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以青番木瓜总RNA为模板,采用RT-PCR技术扩增出木瓜凝乳蛋白酶基因CHY(chymopapain),构建带有His-tag的原核表达载体p EASY-E1-CHY。将该重组载体转化到大肠杆菌BL21(DE3)菌株中,重组菌株用IPTG诱导表达,在诱导6 h,IPTG浓度为0.05 mmol/L时重组蛋白表达量最高。SDS-PAGE凝胶电泳和West-ern-Blot杂交分析结果表明蛋白大小为45 k Da。用脱脂奶粉进行凝乳分析,证明此木瓜凝乳蛋白酶具有凝乳活性,此结果为今后利用生物技术进行木瓜凝乳蛋白酶工程化生产奠定了基础。
Chymopapain was amplified by RT-PCR using total RNA of Papaya as template. The prokaryotic expression vector pEASY-E1-CHY with His-tag was constructed. The recombinant vector was transformed into Escherichia coli BL21 (DE3) strain. The recombinant strain was induced by IPTG. The recombinant protein was expressed at the concentration of 0.05 mmol / L IPTG for 6 h. SDS-PAGE gel electrophoresis and West-ern-Blot hybridization analysis showed that the protein size was 45 kDa. Curdling analysis with skimmed milk powder showed that this papain had curd activity, which laid the foundation for biotechnological engineering of chymopapain engineering in the future.