Creation And Application Of Monoclonal Antibodies Against Pepino Mosaic Virus And Alfalfa Mosaic Virus Virus

Tomato is an important vegetable and fruit crop,while soybean is an important vegetable and oil crop,and also a high quality protein source for human and livestock.However,tomato and soybean plants are easily infected with a variety of plant viruses,which leads to significant yield and economic losses.Pepino mosaic virus(PepMV)and alfalfa mosaic virus(AMV)are separately important viral pathogens on tomato and soybean crops worldwide,and seriously threaten the production of tomato and soybean.So far,crop viral diseases are lack of effective preventive and therapeutic agent.Thus,establishment of the simple,rapid,sensitive,specific,accurate and practical virus detection technology is critical to monitoring,early warning and green prevention and control of plant viral diseases.Therefore,this study respectively developed highly sensitive and specific anti-PepMV and anti-AMV monoclonal antibodies,and developed highly sensitive and specific serological methods to detect these two viruses based on the created monoclonal antibodies.These research result will provide reagents and technical support for diagnostic test,viral epidemiological research and scientific prevention and control of PepMV and AMV.(1)Creation of monoclonal antibodies against pepino mosaic virus and their serological detection methodsPepMV particles were extracted from PepMV-infected tobacco plants through differential speed centrifugation,and used as the immunogen to immunize five BALB/c female mice.Hybridioma cell lines secreting monoclonal antibodies(MAbs)against PepMV were created by the murine hybridioma technique.Six hybridioma cell lines,i.e.15B2,8H6,23D11,20D9,3A6 and 8E3,secreting highly specific and sensitive anti-PepMV MAbs were obtained via cell fusion,antibody detection,and cell cloning.The isotypes of all the six MAbs were determined to be IgG1 and?light chain and indirect-ELISA titers of all ascites containing MAbs were 10^-7.Western blot assay analysis discovered that all the six MAbs specifically recognized PepMV coat protein.The ACP-ELISA analysis demonstrated that all the six MAbs could mightily react with PepMV,and had no cross-reaction with TYLCV,ToMMV,ToMV,TSWV,PVX and healthy plant tissues.Four serological assays,ACP-ELISA,dot-ELISA,Tissue print-ELISA and DAS-ELISA for detection of PepMV were established using the created MAbs as the detection antibodies.The sensitivity analysis assays showed that sensitivities of ACP-ELISA,dot-ELISA and DAS-ELISA for PepMV detection were up to 1:163,840,1:20,480 and 1:1,310,720(weight/volume,g/m L)with the crude extract from diseased leaves,respectively.The established serological methods were used to detect PepMV in field samples.RT-PCR detection,DNA sequencing and nucleic acid sequence alignment verified the reliability and effectiveness of the four serological methods for PepMV detection.(2)Creation and of monoclonal antibodies against alfalfa mosaic virus and their serological detection methodsAMV virions were extracted from AMV-infected soybean plants through differential speed centrifugation,and used as the immunogen to immunize five BALB/c female mice.Hybridioma cell lines secreting MAbs against AMV were prepared by the murine hybridioma technique.Five hybridioma cell lines i.e.22G7,7G12,13G1,21D5 and 22D2,secreting highly specific and sensitive anti-AMV MAbs were obtained.The indirect-ELISA titers of ascites were determined to be 10^-7.Western blot assay analysis found that all the five MAbs specifically recognized AMV coat protein.The ACP-ELISA analysis demonstrated that all the five MAbs could mightily react with AMV,and had no cross-reaction with SMV,SSV,CMV,PNRSV,ApNMV and healthy plant tissues.Three serological assays,ACP-ELISA,dot-ELISA and Tissue print-ELISA for detection of AMV were established using the created MAbs as the detection antibodies.The sensitivities of ACP-ELISA and dot-ELISA for AMV detection were up to 1:81,920 and 1:20,480(weight/volume,g/m L)with the crude extract from diseased leaves,respectively.The established serological methods were used to detect AMV in field samples.RT-PCR detection,DNA sequencing and nucleic acid sequence alignment demonstrated the accuracy and effectiveness of the three serological methods for AMV detection.