Establishment Of ELISA Method With Recombinant Hantaviral Nucleocapsid Protein As Antigen And Its Application For Hantavirus Serologic Typing

Hemonhagic fever with renal syndrome (HFRS) is a worldwide epidemic disease caused by hantavirus. According to the reported case, China account for more than 90 percent of the world HFRS case. In recent years, HFRS incidence take on an accelerating tendency. The purpose of this study is to establish a rapid accurate and easy applied method to monitor the acute infection and used for clinical diagnosis in the HFRS region.Other studies have shown that the amino termini of the hantavirus nucleocapsid proteins (Ns) are sensitive tools for the detection of specific antibodies in hantavirus disease. Based on the Flgh's studies, we expressed truncated Ns (amino acids 1 to 117) in E.coli from the five hantaviruses known to be pathogenic to man. For expression, the plasmids were transformed into BL21-codonplus instead of E.coli BL(DE3). And added some genes which can recognize the tRNA of Arginine, Leucine and Isoleucine. Thus, we can improve the quantity and stability of the products. The proteins were purified by polyhistidine-metal chelare affinity. Five hantavirus nucleocapsid proteins expressed inE.coli were available, they are Hantaan(HTN), Seoul(SEO), Dobrava(DOB), Sin Nombre(SN) and Puumala(PUU) nucleocapsid proteins. We used these recombinant nucleocapsid proteins as coating antigen to establish enzyme-linked immunosorbent assay (FJTSA) method. Both ELBA and indirect irnmunofluoorescence antibody test (IFA) were used to test the 8 postive serum specimens and 3 negative serum specimens. We found the coincide rate was 90.9%. FT ISA was also used to detect antibody of the 241 HFRS patient sera from Shaanxi, Shandong, Hebei and Beijing. We found the sera reacted mainly to HTNV and SEOV nucleocapsid antigen, there are 40 multitype. What's more, we found 4 sera show high reactivities to the antigen of serotype DOB. This was the serological evidence that showed serotype DOB exsisted in China, so far, no other report has been found.Compared with other detective methods, such as IFA and PRNT(plaque reduction neutralization test), this kind of ELLS A is more reliable, economical and easy applied. Further studies need to be done to find a resonable explanation of the mutitype. The finding of serotype DOB in our country will have significant influence on the monitoring acute infection and the studies of the vaccination.