Expression and purification of intestinal microflora protein hypothetical CE2188 in dust mites
YANG Xiao-meng1,YUAN Xie-fang1,2,CHEN Tao1,YANG Ping-chang1,3,LIU Zhi-gang1,3
1. Institute of Allergy and Immunology, School of Medicine, Shenzhen University, Shenzhen 518060, China; 2. Respiratory Department, the Affiliated Hospital of Luzhou Medical College, Luzhou 646000, China; 3. Shenzhen Key Laboratory of Allergy and Immunology, Shenzhen 518060, China
摘要目的 研究尘螨肠道微生物蛋白Hypothetical protein CE2118的表达和纯化,为研究其在尘螨疫苗免疫治疗中的作用奠定基础。方法 采用生物信息学方法,根据GenBank中Hypothetical protein CE2118蛋白的基因序列,将其中稀有密码子改造为大肠杆菌常用密码子并进行二级结构优化,合成Hypothetical protein CE2118基因,构建原核表达载体pGEX 6P-1-hypothetical protein CE2118并经酶切鉴定,在大肠埃希菌Rosetta(DE3)中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,重组产物采用GST亲和层析纯化柱纯化。结果 经密码子改造和二级结构优化后Hypothetical protein CE2118基因长度为588bp,其编码蛋白理论分子量为21 kDa。重组表达载体经酶切鉴定与理论推测结果相符,该基因经IPTG诱导在大肠埃希菌Rosetta(DE3)中得到高效的可溶性表达,纯化后的重组蛋白分子量约为21 kDa,其单一蛋白纯度达95%以上。结论 本研究成功构建了Hypothetical protein CE2118基因的pGEX6P-1原核重组质粒,获得的可溶性重组蛋白为进一步研究肠道微生物蛋白Hypothetical protein CE2118在尘螨疫苗免疫治中的作用机理奠定基础。
Abstract:To express and purify intestinal microflora protein hypothetical CE2188 in dust mites, the gene order of hypothetical CE2118 was obtained from the GenBank, the rare codon in the gene was changed to the commonly used codon of Escherichia coli (E. coli), and the secondary structure was optimized by means of bioinformatics. Then the DNA sequence was synthesized and its prokaryotic expression vector pGEX 6P-1-hypothetical protein CE2118 was constructed. The vector was guided into E. coli Rosetta (DE3) and induced by IPTG. Finally, the recombine protein was purified by means of GST affinity column. The gene length of the reformed and optimized hypothetical CE2188 was 588 bp and the theoretic molecular mass of the coding protein was about 21 kDa. The enzyme identification of the recombinant vector was agreed with the theoretic value. Soluble hypothetical protein CE2118 could be expressed efficiently in E. coli Rosetta (DE3) by IPTG. Molecular mass of purified recombine protein was about 21 kDa. The prokaryotic expression vector pGEX 6P-1-hypothetical protein CE2118 was constructed successfully in our research. Efficient expression and soluble hypothetical protein CE2118 could be used to investigate the role of hypothetical protein CE2118 in dust mite vaccine immunotherapy.
杨小猛,袁谢芳,陈涛,杨平常,刘志刚. 尘螨肠道微生物蛋白Hypothetical protein CE2118的表达及纯化[J]. 中国人兽共患病学报, 2014, 30(9): 879-883.
YANG Xiao-meng,YUAN Xie-fang,CHEN Tao,YANG Ping-chang,LIU Zhi-gang. Expression and purification of intestinal microflora protein hypothetical CE2188 in dust mites. Chinese Journal of Zoonoses, 2014, 30(9): 879-883.
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