| C-reactive protein(CRP)is an acute phase protein.When the body is damaged or infected,its content in the plasma will rise sharply.It plays a regulatory role by activating complement and enhancing macrophage phagocytosis.Remove the pathogenic microorganisms invading the body and the tissue cells of the body’s damage,necrosis,and apoptosis.As the most important,most sensitive and systematic inflammation marker in the body,CRP levels in plasma rapidly increase with the occurrence of large amounts of foreign body invasion,infection,tissue damage,kidney disease and cardiovascular disease,and can be used for a variety of diseases The diagnosis provides a reliable basis.With the passage of time,the clinical diagnosis of CRP has received more and more attention,and it has been used in the diagnosis,treatment,and prognosis of various diseases.Therefore,the diagnostic method for CRP is also constantly innovating.In the CRP detection method,most methods still rely on specific antibodies as diagnostic tools,but the traditional mouse monoclonal antibody production cost is relatively high,time-consuming,low antibody yield and other shortcomings limit its application,The egg yolk antibody(IgY)found in poultry has low production cost,can produce a large number of high-specific antibodies and is easy to prepare;the single-domain antibodies found in camel bodies can now play a good role in biological diagnosis and are derived as genes Hot research direction of engineering antibodies.In this study,CRP antigen was used to immunize laying hens,collect eggs,extract IgY,conduct long-term titer monitoring,and verify by Dot blot It can reach 1:128 000,Dot blot results show that IgY specifically binds to CRP,proving the high specificity and affinity of IgY.Immunize the Bactrian camel with CRP antigen,collect peripheral blood after final immunization,isolate lymphocytes and extract RNA from reverse transcribe into cDNA,design related primers,use cDNA as a template,and use PCR to amplify the purpose of encoding single-domain antibodies Gene fragment.The target gene fragment was cut by double enzyme digestion,and the target gene was ligated to the T7 phage carrier arm using T4 ligase.The phage library containing the target fragment was obtained by in vitro packaging.The original library capacity was 1.08×108 cfu.After amplification,the library capacity Reached 3.4×1012 cfu.Subsequently,CRP was used as a screening source for four rounds of affinity biopanning,and screening was performed by decreasing the amount of antigen to make the phage library specifically enriched After the fourth round of screening,the monoclonal plaques with high specificity were selected by Phage-ELISA,and 10 monoclonal plaques were successfully selected.Four of the 10 monoclonal plaques with the highest positive results were subjected to subsequent expression experiments.The target fragments were obtained by double digestion and ligated into the pET-28a vector to construct BL21 expression strains,which were transformed into coated plates for sequencing and PCR verification.Results The four expression bacteria were successfully established.The expression conditions were explored,and finally the induction conditions of 20℃,180 rpm,12 h,and the final concentration of 0.8 mmol/mL IPTG successfully expressed many single-domain antibodies.Indirect ELISA and Western blot methods were used to verify antibody activity,showing high specificity and affinity.I hope that specific IgY and single-domain antibodies can establish sandwich ELISA detection methods in subsequent experiments,and high-quality antibodies can also provide a reliable source of antibodies for other CRP detection methods. |
PDF Full Text Request