Tạo dòng, biểu hiện và tinh sạch nhân tố tăng trưởng nguyên bào sợi 1 (FGF-1) người trong Escherichia Coli

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Tác giả: Tất Trường Đặng, Văn Hiếu Trần, Minh Thượng Trịnh

Ngôn ngữ: vie

Ký hiệu phân loại: 571.538 *Tissue culture

Thông tin xuất bản: Công nghệ Sinh học, 2014

Mô tả vật lý: 615-621

Bộ sưu tập: Metadata

ID: 505095

FGF-l (Fibroblast growth factor-I) is a pluripotent growth factor which can possess broad mitogenic and cell survival activities, and is involved in a variety of biological processes, including embryonic development, cell growth, tissue repair, cellular regeneration, angiogenesis, and so on. With such potentials, FGF-l has now been applied to many fields such as cosmetics, treatment to cardiovascular diseases, wound healing. In this study, the authors proceeded to overexpress recombinant protein FGF-l in E. coli for low-price FQ.F-l production towards applications in Vietnam. The fgfl gene of 423 bp encoding for human FGF-l was synthesized by PCR using plasmid pIDT -fgfl as template and cloned into plasmid pET -His under the control of strong T7 promoter. The gene in correction of recombinant plasmid, termed pET -fgfl, was confirmed by PCR, restriction enzyme digestion and DNA sequencing. After that, plasmid pET-fgfl was transformed into E. coli BL21(DE3) to establish the recombinant E. coli BL21(DE3)/pET-fgfl. When the recombinant E. coli BL21(DE3)/pET-fgfl was shakily cultured in LB medium containing ampicillin, subsequently added with IPTG to ,induce the T7 promoter, FGF-l was over-expressed and accumulated as inclusion body in cytoplasm with content accounted for 22 percent of total protein after 16 h induction using SDS-PAGE, Western blot, and Quantity-One softwave analyses. Inclusion bodies harboring FGF-l was solubilized by Guanidin 4M and was refolded using diluted method to isolate solubilized FGF-l. Heparin-affinity chromatography was conducted to purify FGF-l. Results indicated that the recombinant protein was eluted at NaCl 1.2M and NaCl 0.4M with the purity of 93.4 percent and 50.1 percent, respectively. The former purity had in vitro NIH 3T3 stimulating capacity equal to that of commercial product, having EDso = 657 pg/ml.
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