The Cloning And Expression Of Derf 2, The Main Allergens Of Dermtophagoides Farinae, And The Pilot Study On Molecular Reconstruction

Background: The difficulties in quantification and standardization of allergen extracts have been hindered their clinical utilization. On the other hand, our research on allergen proteins showed that there was a discrepancy in mite allergens between native and abroad mite materials; immunotherapy with abroad mite allergens sometimes would take side-effects on patients.Objective: To reduce the allergenicity of the main allergens ofDermtophagoides farinae, Der f2, Der f1 and improve the security in clinical application, gene reconstitution on Der f2, Derf1 will be done.Methods: The genes were cloned from house dust mite by RT-PCR. The antigenicity of Der f2 and Derf1 was analyzed by using bioinformatics software SYFPEITHI on-line to find the fragments with high antigenicity; then amino acid replacement was donetheoretically. The overlapping PCR and bridge PCR were used to change the gene order. Finally, comparison of the spatial structures between mutant and original allergen were made by software.Results: The main allergen of mites, Der f2, has been cloned successfully; The antige icity of Derf2 and Derf1 were analyzed to determine the segments which have more contribution to antigenicity, then replacement mutation were done in these segments in computer level; it is showed that there are some differences between original and altered proteins in tertiary structure. The mutations were done by overlapping primers and bridge PCR at the same time. Some altered segments have been obtained, and the construction of expression vector for original derf2 was also carded out.Significance: A methodological system for gene cloning and antigenicity estimating was established and commenced an attempt to modify an individual allergen, which can be used in future research. In addition, the post translation modification maybe contribute to allergen protein function diversity, although a little difference was found in the bases between local Dermtophagoides farinae allergens and abroad allergens gene.