Screening And Preparation Of Single Chain Variable Fragment Against Soybean Gly M Bd 28K Protein

Soybean(Glycine Max),because of its rich nutrition these soy products as food ingredients Feed ingredients are widely used.However,soybean allergen can cause a severe allergic reaction to sensitive individuals.Error label of food product,cross contamination in food processing and component contation uncharted soybean ingredients may cause allergic individual contact allergens and threat to health.In order to minimize the accident allergic food intake,label must provide accurate information about the composition of food product.Some countries have developed labeling guidelines for the protection of individuals with allergies,but the enforcement of these regulations depends on the sensitivity and accuracy of the analytical methods.The allergen detection methods mainly include DNA and immunochemical technical based on protein.According to this,DNA molecules are more stable than protein,and it is not affected by geography and seasonal change.However,although its sensitivity is very high,but using DNA analysis is controversial in the allergen detection,because it does not directly detect allergen or any specific protein.Tests target can be allergen or marker proteins based on the interaction between antibody and antigen,Enzyme-linked immunosorbent assay(ELISA)is the most widely used technique for routine screening of allergens in food.Phage display technology is an alternative to the traditional method of antibody production.It can produce a large number of antibody of which amino acid sequence is clear and stable.After construction of recombinant phage library,high affinity antibodies will gain through vitro screening,phage display technology has been successfully applied to the monitoring in the food of salmonella typhi or detection of clostridium difficile spores in milk and so on.The aim of this work was to isolate specific recombinant antibodies against soybean allergen Gly m Bd 28 K employing a phage-display library of single chain variable antibody fragments(sc Fv),and to screen the high affinity scFv using the recombinant Gly m Bd 28 K.Gly m Bd 28 K recombinant proteins were prepared through E coli expression system.Chicken was immunized with Gly m Bd 28 K.After five immunizations,spleen was collected,and then RNA was extracted and reversed into cDNA.First,we amplified VH and VL using cDNA as template;second,the scFv fragment was prepared by overlap PCR.After ligation with arm T7 Select10-3b,ligation reactions can be added directly to T7 Packaging Extracts for in vitro packaging.In this paper,Phage library was used for biopanning,and the positive monoclonal phage was screened in the forth elution phage library,and the high specific antibody genotype was determined by Phage-ELISA.We amplified the sc Fv fragment using positive phage as template.We constructed the pET-30a/scFv recombinant plasmid.The plasmid was transformed into the BL21(DE3)to express the protein.The protein was identified by WB and SDS-PAGE.