| Bacteriacidal/permeability-increasing protein is a cationic protein with 55KD (is called BPI55), which possesses antibacterial, endotoxin-neutralizing, opsonic activity and antiinflammatory against Gram-negative bacteria so on. A recombinant 23KD protein (rBPI23) derived from human bactericidal/permeability-increasing protein (BPI) possesses all biological activity of natural BPI (nBPI55). The ability of rBPI23 to bind smooth-typed lipololysacchrides (LPS) is as 30 times as the ability of BPI55. Human acidic fibroblast growth factor (haFGF) is a member of fibroblast growth fac-tor (FGF), which can induce a kind of cells differentiation and proliferation, with car-dio- and neuroprotective characters, tissue-injury repair properties, promoting ische-mia-induced angiogenesis, promoting wound healing and so on. The wound heals is a long process, which has many barriers in the clinical treatment. It is infected easyly by bacterium, cause the bacteriemia or the pyemia, Systemic Inflammatory Response Syndrome(SIRS), result into the multi- organs dysfunction syndrome (MODS). To splice BPI23 gene, the functionality N-terminal gene of the BPI, and haFGF gene via a non-polar flexible hydrophobic linker sequence (Gly4Ser)3 into BPI23-haFGF fusion gene, and clone the fusion gene into secretion expression vector pPICZαA, then ex-press recombinants in Pichia pastoris X-33 and purify target proteins to analyze their biological functions. Which laid a solid foundation for the further research to develop a new difunctionality polypeptide drug which not only shorted wound healing time but also antibacterial activity.Methods and results①A pairs of specific primers were designed with Primer Premier 5.0, PCR obtained a DNA fragment about to 1.1kb, The fusion gene BPI23-haFGF with signal peptide which was preserved in the laboratory as a template, then the amplified fragment was cloned into the vector pUCm-T, to construct pUCm - BPI23-haFGF recombinant plasmid. The sequencing results demonstrated that the amplified sequence in-cluding non-coding signal peptide sequence BPI N-terminal 199 amino acid gene, Coding (Gly4Ser)3 Linker gene and the non-terminator haFGF gene, 1107bp size. BLAST results showed: Anterior fusion gene withthe BPI Genebank mRNA sequence (Login : 4502446) 98% homology; Integration of the gene with the haFGF Genebank mRNA sequence (Login : 15055546) completely identical.②Amplified pUCm-BPI23-haFGF plasmid , BPI23-haFGF gene fragments, which was digested by Kpn I / Not I, was inserted into the eukaryotic expression vector pPICZαA digested by those two endonucleases to construct recombinant vector pPICZαA-BPI23 -haFGF. The results of sequencing demonstrated that the construction was correct. Bioinformatics software predicted that the fusion gene codes a fusion protein: 397 amino acids, the molecular weight 43.5KD, pI of 9.24, and with BPI23 and haFGF two conservative domain, BPI23 conservative 99% sequence homology, haFGF conserva-tive 100% sequence homology.③After linearized by Sac I, the pPICZαA-BPI23-haFGF plasmid was transformed into X-33. After selected by Zeocine and identified by PCR, the steady engineered stains X-33/pPICZαA-BPI23-haFGF was obtained. SDS-PAGE(15 % gel) and Western Blot (6×His-labeling Monoclonal antibody as first antibody ) analyzed the expression su-pernatant induced by 0.5% methanol, the results showed that the concentrate superna-tant had the Specific band in 43.5KD according with the predicted size, the yield of the fusion protein is 10～20μg /ml.④Optimal conditions for inducible expression to explore different induction time, temperature, different methanol concentration and pH for the expression impact, the results showed that : 28°C, pH 5.0, Methanol final concentration of 1.0%, PMSF fi-nal concentration of 0.5M, 4d-induced expression was the best conditions , the ex-pression was up to～25μg /ml. Purified by HisTrap ? HP Affinity chromatography to separate and purify the product.The fusion protein purity is about 90%, the recov ery rate is about 50%.⑤Biological function evaluation1. In Vitro: Agar-diffusion method results showed that the fusion protein has a cer-tain inhibition and anti-gram-negative bacteria; The improving proliferation of NIH3T3 was measured by MTT assay, When the concentration is 0.40mg/ml, pro-moting the proliferation of NIH3T3 cells is most obvious, and above or below 0.40mg/ml, the impact is lower.2. In Vivo: To medicate on burn model in mice (90°C, 15 s)daybreak, to aFGF standard as a positive control, to 0.9% saline as a negative control, pathological biop-sy was made for mice damaged skin after burned 7, 14, 21 days respectively. The re-sults showed that the sample group wound has basically healing after scalded for 14 days, the capillaries and fibroblasts is more dense than negative control group, and as the standard aFGF; the sample group healed 2 or 3 days in advance than the control group.ConclusionsThe experiments constructed the secretory yeast expression vector pPICZαA -BPI23-haFGF successfully, screened of a stable engineering stain X-33/pPICZαA -BPI23-haFGF, and expressed the target protein BPI23-haFGF. In vitro, fusion pro-tein has the dual function with bactericidal and promote cell proliferation; In vivo, fusion protein can promote capillary formation and fibroblasts, and improve wound healing. It laid the foundation for the clinical application of the BPI23-haFGF.
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