Generation And Primary Selection Of Antibody Library Specific For Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum(Lib) de Bary is a filamentous Ascomycete fungus with a wide host range and geographical distribution,which is one of the most non-specific, omnivorous and successful plant pathogen.In China,Sclerotinia sclerotiorum causes the number one disease of the three most serious diseases of rapeseed plants and affects the rapeseed production greatly.After long time screening,no completely resistant cultivars have been identified in rapeseeds,as well as in the closely related species.Resistantance to Sclerotinia sclerotiorum in rapeseeds is quantitative trait and easily affected by environment condition.There is no accepted widely identification resistance method. These circumstances make breeders difficult to select stable resistance plants.Antibody and their fusions have been reported to create fungus-resistant plants as a novel approach developed recently.Fungal cell wall and cell wall protein antigens prepared from S.sclerotiorum were used for immunization of chickens and mouse in this study.Eggs of the chickens were collected at the fifth day after final immunization with antigens.Polyclonal antibodies were purified from the chicken egg yolks,and their reactivity and specificity towards the corresponding fungal antigens were evaluated under optimized conditions.Chicken antibodies were shown a high specificity and reactivity,but there were cross reaction between antigens.Antisera of the mouse were isolated at the fifth day after last immunization with antigens and their titer of the polyclonal antibodies were tested.The result showed that polyclonal antibodies had high reactivity and better specificity.Total RNAs from immunized animals were isolated and mRNA were purified that were then subjected to cDNA synthesis.Heavy-chain and light-chain fragment of variable were amplified by PCR using the synthesized cDNA as templates.Then,heavy-chain and light-chain fragments of variable domains were cloned into phage display vector, resulting in V_H and V_L libraries both of which contained about 5×10~4 independent clones with an inset size of 400 bp.Single-chain fragments of variable library were constructed by recovering V_H fragments from V_H library and cloning into the V_L library or vice versa to generate the V_L to V_H library.The total capability of both libraries was about 1×10~6 and used for selection of antibodies by phage display.Several rounds of panning were performed by phage display,resulting in selection of one single-chain antibody with high affinity.The current study provided an antibody that would be used for construction of antibody fusion for enhancement of resistance to Sclerotinia sclerotiorum in plants.