known to cause HFRS. They are defined as different serotypes: Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava/Belgrade virus(DOBV), and Puumala virus (PUUV). While Sin Nombre virus(SNV) and related viruses cause HPS. In China, two pathogenic hantaviruses, HTNV and SEOV, are present. The severity of the disease ranges from a mild or moderate form to severe and fatal cases, depends on the viral serotype, so it is important to find a safe, rapid and specific serotyping diagnosis for hantavirus infection, not only for controlling hantavirus infection but also for using specific prophylactic vaccines and effective therapeutic drugs.Currently, the plaque reduction neutralization test (PRNT) is the onlyavailable serological assay to define the serotype of a causative hantavirus. However, PRNT is time consuming, which usually spends more than 1 week to perform and needs specialized techniques and equipment, and requires a containment laboratory for virus manipulation. Therefore, It is essential to establish a simple, safe, and rapid diagnostic method to distinguish HTNV from SEOV infections serologically.As we know, Hantavirus nucleocapsid protein (NP) possesses immuno-dominant, linear, cross-reactive epitopes within the first 100 amino acids of the N terminus. The C terminus of NP contains serotype -specific epitopes, which occupys about half of the C terminus. Some studies suggested that Recombinant NPs of HTNV and SEOV,which were truncated 154aa from the N terminus of NP, were able to differentiate HTNV from SEOV infections serologically by indirect immunofluorescent antibody assay (IFA).In this research, firstly, we constructed the prokaryotic expression vectors pRSETA-76-S,pRSET A-76-50, pRSET A-76-100,pRSET A-76-155, pRSET A-L-S,pRSET A-L-50, pRSET A-L-100 and pRSET A-L-155, which contain S segment and different partial segments of HTNV S gene and SEOV S gene. Secondly, these plasmids were transformed into BL21(DE3)pLySs E.Coli. and induced by IPTG to express target protein respectively. Thirdly, using the B-Per 6xHis fusion protein purification kit (Pierce company), we purified these recombinant NP(rNP): 76-S-P, 76-50-P,76-100-P, 76-155-P and L-S-P, L-50-P, L-100-P, L-155-P. Finally, by analyzing the antigenicity of these purified rNP, we examined their applicabilities as serotyping antigens, particularly for using in ELISA. The results are as following:1. The prokaryotic expression vectors ( pRSET A-76-S,pRSET A-76-50, pRSET A-76-100, pRSET A-76-155, and pRSET A-L-S, pRSET A-L-50 , pRSET A-L-100, pRSET A-L-155) were successfully constructed and identificated with special PCR, restriction enzyme digestion and sequencing.2. SDS-PAGE and Western-blot analysis were carried out to detect these recombinant fusion proteins. SDS-PAGE results showed that all these fusion proteins were efficiently expressed in BL21 (DE3) pLySs , the molecular weight of expressed fusion proteins encoded by HTNV and SEOV S gene 1287bp, 1140bp, 990bp and 825bp were about 55KD ,49KD, 43KD and 37KD, respectively. Western-blot analysis showed that the fusion proteins 76-S-P, 76-50-P, L-S-P and L-50-P could distinctly react with the mixture of mAb A3 5 and 5H5, however the other four fusion proteins could not. But all these eight fusion rNPs could react with the immune mice sera against HTNV or SEOV.3. After all these eight recombinant fusion proteins purified by using fusion tag technology, their antigenicity were analyzed with ELISA. The results showed that the eight fusion rNPs could react with the immuned mice sera against HTNV or SEOV, but immune responses of the rNPs 76-100-P, 76-155-P, L-100-P and L-155-P were very weak. In addition, we found that the rNPs 76-S-P and L-S-P had strong cross reaction with the both immune mice sera and could not distinguish them from each other, while rNP 76-50-P showed more distinct reaction with the immune mice sera against HTNV than rNP L-S-50 , at the same time, rNP L-S-50 also showed more distinct reaction with the immune mice sera against...