• 1.瀘州醫(yī)學院附屬醫(yī)院血管外科（四川瀘州 646000）;;
  • 2.復旦大學遺傳工程國家重點實驗室（上海 200433）;;
  • 3.上海第二醫(yī)科大學附屬第九人民醫(yī)院血管外科（上海 200011）;

目的  在大腸桿菌高效表達人血管內(nèi)皮生長因子蛋白質(zhì)。
方法  用PCR從人胎兒腦cDNA文庫擴增血管內(nèi)皮細胞生長因子（VEGF）cDNA，得到517bp的DNA片段。擴增片段重組到M13mP18中，經(jīng)測序證實為VEGF165cDNA。將該片段重組到PRL621溫控表達質(zhì)粒中，在大腸桿菌表達20kd的重組蛋白。
結果  該表達產(chǎn)物占菌體總蛋白的35%，其N-端15個氨基酸序列與天然VEGF165蛋白相應序列一致。
結論  工程菌TG1/PRL621/VEGF高效表達人VEGF165蛋白質(zhì)。

引用本文： 何延政,劉建平,謝毅,張培華. 人血管內(nèi)皮細胞生長因子cDNA克隆和在大腸桿菌的高效表達. 中國普外基礎與臨床雜志, 2001, 8(2): 72-74. doi: 復制

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  1. 1. Anne A, Knud SC. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia [J]. Circulation, 1999; 99(17)∶2967.
  2. 2. 何延政，張培華，劉建平等.重組血管內(nèi)皮生長因子在大腸桿菌的高效表達 [J].中華實驗外科雜志，1997； 14(5)∶263.
  3. 3. 何延政，張培華.血管內(nèi)皮細胞和血管病的基因治療 [J].中華實驗外科雜志，1995； 12(增刊)∶119.
  4. 4. 何延政，張培華.腺病毒介導的體內(nèi)基因轉移 [J].中華實驗外科雜志，1995； 12(增刊)∶121.
  5. 5. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, a laboratory manual [M]. Second edition, New York:Cold Spring Laboratory Press, 1989∶16-688.
  6. 6. Leung DW, Cachianes G, Kuang W J, et al. Vascular endothelial growth factor is a secreted angiogenesis mitogen [J]. Science, 1989; 246(4935)∶1306.
  7. 7. Shen, BO. Homologous upregulation of KDR/Flk1 receptor expression by vascular endothelial growth factor in vitro [J]. J Biol Chem, 1998; 273(6)∶29979.
  8. 8. Mohanraj D. Expression of biologicaly active human vascular endothelial growth factor in yeast [J]. Growth Factor, 1995; 12(1)∶17.
  9. 9. Perets D, Goren HG, Safram M. Glyosylation of vascular endothelial growth factor is required for its mitogenic activity [J]. Biochem Biophys Res Commun, 1992; 182(3)∶1340.
  10. 10. Yeo TK. Glyosylation is essential for efficient secretion but not for VEGF [J]. Biochem Biophys Res Commun, 1992; 179(3)∶1568.