Monoclonal Antibody Preparation Of Impatiens Necrotic Spot Virus(INSV) And Virus Detection In Yunnan Samples Of Tomato Spotted Wilt Virus(TSWV)

Both of Impatiens necrotic spot virus(INSV)and Tomato spotted wilt virus(TSWV)belong to the family Bunyaviridae and genus Tospovirus.Boh of the two virus have wide host range and are mainly transmitted by thrips.The symptoms of the plants when infected by two of these virus are similar such as:dwarf,ring spot,stunting,mottling,and dark streaking of the stem.INSV mainly infects ornaental plants while TSWV infects vegetables and crops.Both of these virus are transmitted quickly throughout the world.They bring huge economic losses to growers.Myeloma cells in mice can immortalizate.The immune antigen could be injected into abdominal cavity of mice to make the spleen cells produce antibodies.But the spleen cells do not have the ability of immortalization.If myeloma cells and spleen cells could fuse together in the role of fusion agent,hybridoma will have the ability of two kinds of the cells.We must screen the cells to have a separate cell which can produce antibody.The antibody is what we called monoclonal antibody.Monoclonal antibody has high specificity and is widly used in cancer treatment.In this experiment,I extracted the total RNA from the infected plants and used the reverse transcription technology to amplificate cDNA.Then,I used cDNA as a template to build the expression vector pET28a-INSV-N,and then the protein of the INSV-N was expressed as mice immune antigen.After the dialysis for protein,we emulsificated the protein that the concentration was lmg/mL and freund's adjuvant together.The mixture was then injected into mice abdominal cavity.After 5 times of immunity,we took tail blood of mice to detect the titer by indirect ELISA.The mouse with the highest titer was taken to do cell fusion.Spleen cells in mice and SP2/0 were fused in the role of PEG1500.Seven to ten days later,I carried on the positive clone selective cultivation.At the end,we used limited dilution method to screen positive clones.1E1 and 2D9 were two of the positive clones.So,we must take them to cryopreserve.To get the antibody,I put the hybridoma into the abdominal cavity of mouse.Seven days later,I could get the ascites.The ascites must be purificated to get monoclonal antibodies.I detedted the titer of both of the MAbs by indirect ELISA.The titer of both MAbs were 1:250.Tomato spotted wilt virus(TSWV)is an important member of the genus Tospovirus.In this experiment,I successfully inoculated TSWV to benthamiana.At the same time,I used two of the methods:DOT-ELISA and RT-PCR to detect TSWV.I detected eleven samples:YN-18,YN-11,YN-34,YN-26,YN-51,YN-61,YN-80,YN-65,YN-53,YN-42 and LE.Six samples of these were diseased,and they wereYN-18,YN-11,YN-34,YN-53,YN-65 and LE.The testing results of the two methods were consistent.At last,Idetected TSWV by MAbs 1E1 and MAbs 2D9,but the results were both negative.