Eucaryotic Expression And Bioactivity Evaluation Of Fused Protein HLY-hbFGF

Human lysozyme (EC3.2.1.17) is a bacteriolytic enzyme that is widely distributed in avariety of tissues and body fluids. It hydrolyzes preferentially the β-1,4glycosidic linkagesbetween the N-acetylmuramic acid and N-acetylglucosamine groups that occur in thepeptidoglycan cell wall structure of certain microorganisms and lead to bacterolysis,particularly of Gram-positive bacteria, and therefore appears to have a role in host defense.Furthermore, it could bond viral protein with negative charge direcdly. For this reason, studyhuman lysozyme on antibiosis, dephlogisticate and antivirus to have considerable significanceon exploit original antibacterial.Human basic fibroblast growthfactor(hbFGF) is a biotic activator which reside in variouskinds tissue of human body extensively. It is an important member of the FGF family, a potentangiogenic molecule in vivi and in vitro stimulates wound healing, and tissue repair.is one ofimportant wound healing factor in vivo.On the wound healing process, in order to full play the function of human lysozyme andhuman basic fibroblast growthfactor, at the same time, to consider their spatial structure andhave more thrombase in human body wound. We expectly fused huaman lysozyme andhuman basic fibroblast growthfactor. Between the two proteins, a thrombase cleavage sitepeptide(LVPRGS) was introduced.We construct a multifunctional fusion protein in humanbody wound.The major achievements in this study were as follows:1. A human total mRNAwas acquired from human leukocyte, and the cDNA which encodes human lysozyme wasobtained by RT-PCR. Eukaryotic expression vector was onstructed and secreted expressed inPichia pastori. The expressed rhLY activity was up to533U/ml by using improved Shugarmethod under the optimized conditions；2. hLY gene and hbFGF gene were fused withspecific primers by RT-PCR, between the fusion protein introduce a polypeptide whichrecognized by thrombase. Fusion gene was ligated into pPIC9K vector and expressed in Pichiapastoris. Determination of the supernatant activity was to42U/ml by using improved Shugarmethod；3. Fusion protein hLY-hbFGF was extracted and purified by means of ammoniumsulfate precipitation and Sephadex G-75gel chromatography. After thrombase cleavage, theactivity analysis indicated that lysozyme activity was416U/ml,which had considerableimprovement.