Monoclonal Antibody-based Serological Detection Techiques Of Near Isolate Of Apple Stem Pitting Virus And Apple Stem Grooving Virus

In recent years,fruit tree viral diseases are widely occurring and epidemic worldwide,leading to a serious decline in the yield and quality of fruit industry,and even causing devastating economic losses.Among them,Apple stem pitting disease caused by Apple stem pitting virus(ASPV)and Apple stem grooving disease caused by Apple stem grooving virus(ASGV)are two important virus diseases of furit trees.Once the fruit tree is infected with the virus,it will be viruliferous permanently during its life cycle,and no medications can cure the disease.Therefore,establishments of the virus detection technology and viral monitoring systemare crucial to the prevention and control of fruit tree virus diseases.Serological assays based on monoclonal antibody for plant virus detection is specific,rapid,sensitive,simple and is suitable for detecting large-scale samples,thus is considered to be the most practical method of detecting virus on fruit trees.In view of the fact that the serological detection technology specific for ASPV and ASGV has not been established in domestic country.Monoclonal antibodies(MAbs)against ASPV and ASGV were respectively produced by Hybridoma technology,and several serological assays based on MAbs were respectively developed for rapid and sensitive detection of ASPV and ASGV in this study.These study achievements will provide material and technical support for orchard disease identification and virus detection,epidemiological analysis as well as the establishment of scientific prevention and control system of these two apple virus.1)MAbs against ASPV and their detection application:The major coat protein gene(CP)of ASPV isolated from pear was expressed in Escherichia coli BL21(DE3)using the expression vector pET-28a.The recombinant protein was purified through Ni2+-NTA affinity column and used to immunize BALB/c rats and rabbits.Four hybridoma cell lines(2H2,14C3,20F9 and 20G6)secreting monoclonal antibodies(MAbs)fluid against ASPV were obtained via cell fusion?cell screening and cell cloning.The titers of ascetic fluids of MAbs secreted by the prepared hybridomas were up to 10-6by indirect-ELISA.Western blot analyses indicated that all the four MAbs could specifically react with coat protein of ASPV and ASPV-infected leaves from pear tree,while leaves infected with ASGV,ACLSV and leaves from healthy fruit trees did not occur immune response.Based on the prepared MAbs,three serological assays which are respectively dot enzyme-linked immunosorbent assay(dot-ELISA),triple-antibody sandwich enzyme-linked immunosorbent assay(TAS-ELISA),double-antibody sandwich enzyme-linked immunosorbent assay(D AS-ELISA)were developed to detect ASP V in fruit trees,the crude extracts from leaves of pear trees infected with ASPV diluted up to 1:1,280,1:10240,1:10240(w/v,g/ml),respectively.168 samples of suspected diseased fruit leaves collected from Wuhan,Beijing Xi'an and Hangzhou were detected by the method of serology,the results indicated that 96 of 168 samples were infected with ASPV,the coincidence rate of serological results and RT-PCR test results reached more than 94.8%which demonstated the established serological method could detect ASPV in fruit trees effectively and accurately,and the virus was common epidemy in our country.The preparation of ASPV MAbs and three developed detection assays have great significance in ASPV diagnosis,detection as well as prevention and control of ASPV.2)Preparation and application of MAbs against ASGV:Capsid protein(CP)of ASGV was prepared using a prokaryotic expression system and used as the immunogen to immunize rats and rabbit,two highly specific and sensitive murine MAbs(9B10 and 19B10)and one polyclonal antibody(PAb)were produced in this thesis.The titers of ascetic fluids of MAbs(9B10 and 19B10)secreted by the prepared hybridomas were up to 10-6 by indirect-ELISA.Western blot indicated that all the two MAbs could specifically react with coat protein of ASGV and leaves from fruit trees infected with ASGV.Specificity analyses of the two developed serological assays demonstrated that all the two MAbs could specifically react with leaves from fruit trees infected with ASGV,but show no response of any immune result to the fruit tree leaf tissues infected with ASPV or ACLSV,as well as healthy apples,pears and citrus leaf tissue of crude body fluids.Based on the prepared MAbs,two serological assays,dot-ELISA and TAS-ELISA,were developed to detect ASGV in fruit trees.Among them,the sensitivity of dot-ELISA method for detection of crude extracts from apple and pear leaves infected with ASGV diluted was up to 1:640(w/v,g/ml),and the detection sensitivity of ASGV citrus disease leaf crude extracts diluted was up to 1:2,560(w/v,g/ml).The sensitivity of TAS-ELISA for ASGV-infected apple and pear leaves crude extracts diluted was up to 1:5,120(w/v,g/ml),and ASGV-infected citrus disease leaves crude extracts diluted was up to 1:10,240(w/v,g/ml).187 fruit tree leave samples from Wuhan,Beijing Xi'an and Hangzhou were detected for presence of ASGV by the developed serological assays,79 of 187 were shown ASGV-postive.The coincidence rate of dot-ELISA and TAS-ELISA and RT-PCR was up to 96.3%which indicated the established serological method can effectively and accurately detect ASGV in fruit trees.The preparation of ASGV monoclonal antibody and the establishment of its serological technique provide technical support for the diagnosis and detection of ASGV,as well as epidemiological research and production of virus-free seedling.