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目的在毕赤酵母中表达人溶菌酶(Human lysozyme,hLY)-人碱性成纤维细胞生长因子(Human basic fibroblast growth factor,hbFGF)融合基因,并分析融合蛋白的hLY活性。方法从人全血中提取总RNA,以其为模板,采用RT-PCR法扩增编码hLY成熟肽的cDNA。利用重叠PCR技术将hly基因与hbfgf基因融合,中间引入凝血酶识别位点序列;将融合基因克隆至载体pPIC9K中,构建重组表达质粒pPIC9K-hly-thr-hbfgf,电转化毕赤酵母GS115,通过G418抗性筛选高拷贝重组子GS/hly-thr-hbfgf,甲醇诱导表达融合蛋白,Bradford法测定重组子发酵液上清中总蛋白含量,改良舒加法检测融合蛋白hHLY-THR-hbFGF的hLY活性。结果获得的融合基因序列与理论序列一致;经PCR鉴定,融合基因hly-thr-hbfgf已整合入GS115基因组内;表达的融合蛋白相对分子质量约为33 000,发酵液上清中总蛋白含量为196.20 mg/L,融合蛋白的hLY活性为40 U/ml。结论已成功地在毕赤酵母GS115中表达了具有hLY活性的hHLY-THR-hbFGF融合蛋白。
Objective To express human lysozyme (hLY) - human basic fibroblast growth factor (hbFGF) fusion gene in Pichia pastoris and analyze the hLY activity of the fusion protein. Methods Total RNA was extracted from human whole blood and used as template to amplify cDNA encoding hLY mature peptide by RT-PCR. The recombinant plasmid pPIC9K-hly-thr-hbfgf was constructed by the fusion of hly gene and hbfgf gene and the thrombin recognition sequence in the middle by overlapping PCR. The fusion gene was cloned into vector pPIC9K and transformed into Pichia pastoris GS115 G418 resistance screening of high copy recombinant GS / hly-thr-hbfgf, methanol induced expression of fusion protein, Bradford assay of recombinant protein content in the supernatant of fermentation broth, modified Shu Jia method to detect the hLY activity of the fusion protein hHLY-THR-hbFGF . Results The fusion gene sequence was consistent with the theoretical sequence. The hly-thr-hbfgf fusion gene was integrated into the genome of GS115 by PCR. The relative molecular mass of the expressed fusion protein was about 33 000. The total protein content in the supernatant of the fermentation broth was 196.20 mg / L, hLY activity of the fusion protein was 40 U / ml. Conclusion The hHLY-THR-hbFGF fusion protein with hLY activity has been successfully expressed in Pichia pastoris GS115.