Construction, Expression And Characterization Of Disulfide Stabilized Fv Fragment Antibody Against Human Lipopolysaccharide Binding Protein

Septic shock and multiple organs dysfunction syndrome (MODS)are the two fundamental reasons which lead to death in Gram-negative bacteria infection. It has been proved that lipopolysaccharide(LPS)is the main causative agent. Lipopolysaccharide Binding Protein(LBP)is a kind of emergency reaction proteins that conduct the binding of lipopolysaccharide to its receptor on the target cells. Therefor LBP plays an important role in inflammatory reaction.It has been known that LBP can promote the binding between lipopolysaccharide and membrane CD14 (mCD14) of mononuclear/macrophage cells,to form the complex of LPS-LBP-mCD14 and to activate mononuclear/macrophage cells by TLR4-MD2-MYD88 and NF-kappa B , which also can accelerate the occurrence and development of inflammation. The N-teminatal fragment of human LBP (NH-LBP) combineds LPS,So if the antibody against LBP especially NH-LBP can be obtained,the binding between LBP and LPS could be blocked,too; which will lighten the damage of infection and reduce the death rate.In this study,we screened human phage libraries by human LBP and NH-LPB and got one of the clones producing antibody with better activity against LBP and NH-LBP. After PCR and vector construction, mutation was introduced and dsFvVH / dsFvVL fragment was expressed by Escherchia coli BL21 star. Then the inclusion bodies of dsFvVH and dsFvVL were isolated. The inclusion bodies were purified and the protein was folded by disulfide bind and the activity of disulfide stabilized Fv fragment antibody(dsFv) was characterized at last.The main resμlts are as following:1,After 5 rounds of screening and enrichment of human antibody library(Fab)using human LBP as antigen, the tite of the phage library was raised from 3.8×10⁸ to 3.1×10¹³. The phage was concentrated to 8.15×10⁴.2,One clone of Escherchia coli XL1-Blue which was called NO.4 producing antibody...