A Study Of Cloning, Expression Of Endotoxin Binding Peptide Gene And Its Antagonistic Effect

It was reported that N-terminal of lipopolysaccharide binding protein (LBP) binds lipopolysaccharide (LPS) and its C-terminal binds the CD14, then forms a triad-complex of LPS-LBP-CD14. LBP enhances the effect of LPS and initiates the subsequent release of inflammatory mediators and cytokines from monocytes/ macrophages triggered by LPS. A truncated form of human LBP, NH-LBP is a 27-kD peptide containing 197 amino acid residues of the parental molecules. NH-LBP can bind LPS efficiently but not transfer LPS to CD14. Analysis of the sequence of the NH-LBP gene showed that the LPS specific binding domain is located between the 109th and the 125th amino acid residues, comprising of 17 amino acid residues. In order to find a more effective and safer endotoxin antagonist, we have modified and truncated the NH-LBP molecule appropriately and designed a new endotoxin binding peptide (EBP) gene based on the reported gene sequence of NH-LBP. Two terminators favored by E.coli were added to the new gene. We isolated and purified EBP effectively, and obtained EBP with biological activity for the first time with gene cloning method and prokaryotic expression system.Based on the evaluation of its activities in vitro, we have observed its protection effects on a rat model of burn combined with endotoxemia.The main results are as follows:1. Recombination and expression of endotoxin binding peptide gene(a) A pair of primers were designed based on the LPS-binding site of NH-LBP amino acid sequence and the based on rules of primer design. The upstream primer contains a HindⅢ site; the downstream primer contains a BamHⅠsite and two terminators favored by E.coli.(b) The 93bp endotoxin binding peptide gene was cloned with PCR from the amino-terminal of human LBP cDNA. PCR products were analyzed with electrophoresis in 2% agarose gel and the gene fragments were recovered with DNArecovery kit. The results showed that the band of the PCR product was single and accordant with the predicted size.(c) The 93bp target fragment was cut with endonuclease BamHⅠand HindⅢ, and then inserted into expression plasmid Pinpoint Xa-3 processed in the same manner.(d) After the constructs being transformed into E.coli DH5α, the positive recombinants were identified with restriction enzyme analysis and DNA sequencing analysis. The results showed that the Pinpoint Xa3-EBP recombinant plasmids were constructed successfully.(e) To express the recombinant fusion protein, we have used E.coli DH5α as the expression host. Under the induction of IPTG, a 16.5kD EBP fusion protein was expressed in E.coli and could be detected by SDS-PAGE and Western-blot. The appropriate inducing condition is 0.1mmol/L IPTG at 37℃ for 4 hours. The fusion protein was expressed in the form of inclusion. The yield of the fusion protein was 33% of the total bacterial protein.2. Isolation, purification and biological activity determination of EBP(a) The inclusion bodies of fusion protein were extracted from cultured engineering E.coli DH5α and dissolved in 8mol/L urea. The EBP-biotinylated fusion protein was captured and purified with SoftlinkTM Soft-release avidin resin.(b) After cleavage with factor Xa, the target protein EBP was separated and purified with Sephdex G-25 gel and reverse phase HPLC. The purity of EBP detected with RP-HPLC was higher than 99%.(c) The activity of EBP was recovered in GSH oxidation/deoxidization system. The concentration of EBP was 1.28mg/ml and its total product was 13mg.(d) We have observed the effect of EBP on cultured human monocytes U937 incubated with FITC-LPS. The fluorescence intensity on the surface of U937 cell was detected with a flow cytometer. The results showed that EBP partly neutralized the FITC-LPS bound to the U937 cells.(e) We have also observed the effect of EBP on LPS-dependent activation to produce tumor necrosis factor (TNFα) and interleukin-6 (IL-6) on U937 cell at different dose and time points. The contents of TNFα and IL-6 in the supernatant of cultured cells was assayed by ELISA...