人血小板衍生生长因子B链基因酵母表达载体的构建

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (03): 273-276.

人血小板衍生生长因子B链基因酵母表达载体的构建

杨春露¹, 陈建庭¹, 邓凡², 唐焕章¹, 谭小云¹, 罗深秋²

1. 第一军医大学南方医院脊柱骨病科, 广东, 广州, 510515;
2. 第一军医大学细胞生物学实验室, 广东, 广州, 510515

出版日期:2004-03-20 发布日期:2004-03-20
基金资助:
收稿日期:2003-9-12。
基金项目:国家自然科学基金(30271321);广州市科技攻关基金(2001-Z-035-1-3)
作者简介:杨春露(1971- ),医师,第一军医大学在读硕士研究生,E-mail:yclj002@163.com

Construction and identification of yeast expression vectors containing human platelet-derived growth factor B-chain gene

YANG Chun-lu¹, CHEN Jian-ting¹, DENG fan², TANG Huan-zhang¹, TAN Xiao-yun¹, LUO shen-qiu²

1. 第一军医大学南方医院脊柱骨病科, 广东, 广州, 510515;
2. 第一军医大学细胞生物学实验室, 广东, 广州, 510515

Online:2004-03-20 Published:2004-03-20

摘要/Abstract

摘要： 目的构建带信号肽和不带信号肽的人血小板衍生生长因子B(PDGF-B)链基因酵母表达载体,探讨该基因在酵母中的表达效率和活性。方法利用逆转录-聚合酶链反应(RT-PCR)从人血管内皮细胞总RNA中扩增出PDGF-B的cDNA,克隆入pGEM-T载体,进行序列测定。进而克隆至酵母表达质粒pMETB和pMETαA并进行重组子双酶切鉴定。结果扩增获得了含信号肽(578 bp)和不含信号肽(340 bp)的编码PDGF-B链基因;构建了PDGF-B的酵母表达载体pMETB-PDGFB₁,pMETαA-PDGFB₂。测序结果显示,重组载体中目的基因序列与GENE BANK公布的序列一致。结论人血小板衍生生长因子B链基因酵母表达载体构建成功,为进一步在酵母中的高效表达奠定基础。

Abstract: Objective To investigate the expression efficiency of human platelet-derived growth factor B-chain (PDGF-B) gene in yeast and assess the activity of the expressed product. Methods A full-length complementary DNA of human PDGF-B gene was amplified from the total RNA extracted from human vascular endothelial cells using reverse transcription (RT)-PCR and then cloned into pGEM-T vector. The PCR products with specific primers were recombined into yeast expression plasmids pMETB or pMETαA, followed by identification with restriction endonuclease. Results The 578 bp fragment encoding mature PDGF-BB peptide with signal peptide and the 340 bp fragment without signal peptide were identified and verified by restriction endonuclease mapping and sequencing, and two yeast expression vectors pMETB-PDGFB1 and pMETαA-PDGFB2 were reconstructed. Conclusion The yeast expression vectors containing human PDGF-B gene have been successfully constructed for further studies of gene expression in yeast.

中图分类号:

R392.12

杨春露¹, 陈建庭¹, 邓凡², 唐焕章¹, 谭小云¹, 罗深秋². 人血小板衍生生长因子B链基因酵母表达载体的构建[J]. 南方医科大学学报, 2004, 24(03): 273-276.

YANG Chun-lu¹, CHEN Jian-ting¹, DENG fan², TANG Huan-zhang¹, TAN Xiao-yun¹, LUO shen-qiu². Construction and identification of yeast expression vectors containing human platelet-derived growth factor B-chain gene[J]. Journal of Southern Medical University, 2004, 24(03): 273-276.

