Production Of Monoclonal Antibodies Against Southern Rice Black-streaked Dwarf Virus And Chinese Wheat Mosaic Virus And Their Application

Rice and wheat are the main food crops in China, their production safety related to people’s livelihood. In recent years, Southern rice black-streaked dwarf virus (SRBSDV) is prevalent in southern rice production region of China, causing serious losses in rice production. The occurrence of Chinese wheat mosaic disease caused by Chinese wheat mosaic virus (CWMV) has an upward trend in recent years and affects on the production and quality of wheat. In order to grasp the occurrences, epidemic regularities of two plant virus diseases and establish early detection and warning systems, methods should be urgently developed for rapid, efficient detection of those two viruses. In this study, monoclonal antibodies (MAbs) against SRBSDV and CWMV were produced, and two serological methods based on the MAbs were developed for rapid, specific, sensitive detection of SRBSDV and CWMV. The result of this study has provided technology and materiel for diagnosis, forecast and early warning and establishment of scientific prevention and control systems of the two virus diseases.(1) MAb against SRBSDV and its application:One hybridoma cell line (3F1) secreting MAb against SRBSDV and Rice black-streaked dwarf virus (RBSDV) was produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c mouse, which immunized by a polypeptide containing12amino acids of the C-terminus of SRBSDV coat protein coupled with bovine serum albumin (BSA). selecting and cloning. The indirect-ELISAtitre of ascitic fluid of the MAb is10"6. The MAb has IgG1isotype with κ light chain. Western blot analysis indicated that the MAb could specifically react with the coat protein of SRBSDV and RBSDV. Based on the produced MAb, a dot-ELISA was established for detection of SRBSDV and RBSDV in rice planthoppers and rice samples. The dot-ELISA could detect minimum SRBSDV in infected rice tissue extracts diluted at1:320(w/v, g/mL) and in individual viruliferous rice planthopper extracts diluted at1:6400(individual leafhopper/μL), respectively. A detection kit of SRBSDV was developed based on the dot-ELISA. (2) MAb against CWMV and its application:Using the purified CWMV particles as the immunogen. four MAbs against CWMV were produced by hybridoma technique. The titres of ascitic fluids of MAbs were up to10-6in indirect-ELISA. All MAbs have IgG1isotypes and κ light chain. Western blot analysis indicated that each MAbs could specifically react with the coat protein (about19kDa) of CWMV. An antigen-coated plate ELISA (ACP-ELISA) and a dot-ELISA for detecting CWMV were established with the MAb. The ACP-ELISA and dot-ELISA could specifically detect CWMV in infected wheat tissue extracts diluted at1:40960and1:1280(w/v, g/mL).