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Development of E2 Neutralizing Monoclonal Antibody Against CSFV

Author: ChenJingYan
Tutor: QianYongHua
School: Northwest University of Science and Technology
Course: Preventive Veterinary Medicine
Keywords: Classical swine fever virus Fake virus Pseudotyped virus And monoclonal antibodies
CLC: S852.65
Type: Master's thesis
Year: 2009
Downloads: 84
Quote: 0
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Abstract


This study to express major antigen E2 protein of classical swine fever virus replicating vesicular stomatitis the fake virus immune Balb / c mice, using the conventional method of cell fusion, hybridoma screening introduced to express luciferase swine fever The pseudotyped virus E2 protein non-replicating HIV virus detection system preparation and the role of anti-CSFV E2 envelope glycoprotein monoclonal antibodies and analysis of its biological activity, for classical swine fever virus neutralizing monoclonal antibody Preparation of the introduction of a new viable method and provide a new tool for the study of the swine fever vaccine. The study included mainly the following three parts: a rescue capsule surface expression of the E2 protein of classical swine fever virus replicating vesicular stomatitis fake virus vesicular stomatitis virus reverse genetics system, co-transfection of expression of the vesicular stomatitis virus nucleocapsid protein phosphoprotein and polymerase protein plasmid carrying CSFV E2 gene replication of vesicular stomatitis virus vectors in BHK-21 cells and false use of Western Blot, indirect ELISA, and in vitro microneutralization test for detection and identification of rescue virus. The results prove that succeeded in saving the stable expression of high titer positive serum anti-CSFV E2 protein E2 protein-replicating false vesicular stomatitis virus, immune Balb / c mice. 2 Preparation of the secreted E2 protein using PCR amplified gene of the CSFV strain the F114 cell strain CF114 E2 non-transmembrane region sequence, to obtain a gene fragment of approximately 1000 bp size, with the Flag and His tag These genes were cloned into the eukaryotic expression vector pLL3.7-FH, digestion, PCR and DNA sequencing analysis confirmed correctly inserted into the expression vector in the correct reading frame to build into a recombinant expression vector pLL3.7-FH-E2. Calcium phosphate method, the recombinant plasmid was transfected to 293T cells collected 48 hours after the cell supernatant after Flag purified Cheongwon cells and expressed proteins analyzed by SDS-PAGE electrophoresis detection. The results showed that the anti E2 gene expression product of a molecular weight of about 50kDa, proved by Western-blotting, the expression of the E2 protein may be CSFV positive sera recognize, can be used as coating antigen screening E2 monoclonal antibody. 3 Preparation HIV-luc/CSFV E1 E2 pseudotyped virus and determine the fake viral load in vitro microneutralization test using the calcium phosphate method expression plasmid pcDNA3.1-E1 E2 of classical swine fever virus E1 and E2 envelope protein expression The luciferase plasmid HIV-Luc was transfected into 293T cells, 48 ??hours after the cells were collected on Cheongwon. Take a certain amount of virus pK-15 cells 48h value determination of relative luciferase activity (Relative Luciferase activity Units, RLUs), results showed that the HIV-luc/CSFV E1 E2 fake virus can efficiently infect pK-15 cells, but the reporter gene fluorescein The level of expression of the enzyme and the amount of virus added was a dose-dependent relationship and then determine vitro microneutralization and systems sham virus dilution of 1:2, and the system taken in 100μl 50μl pseudotyped virus (RLUs value of approximately microneutralization test for 1 × 104). The 4 preliminary identification of monoclonal antibodies and biological characteristics to express the the CSFV E2 envelope glycoprotein of vesicular stomatitis virus (Vesicular Stomatitis Viruses, VSV) that VSV / CSFV E2 immunized BALB / c mice spleen cells with myeloma cells SP2 / 0 fusion; indirect ELISA method and carrying firefly luciferase (Luciferase, Luc) reporter gene HIV-luc/CSFV-E1 E2 fake virus system screening the secreting and sexual E2 monoclonal antibody hybridoma cell; determination of monoclonal antibody subtype and purified, indirect ELISA method determination of monoclonal antibody titer; a specific affinity monoclonal antibodies using Western Blotting identification; vitro trace HIV-luc/CSFV-E1 E2 fake virus and testing, and ultimately obtained a secreted sexual monoclonal hybridoma 9C8, can be combined with E2 protein-specific relative titer greater than 1:25 600, in-vitro and test IC90 gt; 1:25600.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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