论文部分内容阅读
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 and express the anti-tyrosinase protein TmAFP84 and obtain the target protein. Methods The target afp84 gene was amplified by PCR and cloned into pUCm-T vector and sequenced. The afp84 gene was double-digested by restriction enzyme digestion to Pichia pastoris expression vector pPIC9K. The recombinant plasmid was transformed into Pichia pastoris GS115 by lithium acetate conversion method, and the recombinant strain was screened out. The target protein was induced with 0.5% methanol, and the target protein was identified by polyacrylamide gel electrophoresis and silver staining. Results The target gene fragment afp84 was obtained by PCR amplification. Plasmid pUCm-T-afp84 was obtained by cloning plasmid pUCm-T and afp84. The recombinant eukaryotic expression vector pPIC9K-afp84 was constructed and the target anti-frozen protein TmAFP84 was expressed after induction. Conclusion The eukaryotic expression vector pPIC9K-afp84 was successfully constructed, and the target antifreeze protein was abundantly expressed, which lays the foundation for expanding the scale of production and further studying the properties and application of antifreeze protein.