PTD4-Cu,Zn-SOD原核重组表达载体优化及蛋白表达  

Optimization of PTD4-Cu,Zn-SOD protein recombinant expression vector and protein expression

作　　者：程艳 万婷婷 薛荣亮 CHENG Yan;WAN Ting-ting;XUE Rong-liang(Department of Anesthesiology,Shaanxi Provincial People’s Hospital,Xi'an 710068,China;Anesthesia&Comfort Health Center,Xi’an International Medical Center Hospital,Shaanxi 710100,China;Department of Anesthesiology,Peking University Third Hospital,Beijing 100191,China)

机构地区：[1]陕西省人民医院麻醉科,西安710068 [2]西安国际医学中心医院麻醉与舒适化医疗中心,西安710100 [3]北京大学第三医院麻醉科,北京100191

出　　处：《中国医药生物技术》2021年第3期215-221,共7页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(81471131)。

摘　　要：目的优化PTD4-Cu,Zn-SOD原核重组表达载体以增加SOD融合蛋白表达。方法通过基因工程设计引物并优化PTD4-Cu,Zn-SOD原核重组表达载体,扩增并鉴定目的基因,将优化后的原核重组表达载体以热激法转化至E.coliBL21(DE3)中,通过IPTG诱导重组载体进行蛋白表达,通过溶菌酶+超声裂解菌体,离心后收集上清,利用Ni-NTA树脂纯化,10 kD,20k D透析袋浓缩,以获得SOD融合蛋白。利用SDS-PAGE凝胶电泳及Westernblot鉴定SOD融合蛋白。采用BCA与黄嘌呤氧化酶法测定SOD融合蛋白浓度及活力。结果优化后的重组表达载体优化了稀有碱基,增加了His·Tag标签,His·Tag-PTD4-Cu,Zn-SOD序列作为一个整体,在Nco I与Bam H I限制性内切酶作用下定向插入,构建了长度为6213bp的优化质粒,基因测序结果与预先设计的序列对比显示碱基序列正确。优化后的SOD融合蛋白表达量约占菌体的32%,较前(20%)提高,纯化浓缩后纯度为94%,较前(90%)增加。测定浓度为1.8 mg/ml,37℃时SOD总活力为(3065.137±19.75)U/mg prot。结论通过重组表达载体密码子优化,可以获得高产量、高活性的SOD融合蛋白。Objective Optimization of PTD4-Cu,Zn-SOD protein recombinant expression vector to increase PTD4-Cu,Zn-SOD protein expression.Methods PTD4-Cu,Zn-SOD prokaryotic recombinant expression vector were optimized and amplified,and the target genes were identified through designing primers and genetic engineering.The optimized vector was transformed into E.coli BL21(DE3)by heat shock method and induced to express the protein by IPTG.After the E.coli BL21(DE3)cells were cracked by lysozyme&ultrasound,the supernatant was collected after centrifugation,and purified with Ni-NTA resin,followed by concentration with 10 kD and 20 kD dialysis bags to obtain the PTD4-Cu,Zn-SOD proteins.SDS-PAGE gel electrophoresis and Western blot were used to identify the protein.The concentration and vitality of PTD4-Cu,Zn-SOD proteins were determined by BCA and xanthine oxidase method.Results The rare bases in recombinant expression vector were optimized,and His-tag was added.The His-PTD4-Cu-SOD and His-PTD4-Zn-SOD sequence were inserted as a whole into the restriction site of Nco I and Bam H I enzymes for construction.The optimized plasmid has a length of 6213 bp,and the base sequence was correct by gene sequencing.Optimized PTD4-Cu,Zn-SOD protein expression accounted for about 32%of the content in bacterial cells,which was higher than the original one(20%);and the protein purity after purification and concentration was 94%,which was higher than the original protein(90%).The concentration was 1.8 mg/ml,and total SOD activity at 37℃was(3065.137±19.75)U/mg protein.Conclusion High yield and activity PTD4-Cu,Zn-SOD protein can be obtained through optimization of recombinant expression vector.

关 键 词：蛋白质结构域 超氧化物歧化酶 质粒 

分 类 号：Q78[生物学—分子生物学]