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本研究通过反转录ＰＣＲ得到人ＧＭ-ＣＳＦ基因片段后，通过重组插入ｐＢＶ２２０载体质粒的ＥｃｏＲⅠ、ＢａｍＨⅠ之间，构建了重组表达质粒ｐｈＧＭ９１。借助计算机分析，优化了构建质粒的转译起始区，采取定点诱变以消除ＳＤ序列和ＡＴＧ区的强二级结构，提高了表达水平。将ＧＭ-ＣＳＦ基因进行序列测定，与文献报导完全一致，所对应的氨基酸序列与天然氨基酸序列一致。将带有表达质粒ｐｈＧＭ９１的ＤＨ５α菌在４２℃诱导，表达了预期的１５ＫＤ的蛋白，约占菌体总蛋白的２０％，并探讨了不同菌株对表达水平的影响。用ＭＴＴ法测定所表达的ＧＭ-ＣＳＦ生物活性，比活性可达到１×１０~７ＩＵ／ｍｇ，表明所表达的ＧＭ-ＣＳＦ具有生物学作用。
In this study, human GM-CSF gene fragment was obtained by reverse transcription PCR and inserted into EcoRI and BamHI of pBV220 vector to construct recombinant expression plasmid phGM91. Through computer analysis, we optimized the construction of the translation initiation region of the plasmid, and took site-directed mutagenesis to eliminate the strong secondary structure of SD sequence and ATG region and improve the expression level. The sequencing of the GM-CSF gene is exactly the same as reported in the literature, and the corresponding amino acid sequence is consistent with the natural amino acid sequence. The DH5α strain with the expression plasmid phGM91 was induced at 42 ℃ and expressed the expected protein of 15KD, accounting for about 20% of the total bacterial protein, and discussed the effect of different strains on the expression level. The bioactivity of GM-CSF was measured by MTT method. The specific activity of GM-CSF reached 1 × 10 ~ 7 IU / mg, indicating that the expressed GM-CSF has the biological effect.