摘要
【目的】为解决医用超氧化物歧化酶(superoxide dismutase,SOD)来源的限制,利用基因工程方法构建牦牛Cu,Zn-SOD原核表达系统,为重组牦牛Cu,Zn-SOD作为注射用药奠定基础。【方法】RT-PCR扩增牦牛Cu,Zn-SOD编码基因,构建Cu,Zn-SOD/pET22b(+)重组表达载体,并转化到E.coli BL21(DE3)宿主菌,经IPTG进行诱导表达。表达产物经SDS-PAGE、活性染色及邻苯三酚法进行检测,采用BradFord方法测定融合蛋白浓度。【结果】本试验成功获得了牦牛Cu,Zn-SOD cDNA,长456bp,编码152个氨基酸。与普通牛SOD编码基因序列cDNA一致率为99.6%,氨基酸序列一致率为98.7%,表达产物分子质量约为32kD。酶粗提取液比活约为35U·mg-1。重组蛋白浓度0.2772(mg·mL-1)。【结论】本研究成功构建了牦牛Cu,Zn-SOD/pET22b(+)原核表达载体,表达量较高、稳定性强,重组表达产物具有较高生物学活性。
【Objective】 Due to the medical superoxide dismutase（SOD） source is restricted from animals and plants,establishment of the prokaryotic expressing system of yak Cu,Zn-SODpET22b（＋）-E.coli BI21（DE3） by genetic engineering method is a basis for recombinant SOD protein of clinical injection.【Method】 Yak Cu,Zn-SOD gene was amplified using reverse transcription-polymerase chain reaction（RT-PCR）.The PCR product was inserted into the vector pET22b（＋） to construct plasmid Cu,Zn-SOD/pET22b（＋）,then the plasmid expressed in E.coli BL21（DE3） cell that induced by IPTG.The expression product and it＇s activity were tested by the method of SDS-PAGE,native PAGE（enzyme active stain） electrophoresis and pyrogallol（1,2,3-trihydroxybenzene） self oxidation.The level of expression protein was determined by Brad Ford.【Result】 The length of ORF of yak Cu,Zn-SOD was 456 bp encoding 152 amino acids peptides.The nucleotide sequence and amino acids sequence of Cu,Zn-SOD similarities between yak and bovine were 99.6% and 98.7%,respectively.The activity ratio of the protein crude extract of recombinant Cu,Zn-SOD was 35U·mg-1.The concentration of the recombinant Cu,Zn-SOD is 0.2772（mg·mL-1）.【Conclusion】 The prokaryotic expression vector for yak Cu,Zn-SOD/pET22b（＋） was constructed successfully and it expresses in Escherichia coli stably and highly.The recombinant product was obtained and the product has higher biological activity.
出处
《中国农业科学》
CAS
CSCD
北大核心
2010年第23期4928-4935,共8页
Scientia Agricultura Sinica
基金
国家“973”计划专项(2007CB116204)
