Monoclonal Antibody-based Serological Detection Techniques Of Wheat Dwarf Virus And The PAV Strain Of Barley Yellow Dwarf Virus

Wheat is the most important food crop in the world,but has always been invaded by various epidemic pathogens.Among these pathogens,viral diseases are the key factors affecting the yield and quality of wheat.It has been reported that more than 50 species of plant viruses,including Wheat dwarf virus(WDV)and Barley yellow dwarf viruses(BYDVs),can infect wheat plants.Wheat dwarf disease caused by WDV and wheat yellow dwarf disease caused by BYDVs are widely distributed in wheat-planting areas in China,resulting in a serious yield loss every year.Development of the rapid,accurate and high-throughput detection technology is crucial to research plant virus epidemiology,breed anti-virus varieties and establish a scientific prevention and control system of plant viruses.Compared to other detection technologies,monoclonal antibody(MAb)-based serological assays are simple,rapid,sensitive,specific and suitable to large-scale detection.Therefore,serological assay has been widely used to diagnose and detect plant viruses,and now is one of the most important detection techniques of plant viruses.However,so far there are no reports about the application of anti-WDV and BYDV PAV MAb-based serological detection technologies.For this purpose,MAbs against WDV and BYDV PAV were produced,and effective serological assays were developed for high sensitive and specific virus detection in this thesis.Those research achievements will provide technical support for diagnosis and detection,forecast and forewarning and establishment of scientific prevention and control systems of these two wheat virus diseases.(1)MAbs against WDV and its detection application:The coat protein gene(CP)of WDV was cloned into the multiple cloning sites of prokaryotic expression vector pET-32a to construct the recombinant prokaryotic expression vector,pET-32a-CP.The recombinant protein was successfully expressed in Escherichia coli,and then purified through Ni2+-NTA agarose affinity column.The purified expression protein was used to immunize BALB/c mice,and four hybridoma cell lines(18G10,9G4,23F4 and 22A10)secreting sensitive and specific MAbs against WDV were obtained by the hybridoma technique.The titers of ascitic fluids of MAbs secreted by four hybridoma cell lines were up to 10-6 by indirect-ELISA.All MAbs were isotyped as IgGl,kappa light chain.Western blot analysis indicated that all four MAbs could specifically react with coat protein of WDV,but not with any wheat plant proteins.According to the principle of serology,two serological assays,antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA)and dot enzyme-linked immunosorbent assay(dot-ELISA)were developed.Among them,the sensitivity of ACP-ELISA based on MAbs 23F4 and 22A10 was higher,and it could detect wheat leaf crude extracts diluted at 1:163 840(w/v,g/mL),while ACP-ELISA based on MAbs 9G4 and 18G10 also could detect wheat leaf crude extracts diluted at 1:81 920(w/v,g/mL).The dot-ELISA based on MAb 23F4 has the highest sensitivity,and its detection sensitivity for WDV-infected leaves were up to 1:5 120(w/v,g/mL).The dot-ELISA based on MAbs 22A10 and 18G10 has a detection sensitivity of 1:1 280(w/v,g/mL)for WDV-infected leaves,while the sensitivity of the assay based on MAb 9G4 was lower,just 1:640(w/v,g/mL).A total of 128 wheat samples from Shanxi and Qinghai provinces in China were screened for the presence of WDV using the developed assays,and the detection results showed that 79 of the 128 wheat samples were infected by WDV.Furthermore,the serological detection result was consistent with the results of PCR.Sequencing and sequence comparative analyses of the PCR products proved that the positive samples tested by the serological assays were really infected by WDV.(2)MAbs against BYDV PAV strain and its detection application:The BYDV PAV-infected wheat leaf crude extracts were used to immunize BALB/c mice,and six hybridoma cell lines(6C4,24G4,25C2,15A12,19G7 and 18C2)secreting sensitive and specific MAbs against BYDV PAV were obtained by the hybridoma technique.The titers of ascitic fluids of MAbs secreted by six hybridoma cell lines were up to 10-6 by indirect-ELISA.All MAbs were isotyped as IgGl,kappa light chain.Based the prepared MAbs,ACP-ELISA and dot-ELISA were developed for BYDV PAV detection.The sensitivity and specificity analyses revealed that the developed ACP-ELISA could detect minimus virus in BYDV PAV-infected wheat leaf crude extracts diluted at 1:163 840(w/v,g/mL),and this assay has a negative reaction with healthy wheat leaves and wheat leaves individually infected by BYDV GAV,BYDV GPV,WDV,Wheat yellow mosaic virus(WYMV),Barley yellow mosaic virus(BaYMV)and Chinese wheat mosaic virus(CWMV).The sensitivity of the developed dot-ELISA for BYDV PAV-infected wheat leaves were up to 1:12 560(w/v,g/mL),and this assay has a negative reation with health wheat plant tissues and other wheat viruses.A total of 135 wheat plant samples collected from Shanxi and Qinghai provinces in China were screened for the presence of BYDV PAV with two established assays and the results indicated that 88 of the 135 samples were infected by BYDV PAV.The detection results of RT-PCR of the field samples were consistent with the serological detection results.Sequencing and sequence comparative analyses of the PCR products confirmed that BYDV PAV indeed existed in the positive samples detected by the two serological assays.