Cloning, Expression Of Human N-terminal Truncated Lipopolysaccharide Binding Protein (tLBP) Gene And Protection Of TLBP

The tLBP, appropriately modified and truncated of human LBP, is a 27-kDa polypeptide which is 0.7kb of gene fragment and encodes 197 aa of parent molecule. The tLBP would be cut down 25 aa signal peptide while secreting into endoplasmic reticulum. This molecule can presumably bind LPS due to reserving the binding domain with LPS , but it loses the ability to transfer LPS to the CD14. Subsequently, it is capable to inhibit the LPS-CD14 formation and block the over-secretion of inflammatory mediators and cytokines from monocytes/macrophages. But it is still obscure that tLBP plays a bioactive role and reacts with LPS.In this study we have designed the primers amplifying tLBP cDNA and cloned the target gene fragment from human whole length LBP cDNA. The target gene was inserted into baculovirus vector pFASTBAC and performed successfully specific transposition and virus recombination of target gene. tLBP recombinant protein was expressed and purified by Bac-to-Bac baculovirus expression system, sf21 insect cells and chromatography of TALON metal affinity resin. The protein product was identified by Lowry, SDS-PAGE, Western blot and capillary electrophoresis. The conjugating effect of tLBP against LPS in vitro and in supernatants of U937 cells was determined by enzyme-linked immunosorbent assay(ELISA). The results showed that recombinant tLBP was able to bind LPS in vitro and also bind or neutralize LPS in evaluation of cell bioactivity.Furthermore, the dynamic comparison experiment was observed by LPS injuring and tLBP protecting in Kunming mice, and its mortality was evaluated after lethal LPS challenging and tLBP protecting. The results showed that recombinant tLBP possessed some protection role to mice with LPS damage. They suggested that tLBP presumably bind or neutralize part of LPS and attenuate the release of related inflammatory factors and chemical mediators. Therefore, it was found that recombinant tLBP possesses some bioactivity afterpreliminary experiments of cell activity in vitro and animal model. So we guess that tLBP, expressed in insect cells/baculovirus expression system, may correctly fold and form the advanced structure similar to human natural N-terminal LBP molecule and recombinant protein tLBP may be expressed similar to original molecule. The main results are as following: 1.The 700±bp gene fragment was successfully generated and confirmed by DNA sequencing. The transfer plasmid pFASTBACtLBP and transposition plasmid pBacmidtLBP of baculovirus were also constructed. It was approved that the gene transposition and specific virus recombination were performed from the constructed plasmids.2.It was found that the expressed protein was a 27 kDa , with the purity more than 98%, the total product of 2.7mg using BAC-TO-BAC baculovirus expression system, sf21 insect cells and TALON metal affinity resin chromatography. The bioactivity assay showed the product could bind LPS in vitro.3.Our data suggest that tLBP product may bind or neutralize LPS in cell level and the contents of cytokine were drop dwon in cell supernatant. It was presumed thatthe tLBP product expressed from insect cells may collapse and form correctly the advanced structure similar to the N-terminal structure of natural human LBP.4.The results of animal experiment indicated that the recombinant tLBP was able to bind or neutralize LPS in vivo in which manifested in decrease of death rate, improvement of liver function, alleviation of tissue lesion and dropping down of cytokine in serum. The tLBP may play a protection role in damage or lethal challenge caused by LPS.5.In this study we firstly gained the human recombinant tLBP by newly baculovirus expression system and explored its bioactivity in vivo in animal model with endotoxemia. Therefore, these results will found a basis for clarifying the structure and function of LBP and studying the prevention and cure of trauma (burns) and clinical infection...