cIts857基因的克隆、修饰及温敏诱导表达载体  

Cloning and Modification of cIts857 Gene for Constructing the Regulated Expression Vectors

作　　者：李伯良[1] 江智红[1] 

机构地区：[1]中国科学院上海生物化学研究所

出　　处：《生物化学与生物物理学报》1994年第4期389-396,共8页

摘　　要：从噬菌体λＤＮＡ（ｃＩｉｎｄｌｔｓ８５７Ｓａｍ７）克隆出９９０ｂＰ的ｃＩｔｓ８５７基因，经缺失，点突变等修饰改造和ＤＮＡ序列测定，获得了带有自身启动子区的修饰型ｃＩｔｓ８５７基因（７７０ｂｐ）。进而利用它构建大肠杆菌表达载体ｐＢＬＭＶＬ２和一套含３种不同阅读框架ｌｉｎｋｅｒ区的表达载体ｐＢＬＭＦＶ４、５Ｂ、６。上述表达载体；用于表达人工合成的牛生长激素（ｂＧＨ）、人干扰素－γΔＣ－１３和酵母ｐｈｏ８５等外源基因，都观察到这些基因产物的温敏表达。这些表明，克隆、修饰并组建到表达载体中的λ噬菌体ｃＩｔｓ８５７基因表达出具有功能作用的转录阻遏蛋白，使上述ＰＬ启动子控制下的大肠杆菌表达载体可经温敏诱导而高效表达外源基因产物．The modified cIts857 gene of λ phage was obtained by cloning a 990bp DNA(cIindlts857Sam7),modified by deletion of the 3'-noncoding sequence and mutation of two HindIII sites.It was used to construct expression vectors(pBLMVL2and pBLMFV4,5B,6),which have three open reading frames in linker for inserting the DNA fragments without the start codon ATG. Using the above constructed expression vectors,the high-level products,such as Met-Phe-bGH,Ala-bGH, hIFN-γΔC-13 and pho85 expressed in E. coliharboringtheir expression plasmids induced at 42℃ were observed by analysis of SDS-PAGE and dye staining. This suggests that the modified cIts857 gene in the expression plasmidscan work well and its expressed protein can regulate the PL promoter in the same plasmid.

关 键 词：cIts857基因 基因克隆 载体 

分 类 号：Q7[生物学—分子生物学]