人肌红蛋白原核表达载体的构建、表达及蛋白纯化

发布时间：2018-12-13 05:42

【摘要】：目的:构建人肌红蛋白(Mb)原核表达载体并诱导其表达、纯化及鉴定目的蛋白。pQE方法:根据已报道的人Mb基因序列,采用RT-PCR技术从人骨骼肌组织中获得肌红蛋白cDNA序列。将所得的PCR产物插入克隆载体pGEM-Teasy中,得重组质粒,经酶切、PCR及测序鉴定正确后,将酶切目的片段插入原核表达载体pQpQEE30中并转化大肠杆菌(E.coli)M15,通过IPTG诱导表达出带有6个组氨酸的融合蛋白。经镍固定金属亲和层析纯化。结果:序列分析表明,人Mb基因成熟肽编码区含有465 bp,编码153个氨基酸;与GenBank(NM203378)中已报道的Mb核苷酸序列有98.50%同源性。经IPTG诱导表达,SDS-PAGE电泳和免疫印迹分析显示,表达的融合蛋白占菌体蛋白总量的31%,相对分子质量约为16700,并与商品化的人Mb单抗呈特异性反应。经Ni2+-NTA agarose纯化获得SDS-PAGE电泳下单一条带。结论:在大肠杆菌中获得了人Mb的高效表达。
[Abstract]:Objective: to construct the prokaryotic expression vector of human myoglobin (Mb) and induce its expression, purify and identify the target protein. PQE method: according to the reported human Mb gene sequence, The cDNA sequence of myoglobin was obtained from human skeletal muscle by RT-PCR technique. The recombinant plasmid was obtained by inserting the PCR product into the cloned vector pGEM-Teasy. After restriction endonuclease digestion, PCR and sequencing, the target fragment was inserted into the prokaryotic expression vector pQpQEE30 and transformed into Escherichia coli (E.coli) M15. The fusion protein with 6 histidine was induced by IPTG. It was purified by nickel immobilized metal affinity chromatography. Results: sequence analysis showed that the mature peptide coding region of human Mb gene contained 465 bp, encoding 153 amino acids, and had 98.50% homology with Mb nucleotide sequence reported in GenBank (NM203378). IPTG induced expression, SDS-PAGE electrophoresis and Western blot analysis showed that the expressed fusion protein accounted for 31% of the total bacterial protein, and the relative molecular weight was about 16700, and the fusion protein reacted specifically with the commercial human Mb monoclonal antibody. SDS-PAGE electrophoresis was obtained by Ni2-NTA agarose purification. Conclusion: the high expression of human Mb was obtained in Escherichia coli.
【作者单位】：吉林大学第一医院中心实验室吉林大学第一医院中心实验室吉林大学第一医院中心实验室吉林大学第一医院中心实验室吉林大学第一医院中心实验室吉林大学第一医院中心实验室吉林大学第一医院中心实验室解放军第461医院检验科吉林大学第一医院中心实验室
【基金】：吉林省科技厅社会发展计划项目资助课题(20030450) 吉林省科技厅重点项目资助课题(20030402) 2004年吉林大学创新基金资助课题吉林省杰出青年基金资助课题(20050113)
【分类号】：R346

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