CAJ | 학술논문

目的:用一种简便有效的方法合成hPF4基因,并在大肠杆菌BL21(DE3)中进行高效表达.方法:根据hPF4基因的序列,用touch-down PCR的方法合成hPF4基因,将合成的基因片断克隆到表达载体pCEX-4T-1中,构建了重组质粒pGEX-4T-1-hPF4,并将之转化大肠杆菌BL21(DE3),IPTG诱导表达由谷胱甘肽转移酶和hPF4组成的融合蛋白.结果:用touch-down PCR的方法成功合成了hPF4基因.融合蛋白形成包含体,表达量约为全菌体蛋白的30%.结论:Touch-down PCR方法可作为合成其它目的基因的简便有效方法.BL21(DE3)/pGEX-4T-1表达系统能高效表达合成的hPF4基因,为下一步hPF4的纯化与生物学活性研究打下了基础.
목적:용일충간편유효적방법합성hPF4기인,병재대장간균BL21(DE3)중진행고효표체.방법:근거hPF4기인적서렬,용touch-down PCR적방법합성hPF4기인,장합성적기인편단극륭도표체재체pCEX-4T-1중,구건료중조질립pGEX-4T-1-hPF4,병장지전화대장간균BL21(DE3),IPTG유도표체유곡광감태전이매화hPF4조성적융합단백.결과:용touch-down PCR적방법성공합성료hPF4기인.융합단백형성포함체,표체량약위전균체단백적30%.결론:Touch-down PCR방법가작위합성기타목적기인적간편유효방법.BL21(DE3)/pGEX-4T-1표체계통능고효표체합성적hPF4기인,위하일보hPF4적순화여생물학활성연구타하료기출.
AIM:To synthesize the human platelet factor-4(hPF4) gene with a convenient and effective approach, and high express the hPF4 gene in E. coli BL21 (DE3). METHODS: According to the primary structure of hPF4, the nucleotide sequence was synthesized using touch-down PCR method. The resultant gene fragment containing EcoR Ⅰ and Xho Ⅰ overhangs at 5' and 3' ends was cloned into the expression vector pGEX-4T-1 to construct the recombinant plasmid pGEX-4T-1-hPF4,which was then transformed into the E. coli strain BL21 (DE3). RESULTS: hPF4 gene was successfully synthesized by touchdown PCR method. A fusion protein composed of glutathione S-transferase (GST) and the recombinant hPF4 was expressed in BL21(DE3) by IPTG induction. The expression level of the fusion protein in E. coli was about 30% of the total cellular protein. CONCLUSION: Touch-down PCR may provide a convenient and effective approach to obtain other target genes. The expressed fusion protein forms the inclusion bodies, providing sufficient material for further purification and biological activities process.