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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pGEX-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。
OBJECTIVE: To synthesize hPF4 gene in a simple and effective way and express it efficiently in E. coli BL21 (DE3). Methods: The hPF4 gene was synthesized by touch-down PCR according to the sequence of hPF4 gene. The synthesized gene fragment was cloned into the expression vector pGEX-4T-1, and the recombinant plasmid pGEX-4T-1-hPF4 was constructed Escherichia coli BL21 (DE3) was transformed into E. coli and induced by IPTG to express the fusion protein consisting of glutathione transferase and hPF4. Results: The hPF4 gene was successfully synthesized by touch-down PCR. The fusion protein forms the inclusion body, and the expression level is about 30% of the total bacterial protein. Conclusion: The Touch-down PCR method can be used as a convenient and effective method for the synthesis of other genes of interest. The BL21 (DE3) / pGEX-4T-1 expression system can efficiently express the synthesized hPF4 gene, which lays the foundation for the further purification and biological activity research of hPF4.