抗冻蛋白TmAFP84在毕赤酵母中的表达

Expression of antifreeze protein TmAFP84 in Pichia pastoris

目的通过对黄粉甲抗冻蛋白TmAFP84进行基因克隆、表达,获得目的蛋白。方法 PCR扩增目的 afp84基因,克隆到pUCm-T载体中测序并保存。通过双酶切将afp84基因定向重组到毕赤酵母表达载体pPIC9K中。醋酸锂转化法将重组质粒转化入毕赤酵母GS115,筛选出重组菌株。用0.5%甲醇诱导目的蛋白表达,通过聚丙烯酰胺凝胶电泳和海波银染鉴定目的蛋白。结果经PCR扩增获得了目的基因片段afp84,克隆质粒pUCm-T与afp84重组获得了质粒pUCm-T-afp84。构建成功真核表达载体pPIC9K-afp84,目的抗冻蛋白TmAFP84经诱导后获得表达。结论真核表达载体pPIC9K-afp84成功构建,并大量表达出目的抗冻蛋白,为扩大生产规模及深入研究抗冻蛋白的性质和应用奠定基础。

Objective To clone TmAFP84 by PCR, and express the target protein. Methods AJ^84 gene was amplified by PCR, then cloned onto pUCm - T vector for sequencing and conservation. By double enzyme restriction, aJp84 gene was recom- bined into pPIC9K, and then the recombinant plasmids（ pPIC9K -afp84 ） were transformed into Pichia pastoris GS115 using Li- AC method to screen the Pichia pastoris GSll5 - aJp84. The TmAFP84 protein expression was induced with 0.5% methyl alco hol and identified by polyacrylamide gel electxophoresis and silver staining. Results Alp84 gene was obtained by PCR amplifi- cation, and recombinant plasmid pUCm - T - afp84 was obtained with pUCm - T clone and afp84 recombination. The eukaryotic expression vector of pPIC9K - aJp84 was successfully constructed to induce antifreeze protein TmAFP84 expression. Conclusion The eukaryotic expression vector pPIC9K - aJp84 was constructed successfully to express the purpose antifreeze proteins, laying the solid foundation for expanding the production scale and studying the properties and application of antifreeze proteins.