Preparation And Application Of Monoclonal Antibodies Against Citrus Tristeza Virus And Citrus Yellow Vein Clearing Virus

Every year, citrus virus diseases cause serious economic loss in citrus industry worldwide. Now, Citrus tristeza disease caused by Citrus tristeza virus (CTV) and Citrus yellow vein clearing disease caused by Citrus yellow vein clearing virus (CYVCV) are two important virus diseases of citrus trees. In order to understand the epidemic laws and establish the scientific prevention and control system of the two citrus virus diseases, rapid and efficient detection techniques should be urgently developed. Serological assays for plant virus detection are simple, rapid, sensitive, specific and suitable to detect large-scale field samples. So it has been widely used in plant virus disease diagnosis, epidemic rule analyses, resistive breeding, scientific prevention and control and so on. Therefore, specific and sensitive monoclonal antibodies (MAbs) against CTV and CYVCV were respectively produced, and specific and sensitive serological assays based on the prepared MAbs were developed for rapid and reliable virus detection in this thesis. The result of this study has provided technology and materiel for diagnoses, epidemiological analyses and establishments of scientific prevention and control systems of the two virus diseases.(1) Preparation and application of MAbs against CTV:The major coat protein gene(CP) of CTV Chongqing isolate 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 mice. Four hybridoma cell lines (14B10,14H11,20D5 and 20G12) secreting monoclonal antibodies (MAbs) against CTV were obtained though cell fusion、cell screening and cell cloning. The titers of ascitic fluids of MAbs secreted by four hybridomas ranged from 10-6 to 10-7 by indirect-ELISA. Western blot analysis indicated that all four MAbs could specifically react with CTV CP. Using MAbs, dot enzyme-linked immunosorbent assay (dot-ELISA), tissue blot enzyme-linked immunosorbent assay (Tissue blot-ELISA) and triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) were developed to detect CTV in citrus trees. The developed dot-ELISA and TAS-ELISA methods could detect virus in CTV-infected citrus leaf crude extracts diluted 1:2560 and 1:10240 (w/v, g/mL), respectively. Furthermore, the immune colloidal gold strip was developed to detect CTV in citrus trees using the prepared two MAbs, and could detect virus in CTV-infected leaf crude extracts diluted 1:1280 (w/v, g/ml). The establishment of the immune colloidal gold strip has realized the on-site, fast, fool detection of CTV. The sensitivity of TAS-ELISA is so high that can easy determine virus-free seedling. The field survey revealed that CTV was prevalent in citrus trees in Chongqing municipality, Jiangxi and Zhejiang provinces of China, and the coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.(2) Preparation and application of MAbs against CYVCV:To detect, analyze epidemiology and control CYVCV, capsid protein (CP) of CYVCV were prepared using a prokaryotic expression system and used as the immunogen to immunitze mice or rabbits, and four highly specific and sensitive murine MAbs and one polyclonal antibody (PAb) were produced in this thesis. Titers of the four MAbs in ascites fluids ranged 10-6 to 10-7 by an indirect-ELISA. All four prepared MAbs could strongly and specifically react with CYVCV CP and CYVCV-infected citrus leaf tissue crude extracts. Based on prepared antibodies, four serological assays, dot-ELISA, tissue blot-ELISA, DAS-ELISA and TAS-ELISA were developed. Sensitivities of the developed dot-ELISA, DAS-ELISA and TAS-ELISA for CYVCV-infected citrus leaf tissues had reached 1:2560,1:10240 and 1:20480 (w/v, g/ml), respectively. Using the developed serological assays,125 suspected virus disease citrus samples from groves in Yunnan Province and Chongqing Municipality were detected. The detection results demonstrated that 46 of 125 samples were infected by CYVCV with a 36.8% incidence rate, while none of 158 samples from groves in Zhejiang or Jiangxi provinces had contained CYVCV. The coincidence rate of serological and RT-PCR test results reached more than 99.6%. These four developed detection assays have great signaficance in CYVCV diagnosis and detection, epidemiological studies and production of virus-free citrus seedlings.