Construction And Eukaryotic Expression Of Anti-LPS Fab,BPI And Fab-BPI Fusion Protein

Lipopolysaccharides (LPS) constitute the major cell wall component in all Gram-negative bacterial (GNB) families. It is now believed to be the primary trigger key of pathophysiologic events in sepsis whether or not gram-negtive bacteramia is present. GNB sepsis or septic shock, is a catastrophic and freoriently fatal systemic syndrome characterized by hypotension, inadequate organ perfusion and multiple organ failure. Having been increasing in prevalence since the 1950s, it cause an estimated a mortality rate of 10-15% in children and up to 40% in adults. Aside from antibiotic and supportive care, no specific pharmacotherapy is available for GNB sepsis or associated organ failure.Recently, adjunctive immunotherapy has been studied as a means to neutralize circulating LPS. Anti-LPS monoclonal antibodies (mAbs) have been studied extensively in the laboratories and in large clinical trials since the 1990s. Antibodies directed towards the highly conserved core glycolipid and lipid A of LPS were expected to provide broad cross-protection against LPS from a variety of pathogenic Gram-negative organisms. But a lot of mAbs even E5 and HA-1A mAb which known to be promising and much-anticipated anbodies have ended with essentially negtive results because of their much great specificactivity. Now a broad cross-reactive mAb against LPS has been prepared in our laboratory with LPS-HDL complex that various LPSs will form in vivo. And the mAb may neutralize broad LPSs from a variety of pathogenic GNB in animal experiment.On the other hand, despite those setbacks, recent findings have provided a novel cationic protein named bactericidal permeability increasing protein (BPI), which is an endogenous human protein found principally in the primary granules within neutrophils. This 456 amino acid protein has antibacterial actions and also binds with high affinity to the lipid A component of LPS, The N terminal domain of human recombinant BPI is now in extensive laboratorial even clinical trials in sepsis. But one potential problem with this recombinant protein is its short serum half-life except its limited cross-reactivity to LPS and competitive inhibitory of LBP.So, in our studies, a favourable fusion proein of BPI and the Fab were constructed and expressed in CHO cells on the base of expression of BPI and the Fab respectively.1. Cloning and expressing of BPI N-terminal cDNAA 726bp cDNA of BPI N-terminal was obtained using RT-PCR from total RNA extracted from human PMN, and cloned into pGEM-T easy vector, pcDNA3 vector and pGEX-4T-l vector step by step. The DNA seqnencing results showed that the cloned BPI gene encode a signal peptide and bioactive N-terminal fragment of BPI. After transfected with pcDNA3-BPI recombinanted vector, CHO cells expressed a 25kD protein that could react to anti-BPI antibody in immunofluorescence assay and western-blot. Meanwhile, the expressed products could react to E.coli J5 LPS in ELISA, and BPI mRNA could be dectected in these cells by RT-PCR.2. Cloning and expression of Fab fragment of C3A2 mAb against LPS Fd and light chain (LC) cDNA were obtained using RT-PCR from mRNAof C3A2 cells. On the basis of constructing pcDNA3-Fd and pcDNA3-LC recombinant vector, a 1.7kb fragment was amplified from pcDNA3-LC, containing hCMV, LC and BGH polyA sequence, and inserted into upstream of hCMV in pcDNA3-Fd vector to construct Fab expressin vector pcDNA3-Fab. ELISA and immunof luorescence assay revealed that anti-LPS Fab were expressed in the culture medium and cell cytoplasm, which presented as a 51kD protein in western-blot.3. Cloning and eukaryotic expression vector construction of Fab-BPI fusion proteinAn 180bp fragment containing bioactive center of BPI was cloned and linked to the downstream of Fd sequence in pcDNA3-Fd vector. Then the 1.7kb LC expression sequence was inserted into pcDNA3-Fd vector as above to construct Fab-BPI fusion pretion expression vector pcDNA3-FB. ELISA and immunofluorescence assay revealed that Fab-BPI fusion protein (FB) were expressed in the...