Preparation And Identification Of Four Candidate Antigens For Diagnosis Of Highly Pathogenic Viruses

Objective: Tick-borne Encephalitis Virus(TBEV), Chikungunya Virus(CHIKV),Yellow Fever Virus(YFV)and Ebola Virus are potent highly pathogenic viruses, the experimental research of these viruses needs to be carried out in the high-level of biosafety laboratories, which increasesthe cost and difficulty of experiment. In order to reduce the risk of infection and detect these viruses in low-level biosafety laboratory, we selected the genes whichare suitable for diagnostic antigen according to the characteristics of molecular structure of four highly pathogenic viruses, and expressed the antigen with different expression systems of antigen. Thus, we provided candidate antigens for the serological specificity diagnosis of the four kinds of highly pathogenic virulent virus.Method:1. TBEV pr M-E gene was connected to the p NLF1-sec N plasmid to construct eukaryotic expression vector p NLF-pr M-E, and the recombinant plasmid was transfected into COS7 cells. By measuring luciferase activity of cell supernatant proteins we identified the expression of protein; The binding activity between recombinant protein and TBEV specific antibody was identified by indirect immunofluorescent assay; 20 TBEV positive serum specimenand 3 serum specimen free from TBEV were detectedwith luciferase immunoprecipitation system(LIPS).2. We expressed the E1 、 E2 gene of CHIKV E gene of YFV, NP gene of Ebola V through prokaryotic expression, and recombinant protein was purified by nickel column affinity; We confirmed CHIKV recombinant protein as test antigen, which could detect thespecific antibodies in the base recovery period patient through ELISA;YFV recombinant protein was used as antigen to detect specific antibodies of YF immune mouse serum prepared in our laboratory; We usedrecombinant NP protein of Ebola Vas immunogen to immunize mice and NP protein as antigen to detect specific antibodies in the serum of immunized mice.Result:1. High expression of luciferase can be detected in the cell supernatants of plasmid p NLF-pr M-E; The indirectimmunofluorescent assay showed that the cell transfected by recombinant plasmid p NLF-pr M-E could combine with TBEV antibody specifically; Detected by LIPS, 19 positive serums were obtained among the 20 TBEV positive serums and all of the three serum specimenwere found to be negative using recombinant proteins containing pr M-E as detection antigen.2. We are success in expressing and purifying CHIKV E1-1, CHIKV E2-1 and CHIKV E2-2 recombinant protein of CHIKV, YFV E1-1, YFV E1-2 and YFV E2-1 recombinant protein of YFV, and NP recombinant protein of Ebalo V; we failed in the expression of CHIKV E1-2 and YFV E2-2 recombinant protein; CHIKV E1-1,CHIKV E2-1 and CHIKV E2-2 were all combined specifically with infected serums from convalescent patients and had no cross-reaction with control antigen; YFV E1-1, YFV E1-2, YFV E2-1combined well with the YF antibodies in immunized mice;Although there were some cross reaction with the serums from patients with type DEN3, they could also be recognized from positive serum;We are success in preparing the positive serum of Ebola V immunized mice, which could combine recombinant antigen NP and have no cross reaction with other control serums.Conclusion:1. The eukaryotic expression vector p NLF-pr M-E was successfully constructed, and the pr M-E protein had good immunogenicity, which could be used as detection antigen in the TBEV infection.2. The recombinant expression plasmidof the CHIKV E1-1, CHIKV E2-1, CHIKV E2-2, YFV E1-1, YFV E1-2, YFV E2-2 and NP was successfully constructed, all which could induce protein expression correctly in prokaryotic cells. They were expected to be used as the candidatediagnostic antigen for the detection of CHIKV,YFV and Ebola V.