Molecular Clone And Expression Of Human Plateletderived Growth Factor B Chain Mature Peptide Gene

Background/Objectives: Bone asynechia , bone defect and osteoporosis are difficulties that puzzle the orthopaedics. Bone fracture healing process is long and may lead to many complications, and there are no effective methods to treat the patients with Bone asynechia or bone defect so far. To cope with the problem, researchers have been engaging in the modulation of osseous tissue metabolism apart from the osseous tissue engineering in recent years, including the modulation of growth factors and extro-cell signal transduction. As a group of polypeptide growth factor, PDGF, expressed in many kinds of tissues and plays important roles in the physiology and pathology process of heart, osseous tissue and so on. PDGF can modulate the functional balance between OB and OC directely or obliquely, and promote bone fracture healing and bone defect repairs, which is considered as a unwounded method that replaces some operations to treat such kind of patients. The clinical application of PDGF may relieve such patients distress and reduce the length of stay. PDGF-BB is the most effective subtype in the PDGF family on the metabolism of osseous tissue or the treatment of wound healing, and its clinical application was approved firstly among the growth factors although it was used to treat the decubital ulcer of diabetes. The purpose of this study is to obtain the bioactive rhPDGF-BB by-5-means of gene engineering for the experimental study and clinical application. Human PDGF-B chain mature peptide cDNA was acquired by molecular cloning and constructed into the prokaryotic expression vector; To express human PDGF-B chain precursor in E colL and obtain the rhPDGF-BB after purification and renaturation; The bioactivity of the rhPDGF-BB was detected with in vitro cytology methods and the feasibility of rhPDGF-BB' s clinical application was priminarily accessed; To investigate initially the expression of PDGF-BB in yeast and to search optimize expression methods ofPDGF-BB.Methods: (1) Primary cultured the human vascular endothelial cells (HUVEC) , collected the cells sediment and extracted the total RNA of HUVEC.(2)The full length encoding region of human PDGF-B cDNA from HUVEC was successfully cloned using one step RT-PCR method, and the mature peptide sequence of human PDGF-B was amplified from the ecnoding region cDNA . (3)The mature peptide of human PDGF-B was constructed into T-vector and sequenced, and then was cloned into a prokaryotic expression vector pQE-30 which can expresse PDGF-B precursor fusion protein with six-His tag on the N-terminal in E colLThe recombinant plasmid pQE-PDGF-B was identified by polymerase chain reaction and restriction endonuclease maping. (4) The expression vector pQE-PDGF-B was transformed into E cott. Ml5, which was induced by IPTG to express PDGF-B chain precursor under 37 centi-degree. The expression status of PDGF-B polypeptide was analysed by 15% SDS-PAGE polyacrylamide gel electrophoresis. (5)The immunogenicity of rhPDGF-B chain precursor was identified with Western-blot. (6)The renaturation and purification of rhPDGF-BB. Expression E coli. M15 was induced Abundantly and lysised. then the inclusive bodies were washed and lysised. Purified the PDGF-B precursor with Ni2+NTA affinity chromatography and renarured it by means of GSSG/GSH buffer, thus the rhPDGF-BB was acquired. The objective protein was analysised by SDS-PAGE. (7)Bioactiviry detection in vitro of rhPDGF-BB. Primary cultured the SD rat osteoblasts andinterfered with rhPDGF-BB. Detected its bioactivity of enhancing osteoblasts proliferation using MTT, and verified its effect on osteoblasts DNA reduplication with FCM. (8)The construction and veritification of the yeast expression vectors of human PDGF-B gene. The PDGF-B chain mature peptide cDNA (with or without signal peptide) was amplified from the encoding region cDNA with two pairs of special primers using PCR and was cloned into the yeast expression plasmids pMETB and pMETA, named pMETB-PDGFB2 and pMET a A-PDGFB3.Results: (l)The full length ecnoding region of human P...