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

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

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ＰＦ４ ｃＤＮＡ，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ｐＧＥＸ－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３６ ０００。结论成功地构建融合蛋白表达载体ＧＳＴ－ＰＦ４，并在大肠杆菌中获得有效表达，为进一步研究打下良好的基础。

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.