不同质粒表达大肠杆菌碱性磷酸酶的研究

目的比较以pET-30a和pET-22b两个质粒表达碱性磷酸酶（alkaline phosphatase,AP）的活性差异。方法以质粒pET-30a-AP为模板,克隆大肠杆菌（Escherichia coli,E.coli）ATCC 25922的碱性磷酸酶成熟肽基因,并构建重组表达质粒pET-22b-AP。将这2种表达质粒转化到E.coli BL21（DE3）中,经IPTG诱导表达AP。对温度和甲醇对AP活性的影响进行研究。结果 pET-30a-AP所产AP浓度约为pET-22b-AP 10倍时,才可达到类似催化效果;表达自pET-22b-AP质粒的AP对甲醇的耐受性略好于pET-30a-AP...

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Published in食品安全质量检测学报 Vol. 7; no. 10; pp. 4125 - 4130
Main Author 刘爱平 申文浩 李俊柔 孙海芹 熊清 李文丽 李诚
Format Journal Article
LanguageChinese
Published 四川农业大学食品学院,雅安,625014 2016
Subjects
基因克隆
大肠杆菌
碱性磷酸酶
蛋白质表达
酶活性
gene cloning
蛋白质表达
Escherichia coli
enzymatic activity
大肠杆菌
alkaline phosphatase
酶活性
protein expression
碱性磷酸酶
基因克隆
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Summary:目的比较以pET-30a和pET-22b两个质粒表达碱性磷酸酶（alkaline phosphatase,AP）的活性差异。方法以质粒pET-30a-AP为模板,克隆大肠杆菌（Escherichia coli,E.coli）ATCC 25922的碱性磷酸酶成熟肽基因,并构建重组表达质粒pET-22b-AP。将这2种表达质粒转化到E.coli BL21（DE3）中,经IPTG诱导表达AP。对温度和甲醇对AP活性的影响进行研究。结果 pET-30a-AP所产AP浓度约为pET-22b-AP 10倍时,才可达到类似催化效果;表达自pET-22b-AP质粒的AP对甲醇的耐受性略好于pET-30a-AP。结论本研究可为AP的生产及基因工程抗体-AP融合蛋白在类似ELISA等免疫检测方法的应用提供重要的参考依据。
Bibliography:Escherichia coli; alkaline phosphatase; gene cloning; protein expression; enzymatic activity
Objective To compare the activity of alkaline phosphatase（AP） expressed with pET-30 a and pET-22 b as plasmid.Methods The mature peptide gene,encoding AP from E.coli ATCC 25922,was amplified from plasmid pET-30a-AP,and then recombinant expression vector pET-22b-AP was constructed.These 2 kinds of expression vectors were transformed into E.coli BL21（DE3）,respectively,and AP were expressed by IPTG induction.Afterwards,the effects of temperature and methanol on activity of AP were investigated.Results The reactivity of soluble AP from pET-22b-AP at same concentration was 10 times higher than that from pET-30a-AP,and AP from pET-22b-AP was more tolerant with methanol.Conclusion This study will lay better foundation for production of AP and further application of genetically engineered antibody-AP fusion protein in the field of immunoassays like enzyme-linked immunosorbent assay.
11-5956/TS
LIU Ai-Ping, SHEN Wen-Hao, LI Jun-R
ISSN:2095-0381