| Background and Objectives: VEGF-B is an important cell growth factor, with a variety of important physiological functions.In this study, the cloning of human beings through the VEGF-B cDNA can construct prokaryotic expression vector and high-level expression and purification, using genetic engineering techniques can produce recombinant protein;Materials and Methods: According to GenBank mature human VEGF-B state of cDNA sequences, combined pET-28a expression vector of the characteristics designs and synthesis a pair of specific primers. Method using PCR amplification of mature VEGF-B protein cDNA fragment, and then cloned into pET-28a vector to construct recombinant prokaryotic expression vector pET-28a-VEGF-B; recombinant vector into E. coli DH5αwas amplified, After extraction, using PCR, restriction endonuclease digestion and DNA sequencing methods, such as identification. The positive identification of the correct pET-28a-VEGF-B recombinant plasmid was transformed into E. coli BL21 (DE3), through the K + resistance screening to obtain stable expression of the positive bacteria; IPTG-induced bacteria to high-level expression of recombinant protein VEGF-B, The use of His-tag affinity chromatography, agarose gelatin chromatography of recombinant VEGF-B protein purification, and Western blot detectionResults:1 The success of the construction of the VEGF-B cDNA prokaryotic expression vector pET-28a-VEGF-B; The establishment of a stable into pET-28a-VEGF-B recombinant plasmid of E. coli cell line BL21-pET-28a-VEGF-B, and VEGF-B cDNA in the transformed cells was highly efficient and stable expression.2 Engineering bacteria to establish a high-level expression of recombinant soluble forms of VEGF-B protein in the culture conditions, the establishment of an efficient purification of recombinant protein, VEGF-B process, a better purification of VEGF-B protein, for the future for more in-depth study prepared basic conditions.
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