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Production Of Monoclonal Antibodies To The VD-8 Strain Of Verticillium Dahliae

Posted on:2003-01-29Degree:MasterType:Thesis
Country:ChinaCandidate:P LiuFull Text:PDF
GTID:2120360062995252Subject:Plant pathology
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In this research monoclonal antibodies of VD-8 strain were produced on the base of previous work of our laboratory, and other common fungal pathogens in cotton were with tested the antibodies.In order to produce monoclonal antibodies , first , several V.dahliae isolates were grown in liquid Czapeak medium , after rinsing mycelia and eliminating zoospores , the fungal tissue was homogenized with the pestle in liquid nitrogen and then transferred to test tubes and was centrifuged in Tris-HCL buffer . The suspension was acquired and stored at 4X3 as the PAGE samples.The analysis and comparison of strain VD-8 of V.dahliae Kleb. and the other common fungal pathogens in cotton were taken with the technology of normal PAGE. The other common fungal pathogens included Rhizoctonia solani, Colletotrichu gossypii and Fusarium oxyporum f.sp. vasinfecoum . After many times of electrophoresis, the specific protein band of VD-8 was cutted and the protein was collected as immunogen. The protein density was 240ul/mL with ultraviolet analysis.After the produre of extracting and purifying immunogen , the production of monoclonal antibodies was carried out. Three BALB/C mice , 6-week old , were used for immunization . Two weeks after the second boost, blood was collected and the antisera were tittered using indirect ELISA, the liter was beyond 1 : 1600 and accorded with the demands of producing antisera . After preparation of spleenocytes and myeloma cells, let the two kind of cell suspension fuse in PEG 1000. then fused cell suspension was deposited to 96-well tissue culture plate . after that the plate was placed in a COa incubator. Three days after fusion . the wells which contained hybridomas were noticed . Five days later , replaced half of the medium with HAT-DMEM , and eight days latter with HT-DMEM. Ten or eleven days later. the screening of supernatants was carried out with using indirect ELISA. After subcloning by limiting dilution and selection . five clones of cells with high OD value were acquired and named MVI . MV2. MV3. MV4, MV5 .The V.dahliae and other fungal pathogens in cotton were tested after the positive clones were further incubated . the results showed that the acquired monoclonal antibodies could identify V.dahliae Kleb. with other pathogens at specie level . Non of the30five hybridomas isolates has specie or genus characterization . The MV2 could identify different pathotypes of V.dahliae ,MV1 and MV4 could test V.dahliae to specie level . Serious cross reation existed between V.albo-atrum and MV2, MV3, MV4 . The other pathogen isolates V31 and V32 also had cross reactions, but the reaction was not serious . Because limited number of pathogen isolates were selected , it could not prove that the selected immunogen was widely presentative , more pathogens isolates should be tested to verify the acquired hybridomas cells .
Keywords/Search Tags:cotton, Verticilium dahliae, monoclonal antibodies, ELISA, electrophoresis
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