钙激活酶激活蛋白基因的克隆及其在原核生物中表达及条件的优化

构建钙激活酶激活蛋白基因（UK114）的原核表达载体并优化其表达条件,为其高效表达提供试验依据。以UK114 cDNA为模板,通过PCR方法扩增钙激活酶激活蛋白基因,将其克隆到原核表达载体pGEX-4T-3中,酶切及测序鉴定重组体。将构建好的重组质粒转化大肠埃希菌BL21（DE3）,用IPTG进行诱导表达,在保持菌种不改变的前提下,分别改变IPTG的浓度、培养时间、菌体浓度、培养温度等来优化表达条件。结果显示,原核表达载体pGEX-4T-3-UK114成功构建,可在大肠埃希菌BL21（DE3）中诱导表达,得到相对分子质量约40 kD的GST-UK114融合蛋白。在IPTG浓度为0.3 mmol/L,诱导温度为32℃,诱导时间为4 h,菌体密度OD600为0.6的条件下,目的蛋白表达量最高。试验成功构建原核表达载体pGEX-4T-3-UK114且获高效表达,为研究UK114生物学活性及产品开发提供了试验基础。

Cloning of Calpain Activator Gene and the Optimized Expression Condition in Prokaryotes

The aim of the study was designed to optimize Calpain activator(UK114)expression in prokaryotes for the high effective expression.The UK114 cDNA was obtained by RT-PCR,and cloned into prokaryotic expression vector pGEX-4T-3-UK114,then recombinant vector pGEX 4T-3-UK114 was constructed,and transformed into E.coli BL21(DE3).The expression of fusion protein GST-UK114 was induced with IPTG,then purified and characterized.The recombinant prokaryotic expression vector was successfully constructed by the restriction enzymes digestion and gene sequencing.Fusion protein GST-UK114 was expressed in solubility in E.coli BL21(DE3)in SDS-PAGE gel.The content of fusion protein was 31% and the molecular weight was 40 kD.Through optimization,the satisfactory expression condition was obtained,as follows,including IPTG concentration 0.3 mmol/L;induced time of 4 hours;OD600 0.6;cultured temperature 32 ℃.The plasmid pGEX-4T-3-UK114 was successfully constructed and expressed in prokaryotes with high efficiency.The results provide a foundation for studing the biological activity of UK114 protein.