Design, Expression And Function Study Of IgG Bindinrotein CBD-SPG

Streptococcal Protein G, a bacterial cell wall protein whose C terminal has the affinityfor immunoglobulin G （IgG）. Cellulose Binding Domain of cellulase, which has a wedge-likestructure that is involved in cellulase adsorption without degradation of cellulose. In this study,we attempt to construct a fusion protein composed of CBD and SPG domain, which has boththe function of IgG binding and cellulose affinity. It is a novel tool in immunoassay andaffinity chromatography applied in purification of IgG.We amplified the coding sequence of CBD_closgene from Clostridium cellulovorans andCBD_trigene from Trichoderma viride as template, and then inserted them into thepET28a-SPG, a E.coli transfer vector. The cloned pET28a-CBD_clos-SPG andpET28a-CBD_tri-SPG plasmid was transformed into E.coli BL21（DE3） and screened bykanamycin to get a positive recombinant. Recombinant E.coli BL21（DE3） cells were culturedin LB broth containing kanamycin until the optical density reaching to1at30℃. Then cellswere induced to express CBD_clos-SPG、 CBD_tri-CBD by addition of Isopropylβ-D-Thiogalactoside. The40kDa and30kDa protein can be detected by this method. TheCBD_clos-SPG、CBD_tri-CBD purified using Sgmacell50column exhibited plenty of fusionprotein binding capacity. After elution, the purity was greater than95%as estimated fromthe protein gel. For the binding assays, the purified CBD_clos-SPG、CBD_tri-CBD was boundonto different matrix first to check the combining weight of CBD_clos-SPG、CBD_tri-CBD toseveral different materials, then IgG capacity was examined. The result showed that Avicelph101, the CBD_clos-SPG binding capacity is11.61mg/g（w/w）, while the capacity of IgG is25.73mg/g（w/w）.In addition, our study has explored the application of CBD_clos-SPG. CBD_clos-SPG bindingcellulose is used to purify IgG in rabbit serum and antigen capture. The result showed thatCBD_clos-SPG binding cellulose can be used to purify IgG in rabbit serum and capture fusionprotein IFNβ-HSA by using HSA antibody.In this thesis, we obtained bi-functional protein CBD_clos-SPG、CBD_tri-CBD by designing,constructing vectors and expressing them in E.coli. We showed that the fusion proteinCBD-SPG has dual binding capacities of both IgG and cellulose. It can be used as a novel toolin fields of immunoassay and affinity chromatography.