Generation,Characterization Of Anti-Hantaanvirus Glycoprotein Monoclonal Antibodies And The Expression And Identification Of HTNV-G2 In The Pichia Pastoris Expression System

Hemorrhagic Fever with Renal Syndrome (HFRS), which is caused by hantanvirus, is an acute infectious disease with severe symptoms, such as fever, vascular hemorrhage, kidney dysfunction, and immunologic function disorder. In China, the epidemic situation is severe, the incidence and mortality is high. The prevention and cure task is difficult.Hantanvirus belongs to the Bunyaviridae family. At present, the HV can be classified into ten serotypes and two decades genotypes. Among all of them the HTNV,SEOV,PUUV,SNV pose great threats to human beings.Many researches have indicated that the glycoproteins (GP), which is encoded by M segments of the virus, is a significant constitutive protein of the HV. The neutralized-antigenic determinants of HV mainly exist in the GP, especially in G2. GP plays an important role in stimulating neutralization antibodies and protecting infected animal and human body from HV lethal infection. Moreover, the GP also contains fellowing characteristics: hemagglutinin activity, inducing cell fusion, binding to cellular membrane receptor, and so on. So the GP is generally considered as a protective antigen and a main candidate protein for the HV genetic engineering vaccine. But the immunogenicity of GP is weak and the fixation titer of antibody against GP is low. The expression of GP in the widely-used different kinds of expression systems is not satisfied. So far there is no effective detecting way for GP. As a result, the research on GP is greatly effected.In this research, we based on the fact that the GP not only contains the neutralization situs but also the hemagglutination situs. We extracted the hemagglutinin of HTNV 76-118 strain, immunized mice and generated the monoclonal antibodies (mAb). Than we screened the mAbs by the CHO cells transfected with the eukaryotic vectors—G1-pDisplay,G2-pDisplay—including the coding gene of G1 and G2, respectively, in order to detect which mAbs could specially against G1 and G2. We identified the immunological characteristics of the obtained mAbs initially, either. At the same time, we introdeced the G2 gene fragment into Pichia intracellar and excretion vector, respectively, than transformed in the Pichia engineering bacteria, screened the positive transformants and induced them.1. The generation and characterization of mAbs against HTNV G1,G2 Firstly, we extracted the hemagglutinin from suckling mice brains infected by HTNV 76-118 strain by the sucrose-acetone method, then immunized the BLAB/c mice, and fused the spleen cells of the immunized mice with SP2/0, the hybridoma cells were screened by hemagglutination inhibitory assay (HIA). We obtained four hybridoma cell strains which could stablely secrete antibodies against HTNV GP hemagglutinin, named 1A6,1G12,2B2,2H5, respectively. We screened the mAbs by the CHO cells transfected with the eukaryotic vectors—G1-pDisplay,G2-pDisplay—including the coding gene of G1 and G2, respectively, in order to detect which could special against G1 and G2. The results showed that 1G12 can specially bind to the CHO cells transfected by G1-pDisplay, so maybe this mAb specially against G1; 2B2 and 2H5 can specially bind to the CHO cells transfected by G2-pDisplay, so maybe these two mAbs specially against G2, while 1A6 could bind to the CHO cells transfected by both of them. The hemagglutination inhibition valency of the four mAbs tested by HIA is 1∶4000,1∶16000,1∶16000 and 1∶8000. The result of neutralization test demonstrated that all of them have the neutralization activity, and the highest titer is 1:40.2. The construction and expression of the G2 in the Pichia pastoris expression systemThe G2 gene fragment was cloned into the yeast intracellar vector pPIC3.5K and excretion vector pPICZαA, respectively. The recombinant clones named pPIC3.5K-G2 and pPICZαA-G2 were introduced into the Pichia host strain GS115. The positive transformants were induced by 0.5% methanol, and the expression products were detected by SDS-PAGE and Western-Blot. After induced, there was an interest protein expression band in the expression products of excretion vector, and it could be recognized by the anti-his specific mAb and convalescent patient sera of HFRS, respectively, whereas there was no such result can be observed in the intracellar expression vector.The generating of mAbs against G1 and G2 and successful expression of G2 in Pichia expression system offers some experimental basis for the identification of GP mAbs, it also lays the basis for the researches concerning to GP.