浙江大学学报（医学版）
浙江大學學報（醫學版）
절강대학학보（의학판）
JOURNAL OF ZHEJIANG UNIVERSITY MEDICAL SCIENCES
2010年
1期
57-63
,共7页
杨蕴刘%饶娆%沈健%冯磊
楊蘊劉%饒嬈%瀋健%馮磊
양온류%요요%침건%풍뢰
N-乙酰神经氨酸/遗传学%果糖-二磷酸醛缩酶%大肠杆菌/遗传学%聚合酶链反应%克隆%分子%重组%遗传%转染%遗传载体%基因表达%重组蛋白质类
N-乙酰神經氨痠/遺傳學%果糖-二燐痠醛縮酶%大腸桿菌/遺傳學%聚閤酶鏈反應%剋隆%分子%重組%遺傳%轉染%遺傳載體%基因錶達%重組蛋白質類
N-을선신경안산/유전학%과당-이린산철축매%대장간균/유전학%취합매련반응%극륭%분자%중조%유전%전염%유전재체%기인표체%중조단백질류
N-Acetylneuraminic acid/genet%Fructose-bisphosphate aldolase%Escherichia coli/genet%Polymerase chain reaction%Cloning,molecular%Recombination,genetic%Transfection%Genetic vectors%Gene expression%Recombinant proteins
目的:为了获得N-乙酰-D-神经氨酸醛缩酶基因表达的两重组菌.方法:以大肠杆菌C600染色体DNA为模板,通过引物设计和PCR扩增,克隆获得编码Neu5Ac醛缩酶基因(nanA)的片段;以pET28b构建的nanA基因重组质粒pRY1转化 E.coli BL21(DE3),然后将nanA基因片段组入到pDR540载体的Ptac启动子下游,所得重组菌 E.coli DH5α/pRY3的nanA基因在tac启动子控制下呈组成型表达.结果:DNA序列测定证明,该基因的开放阅读框(open reading frame,ORF)大小为894 bp, 编码298个氨基酸组成的酶蛋白;SDS-PAGE显示,纯化的目的蛋白为33 kD的单一条带.以pET28b构建的nanA基因重组质粒pRY1转化 E.coli BL21(DE3),在得到的转化子 E.coli BL21(DE3)/pRY1中,nanA基因受lac启动子控制,无需IPTG或乳糖所诱导.在N-乙酰(-D)-神经氨酸醛缩酶固定化酶的催化下合成N-乙酰-D-神经氨酸的合成效率为78.3%,结晶回收率为90.2％,总回收率为70.6%.全波长扫描分析表明:本实验室结晶品与Sigma公司商品的全波长扫描图谱有相同的特征.结论:所构建的诱导型产酶菌株 E.coli BL21(DE3)/pRY1和组成型表达菌株 E.coli DH5α/pRY3都可较多量的产酶.合成的结晶品具有N-乙酰-D-神经氨酸的特征.
目的:為瞭穫得N-乙酰-D-神經氨痠醛縮酶基因錶達的兩重組菌.方法:以大腸桿菌C600染色體DNA為模闆,通過引物設計和PCR擴增,剋隆穫得編碼Neu5Ac醛縮酶基因(nanA)的片段;以pET28b構建的nanA基因重組質粒pRY1轉化 E.coli BL21(DE3),然後將nanA基因片段組入到pDR540載體的Ptac啟動子下遊,所得重組菌 E.coli DH5α/pRY3的nanA基因在tac啟動子控製下呈組成型錶達.結果:DNA序列測定證明,該基因的開放閱讀框(open reading frame,ORF)大小為894 bp, 編碼298箇氨基痠組成的酶蛋白;SDS-PAGE顯示,純化的目的蛋白為33 kD的單一條帶.以pET28b構建的nanA基因重組質粒pRY1轉化 E.coli BL21(DE3),在得到的轉化子 E.coli BL21(DE3)/pRY1中,nanA基因受lac啟動子控製,無需IPTG或乳糖所誘導.在N-乙酰(-D)-神經氨痠醛縮酶固定化酶的催化下閤成N-乙酰-D-神經氨痠的閤成效率為78.3%,結晶迴收率為90.2％,總迴收率為70.6%.全波長掃描分析錶明:本實驗室結晶品與Sigma公司商品的全波長掃描圖譜有相同的特徵.結論:所構建的誘導型產酶菌株 E.coli BL21(DE3)/pRY1和組成型錶達菌株 E.coli DH5α/pRY3都可較多量的產酶.閤成的結晶品具有N-乙酰-D-神經氨痠的特徵.
목적:위료획득N-을선-D-신경안산철축매기인표체적량중조균.방법:이대장간균C600염색체DNA위모판,통과인물설계화PCR확증,극륭획득편마Neu5Ac철축매기인(nanA)적편단;이pET28b구건적nanA기인중조질립pRY1전화 E.coli BL21(DE3),연후장nanA기인편단조입도pDR540재체적Ptac계동자하유,소득중조균 E.coli DH5α/pRY3적nanA기인재tac계동자공제하정조성형표체.결과:DNA서렬측정증명,해기인적개방열독광(open reading frame,ORF)대소위894 bp, 편마298개안기산조성적매단백;SDS-PAGE현시,순화적목적단백위33 kD적단일조대.이pET28b구건적nanA기인중조질립pRY1전화 E.coli BL21(DE3),재득도적전화자 E.coli BL21(DE3)/pRY1중,nanA기인수lac계동자공제,무수IPTG혹유당소유도.재N-을선(-D)-신경안산철축매고정화매적최화하합성N-을선-D-신경안산적합성효솔위78.3%,결정회수솔위90.2％,총회수솔위70.6%.전파장소묘분석표명:본실험실결정품여Sigma공사상품적전파장소묘도보유상동적특정.결론:소구건적유도형산매균주 E.coli BL21(DE3)/pRY1화조성형표체균주 E.coli DH5α/pRY3도가교다량적산매.합성적결정품구유N-을선-D-신경안산적특정.
Objective: To obtain the Escherichia coli strains expressing N-Acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase). Methods: The gene (nanA) coding Neu5Ac aldolase was cloned from Escherichia coli C600,and the recombinant plasmid was sequenced and expressed in Escherichia coli. Results: Sequencing data revealed that the open reading frame was 894 bp and predicted to encode a protein consisting of 298 amino acids.The patterns of SDS-PAGE showed that the purified enzyme protein as a single protein band with a molecular weight of 33 kD,which was consistent with those reported in the reference.In the recombinant plasmid pRY1,the expression of nanA gene was controlled by the lac promoter with the induction of IPTG or lactose.The plasmid pRY3 was constructed,in which the nanA gene ws controlled by the tac promoter.The protein of Neu5Ac aldolase was constitutively expressed using the recombinant strain,E.coli DH5α/pRY3 without induction of IPTG or lactose. The crystal was finally obtained with the efficiency of 90.2% of Neu5Ac.The HPLC indicated that the Neu5Ac crystal prepared in this experiment was same as Simga product. Conclusion: The protein products expressed by two recombinant strains E.coli BL21(DE3)/pRY1 and DH5α/pRY3 has the characteristics of Neu5Ac.