参考文献

[1] Carl HH, Bengt W. Mechanism of action and in vivo role ofplatelet derived growth factor [R]. Physiol Rev, 1999, 79(4), 1283- 316.
[2] 陈建庭.牛血小板衍生生长因子的提取及其生物活性研究[J].第一军医大学学报,1991,(2):114-8.Chen JT. The extraction and biology activity study of bovine platelet derived growth factor [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 1991, (2): 114-8.
[3] Li X, Ponten A, Aase K, et al. PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor [J].Nat Cell Biol, 2000, 2(5): 302-9.
[4] William J. LaRochelle, Michael J, et al. PDGF-D, a new protease activated growth factor [J]. Nat Cell Biol, 2001, 3(3): 517-21.
[5] 陈建庭,区伯平,史占军.异种PDGF与复方BMP联合使用促进骨缺损的修复[J].中华骨科杂志,1994,14(12):764-8.Chen JT, Ou BP, Shi ZJ. The PDGF of different species combined with compound BMP enhance bone defect repairing [J]. Chin J Orthop, 1994,14(12): 764-8.
[6] 张忠民,陈建庭,金大地血小板衍生生长因子-BB在成骨细胞-破骨细胞培养体系中的生物学作用[J].中华医学杂志,2001, 81(7):425-8Zhang ZM, Chen JT, Jin DD. The biology activity ofplatelet-de rived growth factor-BB in the osteoblast-osteoclast culture system[J]. Natl Med J China, 2001, 81(7): 425-8.
[7] Zhang ZM, Chen JT. Platelet-derived growth factor (PDGF)-BB stimulates osteoclastic bone resorption directly: the role of receptor beta[J]. Biochem Biophys Res Commun, 1998, 251(1): 190-4.
[8] Collins T, Ginsburg D, Boss JM, et al. Cultured human endothelial cells express platelet-derived growth factor B chain:cDNA cloning and structural analysis[J]. Nature, 1985, 316(6030): 748-50.
[9] Yu X, Hsieh SC, Bao W, et al. Temporal expression of PDGF recep tors and PDGF regulatory effects on osteoblasticcells in mineraliz ing cultures[J]. Am J Physiol, 1997, 272(5): C1709-16.
[10] Yang D, Chen J. Platelet-derived growth factor (PDGF)-AA: a self imposed cytokine in the proliferation of human fetal osteoblasts [J]. Cytokine, 2000, 12(8): 1271-74.
[11] Gangji V, Rydziel S, Gabbitas B, et al. Insulin-like growth factor Ⅱ promoter expression in cultured rodent osteoblasts and adult rat bone [J]. Endocrinology, 1998, 139(5): 2287-92.
[12] Fujii H, Kitazawa R, Maeda S. Et al. Expression of platelet-derived growth factor proteins and their receptor alpha and beta mRNAs during fracture healing in the normal mouse[J]. Histochem Cell Bi- ol, 1999, 112(2): 131-8.
[13] 陈建庭,杨德宏,景宗森,等.bFGF调节PDGF对人成骨细胞的促增殖作用[J].中华外科杂志,2000,38(9):707-10.Chen JT, Yang DH, Jing ZS. Et al. bFGF adjust the osteoblasts pro liferation accelerating ofPDGF [J]. Chin J Surg, 2000, 38 (9):707-10.
[14] 陈建庭,李菊根,金大地.血小板衍生生长因子促进成骨细胞DNA合成的实验研究[J].中华外科杂志,1999,37(7):409-11.Chen JT, Li JG, Jin DD. The experimental investigation about the platelet-derived growth factor enhances the DNA synthesis of os teoblasts[J]. Chin J Surg, 1999, 37(7): 409-11.
[15] Chaudhary LR. The cell survival signal Akt is differentially activated byPDGF-BB, EGF, and FGF-2 in osteoblastic cells[J]. Cell Biochem, 2001, 81(2): 304-11.
[16] Blanquaert F. Cortisol inhibits hepatocyte growth factor/scatter factor expression and induces c-met transcripts in osteoblasts [J]. Phys iol Endocrinol Metab, 2000, 278(3): 509-15.
[17] Park Y, Lee Y, Lee J, et al. Controlled release of platelet-derived growth factor-BB from chondroitin sulfate-chitosan sponge for guided bone regeneration[J]. Biomaterials, 2000, 67(2-3): 385-94.
[18] Park Y, Lee YM, Park SN, et al. Platelet derived growth factor re leasing chitosan sponge for periodontal bone regeneration[J]. Bio materials, 2000, 21(2): 153-9.