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目的 获得可用于探针的非放射性标记的耐热碱性磷酸酯酶。方法 把耐热碱性磷酸酯酶的基因克隆入质粒pJLA5 0 3上 ,在E .coliMph44中表达。表达的酶用聚乙烯亚胺沉淀核酸 ,热变性 ,硫酸铵沉淀 ,层析等方法纯化。结果 经表达纯化后每克细菌可获得 1.5mg的酶 ,酶的纯度为 90 % ,比活为 78.4U/mg。结论 耐热碱性磷酸酯酶可在E .coliMph44中表达 ,表达的酶可用热变性和其它蛋白质纯化方法来纯化。
Objective To obtain a non-radioactive labeled thermostable alkaline phosphatase that can be used for probes. Methods The gene of thermophilic alkaline phosphatase was cloned into plasmid pJLA5 0 3 and expressed in E.coliMph44. The expressed enzyme is purified by polyethyleneimine precipitation, nucleic acid denaturation, ammonium sulfate precipitation, chromatography and the like. Results After purification, 1.5 mg of enzyme was obtained per gram of bacteria. The purity of the enzyme was 90% and the specific activity was 78.4 U / mg. Conclusion Thermophilic alkaline phosphatase is expressed in E.coli Mph44 and the expressed enzyme can be purified by thermal denaturation and other protein purification methods.