尘螨肠道微生物蛋白核苷二磷酸激酶的克隆、表达及纯化

根据 GenBank 中 NDP kinase 的基因序列，采用生物信息学方法将其中稀有密码子改造为大肠埃希菌常用密码子并进行二级结构优化，合成 NDP kinase 基因，构建原核表达载体 pET28a-NDP kinase 并酶切鉴定其序列，在大肠埃希菌 BL21（DE3）中用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，重组产物采用镍离子金属螯合亲和层析柱纯化.经密码子改造和二级结构优化后 NDP kinase 基因长度为490 bp，其编码蛋白理论分子量为21.5 kDa，重组表达载体经酶切鉴定与理论推测结果相符，在大肠埃希菌 BL21（DE3）中该基因经 IPTG 诱导可高效表达，纯化后的重组蛋白分子量约为21.5 kDa，其单一蛋白纯度达95；以上.本研究成功构建了尘螨肠道微生物蛋白核苷二磷酸酶（NDP kinase）基因的 pET28a 原核重组质粒，为进一步研究 NDP kinase 在尘螨疫苗免疫治疗中的作用机理提供了基础.

Cloning,Expression and Purification of Intestinal Microflora Protein Nucleoside Diphosphate Kinase in Dust Mites

To research the cloning,expression and purification of intestinal microflora protein Nucleoside diphos-phate kinase(NDP kinase)in dust mites. The gene order of NDP kinase 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 struc-ture was optimized by means of bioinformatics. Then the DNA sequence was synthesized and its prokaryotic expres-sion vector pET28a-NDP kinase was constructed. The vector was guided into E. coli BL21(DE3)and expressed in-duced by IPTG. Finally,the recombine protein was purified by means of Ni-NTA affinitycolumn. The gene length of the reformed and optimized NDP kinase was 490 bp and the theoretic molecular mass of the coding protein was a-bout 21 kDa. The enzyme identification of the recombine vector agreed with the theoretic value. Soluble NDP kinase could be expressed efficiently in E. coli BL21(DE3)by IPTG. The molecular mass of purified recombine protein was about 21 kDa. The prokaryotic expression vector pET28a-NDP kinase was constructed successfully in our re-search. Efficient expression and soluble NDP kinase could be used to research the role of NDP kinase in dust mite vaccine immunotherapy.