| Summary: | 碩士 === 東海大學 === 食品科學系 === 96 === Toll-like receptor 2 (TLR2), one of TLRs that recognizes a broad range of microbial components, is a membrane receptor which is closely related to innate immunity. Fc fragment of IgG can induce the polarization towards Th1 that may help to relieve hypersensitivity. Previous studies have demonstrated that the signal transduction via membrane TLR2, was decreased by soluble TLR2 (sTLR2) in breast milk and plasma, thereby minimized the risk of allergy. In this study, we intended to express the functional sTLR2-Fc fusion protein to evaluate its potential protective role toward asthma. Full coding sequences of TLR2 was cloned to a T&A cloning vector. After trimming out the TLR2 stop codon and toll/interlukin-1 receptor, TLR2 was combined with Fc fragment which contains hinge, CH2, and CH3. The hybrid construct and wild-type TLR2 control were then subcloned to pIRES2-EGFP for eukaryotic expression in human embryo kidney cell line HEK-293, after DNA sequence verification. The estimated molecular weight of expression proteins are 87, 90, and 112 kDa. The result show that sTLR2 and Fc in breast milk consisted more than one form. Human breast milk obtained in one week and one month after childbirth contained four forms in sTLR2 ranging from 55~95 kDa, and two forms in Fc of IgG ranging from 28~72 kDa. The expressed protein wild-type TLR2 and recombined sTLR2-Fc proteins will be manifested by Western blot analysis.
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