摘要
目的:在大肠杆菌宿主系统中表达血小板衍生生长因子C(PDGF-C)基因全长编码的蛋白以及两个结构域编码的蛋白。方法:用PCR方法从已构建好的质粒中扩增出大鼠PDGF-CcDNA开放阅读框架片段(ORF-PDGF-C,1035bp),以及位于N端的CUB结构域(CUB-PDGF-C,333bp)和位于C端的生长因子结构域(GFD-PDGF-C,348bp),并重组入表达载体pET-24a中,转化大肠杆菌BL21(DE3),用IPTG诱导PDGF-C融合蛋白的表达,经DS-PAGE,表达蛋白采用Western-blot用抗-His和抗-PDGF-C的抗体分别验证融合蛋白表达。结果:PCR扩增的PDGF-C基因序列与GenBank数据库中的序列一致;37℃,0.1mmol.L-1IPTG诱导6h,pET-24a-ORF-PDGF-C,pET-24a-CUB-PDGF-C,pET-24a-GFD-PDGF-C融合蛋白获得优化表达;Western-blot证实融合蛋白的特异性表达。结论:成功构建了pET-24a-ORF-PDGF-C,pET-24a-CUB-PDGF-C,pET-24a-GFD-PDGF-C重组质粒,并在大肠杆菌中获得有效表达,这为进一步纯化该基因表达蛋白以及研究其结构与功能奠定了基础。
Objective：To express the rat full length of platelet-derived growth factor C（PDGF C）and two domains of PDGFC in Escheriehia coll. Methods：PCR methods were conducted to obtain 1035bp cDNA fragment encoding for open reading frame of PDGF C gene and 333bp fragment for the N terminal CUB domain and 348bp fragment for the C-terminal growth factor domain by plasmid constructed. The PCR products were inserted into the prokaryotic expression vector pET-24a for expressing the fu sion proteins in Escherichia col （E. coli） BL21 （DE3） strain under the optimized induction of isopropyl-β-D-thiogalactopyranoside （IPTG）. Western-blot analysis wasperformed using anti-His antibody and anti PDGF-C antibody respectively to confirm the fusion proteins. Results： DNA sequencing showed that the rat platelet derived growth factor C gene nucleotide sequences obtained from PCR were identical with that of PDGF-C cDNA recorded in the GenBank. The maximum quantity of the fusion proteins of pET-24a-ORF PDGF-C, pET 24a-CUB-PDGF C, pET-24a-GFD-PDGF-C were produced at 6h after induction with 0.1 mmol · L ^-1 IPTG at 37℃. Western-blot analysis indicated that the recombinant proteins could react specifically with anti His antibody and anti PDGF-C antibody. Conclusion： The recombinant plasmids of pET 24a-ORF-PDGF-C, pET 24a- CUB- PDGF-C, pET 24a GFD-PDGF C were constructed successfully and the fusion proteins were expressed effectively in E. coli cells. These results would provide a basis for purifying this protein and researching its sructure and functions.
出处
《中国临床医学》
北大核心
2008年第6期811-813,共3页
Chinese Journal of Clinical Medicine
基金
江苏省科教兴卫重点医学人才基金(RC2007087)
关键词
血小板衍生生长因子C
原核表达
融合蛋白
Platelet derived growth factor C
Prokaryotic expression
Fusion protein
