The Expression Of Recombinant Human Tumor Necrosis Factor Receptor-Fc Fusion Protein In Bombyx Mori

Rheumatoid arthritis (Rheumatoid Arthritis, RA) is a common diseases that involves jointautoimmune. Studies have shown that tumor necrosis factor TNFα is a key factor in RApathogenesis, and the recombinant human tumor necrosis factor receptor-Fc fusion protein(rhTNFR-Fc) is a soluble tumor necrosis factor antagonists, which can competitively bind withthe in vivo tumor necrosis factor (TNFα) specifically. So it can inhibit joint immune responseinduced by TNFα, achieving the purpose of treatment of rheumatoid arthritis.Bombyx mori Baculovirus expression system is complete new eukaryotic expressionsystem, which has advantages of low production costs, high security, high efficiency and highlevel of expression products of post-translational modification. It is an ideal expression of theexogenous protein system. In this thesis we try to use the Bombyx mori Baculovirus expressionsystem to express and purify the recombinant human tumor necrosis factor receptor-Fc fusionprotein (rhTNFR-Fc).Firstly， We build a recombinant transfer vector pFastBac Ⅰ-(rhTNFR-Fc) throughBac-to-Bac system，which was used to transform E.coli DH10Bac competent cell, and then getthe recombinant Bacmid. Secondly, we use the recombinant Bacmid to infect the BmN cellthrough the method of lipofectin-mediated, we get a recombinant baculovirusvBm-(rhTNFR-Fc）. When recombinant baculovirus F4infect BmN cell, the target protein can befound in BmN cell, silkworm hemolymph and silkworm pupa. The fusion protein rhTNFR-Fcwas purified from silkworm haemolymph, and after deglycosylation by PNGase F, rhTNFR-Fc is2~3kDa smaller than before, but its weight is larger than expected, which indicate thatrhTNFR-Fc protein is modified with N-linked oligosaccharide chains and has a potentialO-linked glycosalation. Finally, the binding activity of fusion protein rhTNFR-Fc was tested byElisa，and its neutralizing activity was tested by MTT method. The result shows that the targetprotein has a very excellent activity in vitro, which promots the next research of the therapeuticeffects of adjuvant arthritis model in rats.In this study, we demonstrated that the recombinant human tumor necrosis factorreceptor-Fc fusion the protein (rhTNFR-Fc) could efficiently be expressed and purified withbiological activity through the Bombyx mori baculovirus expression system, which provide a new approach for the efficient expression of rhTNFR-Fc protein, and also provide a reference forthe expression of other Fc fusion protein and antibody in the Bombyx mori baculovirusexpression system.