江西师范大学学报（自然科学版）
江西師範大學學報（自然科學版）
강서사범대학학보（자연과학판）
JOURNAL OF JIANGXI NORMAL UNIVERSITY（NATURAL SCIENCES EDITION）
2014年
5期
485-488
,共4页
杨小猛%王俊轶%陈涛%刘志刚%杨平常
楊小猛%王俊軼%陳濤%劉誌剛%楊平常
양소맹%왕준질%진도%류지강%양평상
尘螨%肠道微生物%蛋白核苷二磷酸激酶
塵螨%腸道微生物%蛋白覈苷二燐痠激酶
진만%장도미생물%단백핵감이린산격매
dust mite%intestinal microflora%nucleoside diphosphate kinase
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.
根據 GenBank 中 NDP kinase 的基因序列，採用生物信息學方法將其中稀有密碼子改造為大腸埃希菌常用密碼子併進行二級結構優化，閤成 NDP kinase 基因，構建原覈錶達載體 pET28a-NDP kinase 併酶切鑒定其序列，在大腸埃希菌 BL21（DE3）中用異丙基-β-D-硫代半乳糖苷（IPTG）誘導錶達，重組產物採用鎳離子金屬螯閤親和層析柱純化.經密碼子改造和二級結構優化後 NDP kinase 基因長度為490 bp，其編碼蛋白理論分子量為21.5 kDa，重組錶達載體經酶切鑒定與理論推測結果相符，在大腸埃希菌 BL21（DE3）中該基因經 IPTG 誘導可高效錶達，純化後的重組蛋白分子量約為21.5 kDa，其單一蛋白純度達95%以上.本研究成功構建瞭塵螨腸道微生物蛋白覈苷二燐痠酶（NDP kinase）基因的 pET28a 原覈重組質粒，為進一步研究 NDP kinase 在塵螨疫苗免疫治療中的作用機理提供瞭基礎.
근거 GenBank 중 NDP kinase 적기인서렬，채용생물신식학방법장기중희유밀마자개조위대장애희균상용밀마자병진행이급결구우화，합성 NDP kinase 기인，구건원핵표체재체 pET28a-NDP kinase 병매절감정기서렬，재대장애희균 BL21（DE3）중용이병기-β-D-류대반유당감（IPTG）유도표체，중조산물채용얼리자금속오합친화층석주순화.경밀마자개조화이급결구우화후 NDP kinase 기인장도위490 bp，기편마단백이론분자량위21.5 kDa，중조표체재체경매절감정여이론추측결과상부，재대장애희균 BL21（DE3）중해기인경 IPTG 유도가고효표체，순화후적중조단백분자량약위21.5 kDa，기단일단백순도체95%이상.본연구성공구건료진만장도미생물단백핵감이린산매（NDP kinase）기인적 pET28a 원핵중조질립，위진일보연구 NDP kinase 재진만역묘면역치료중적작용궤리제공료기출.
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.
