Gene Cloning Of Cat Allergen And Expression And Purification Of Recombinant Chimeric Protein

Background and Objective:Cat allergens are very important among the indoor allergens and a common cause of Allergic Rhinitis (AR) and Bronchial asthma (BA). AR and BR are common chronic diseases of respiratory tract, and the two diseases are the allergic inflammation of the respiratory mucosa caused by allergens. Allergen specific immunotherapy (ASIT) is the only treatment for the cause of allergic disease and able to improve the natural course of allergic disease. However, currently the crude allergen extracts used widely have several disadvantages. The extracts may contain unpredictable components, dose of which may be difficult to standardize and can lead to severe side-effects. While, allergens prepared by recombinant DNA technology have highly purified and highly specific advantages, which can enhance the effectiveness of treatment and reduce side effects. Currently, the recombinant protein allergen of allergic diseases has become the new research focus.Feld1 is the major allergen of cat allergens, recognized by 65%-95% of cat-allergic patients, specificity of cat allergy diagnosis with which up to 96%. So, Feld1 is an excellent marker allergen for diagnosis and treatment of cat allergy. Cytokines in allergic inflammation plays a very important regulatory role. Among them, IL-10 is an immunosuppressive factor with a variety of biological activities, which can protect human body from allergic inflammation. In this study, we restructured chimeric protein, including the major cat allergen Feld1 and human IL-10 gene. Expected through IL-10 immune adjustment function, immune response of the body to Feld1 can reduce, and induce immune tolerance, and we will obtain easy-to-standard and safe and efficient cat allergen preparations.Methods:After codon optimization with codon preference in E. coli, with full length gene synthesis, we got the gene of fusion cat allergen, including Feld1 and IL-10 gene. We used a prokaryotic expression system to express recombinant chimeric protein. Through purification by affinity chromatography, we obtained the interest protein, which was identification by Western blotting.Results:We obtained the optimized FIL nucleic acid sequence, and successfully constructed the expression vector pET44a-FIL. The recombinant protein successfully expressed in E. coli can be detected as a band of 38kDa by SDS-PAGE and optimal conditions of induction were achieved. After purification, we obtained the highly purified recombinant protein.Conclusion:1. Codon optimization can improve the expression level of recombinant protein.2. Successfully constructed expression vector pET44a-FIL.3. Recombinant proteins in E. coli was expressed in a high level4. After purification, we obtained the recombinant protein (molecular weight about 38kDa), for the subsequent study of the recombinant protein function etc. laid a good foundation.