Expression And Purification Of Sycamore Pollen Allergens Pla A1 And Pla A3 And Preparation Of Monoclonal Antibodies

Platanus acerifolia(P.acerifolia)is one of the major sources of outdoor allergens for humans,which induce allergic asthma,rhinitis,dermatitis,and other allergic diseases.So far,three major allergens have been identified in P.acerifolia pollen,including Pla a 1,Pla a 2 and Pla a 3.Pla a 1 is a non-glycosylated protein and has a prevalence of 84%among Platanus-allergic patients in Western European cities.Pla a 2 is a glycoprotein and is related to the allergic responses of 84%of patients with Platanus-induced pollinosis worldwide.Pla a 3 is a non-specific lipid transfer protein(nsLTP)and 45%of Spanish patients with P.acerifolia pollen allergies were sensitized to the natural Pla a 3.The aim of this research is to express and purify Pla a 1 and Pla a 3 in Escherichia coli(E.coli)expression systems,to prepare their monoclonal antibodies of recombinant Pla a 1 and Pla a 3.It will provide a basis for clinical diagnosis and vaccine development of P.acerifolia allergy.The paper contains two major parts.Part 1:The expression and purification of Pla a 1 and Pla a 3.Pla a 1 and Pla a 3 genes were sub-cloned into vector and transformed into Arctic Express^TM(DE3)RP E.coli host strain.Isopropyl-b-D-thiogalactopyranoside(IPTG)was added to the final concentration of 0.5 mM and the culture was incubated for further 4 h.The bacterial cells were harvested by centrifugation at 4? for 10 min.The inclusion bodies were collected by centrifugation at 4? for 20 min.The supernatant and the inclusion bodies were loaded on the Nickel column(Genscript,Nanjing,China).Part 2:The purified Pla a1 and Pla a 3 were used to immunize BALB/c mice.When the serum detection was positive,spleen cells from the mice were isolated and fused with SP2/0 myeloma cells.The ascites antibodies were induced and prepared by the positive cell line,and then were purified by protein A-agarose.