谷胱甘肽转硫酶-血小板因子4融合蛋白表达载体的构建、鉴定与表达

目的 构建谷胱甘肽转硫酶－血小板因子4（GST－PF4）融合蛋白表达载体，并研究其编码的蛋白质在大肠杆菌中的表达。方法 通过RT－PCR方法，从HL－60细胞中克隆PF4cDNA，然后克隆至载体pUC19中，序列测定后把PF4基因重组入谷胱甘肽转硫酶融合基因表达载体pGEC-4T-3中，并用IPTG诱导其在大肠杆菌中表达。结果 获得了PF4 cDNA。序列分析表明，该序列与GenBank数据库中的序列一致。重组质粒酶切鉴定表明，PF4基因已正确插入到pGEX-4T-3中。重组融合蛋白表达载体GST－PF4经IPTG诱导表达，在SDS－PAGE后得到1条蛋白表达带，相对分子质量（Mr)约为36000。结论 成功地构融合蛋白表达载体GST－PF4，并大肠杆菌中获得有效表达，为进一步研究打下良好的基础。

Construction,identification and expression of recombinant expression vector of giutathione s-transferase-platelet factor 4 fusion protein

Aim To construct expression vector of giutathione s-transferase-platelet factor 4 (GST-PF4) fusion protein and express it in Escherichia coli.Methods The PF4 cDNA was amplified from HL-60 cell lines by RT-PCR, then cloned into vector pUC19. After sequencing, the PF4 gene was inserted into pGEX-4T-3,the recombinant vector GST-PF4 was identificated by restriction endonuclease digestion. GST-PF4 was expressed proteins in Escherichia coli via induction of IPTG. ResultsS equencing showed that sequence of human PF4 gene was identical with that of PF4 cDNA recorded in the GenBank. The analysis for recombinant plasmid DNA digested by EcoRIand SalIdemonstrated that the PF4 gene was inserted exactly into pGEX-4T-3. SDS-PAGE analysis showed that the ralative molecular mass (Mr)of the expressed protein was about 36 000. ConclusionExpression vector of GST-PF4 fusion protein has been constructed successfully and expressed effectively in Escherichia coli. These results provide an essential preparation for obtaining a larger quantity of recombinant human PF4 for further study.