【摘要】 报道了一个新的含ＰＲＰＬ，Ｐｔａｃ双启动了的大肠杆菌表达载体ｐＮＪ的构建．在ＰＲＰＬ，Ｐｔａｃ分别或共同启动下，报告基因人肿瘤坏死因子（ＴＮＦ）基因获得了较好的表达．双启动共同启动时的表达水平为两启动子分别启动时的表达水平之和．更多还原

【Abstract】 The expression of recombinant genes in E. colt has become an valuable toolfor basic research and industrial applications. Yields of 30% or more of the total cellularproteins can be obtained with some foreign genes in this expression system.Because of its advantages such as economy, effieiency, simplicity of efficient expressionand short growth period, it attracts much attention of researchers to develop new kindvectors to increase gene expression level, or even to express more than one genes in one expression vector.In this article, we report the construction of a new expression vector PNJ, which conjoins PRPL and Ptac promoters. The vector is deviated from PDR540 and PBV220, each ofwhose promoters is Ptac and PRP. Digested by EcoRI, Blunted.by Klenow, then cut byBamHI, a 400hp fragment containing Ptac pronoter is obtained from PDR540 plasmid,PBV220 is partially cut by HindTll, blunted by Klenow, finally digested by BamHI, its larger fragment will be used. After ligation of the two fragments and selection with restrictionenzymes, we generate expression vector PNJ, which has PRP. and Ptac dual promoters.To test the function of pllJ, TNF cDNA is used as a reporter gene. TNF cDNA inserted in pUC18 is isolated by BamHI and HIndlll digestion, then cloned into Bamlll siteof PNJ. It assures that only the head to head ligated TNF fragments can be Inserted intopNJ, and each of two TNF cDNA is controlled by pRPL and Ptac respectively.Induction of TNF is performed under different conditions. As we known, Ptac is induced by IPTG, while PRPL is done at 42C. So when PNJ / TNF is induced by IPTG or at42t, only Ptac or PRPL controlled TNF will be expressed, if when induced by TPTG at42t,’the two TNF genes will be expresed. Comparing the expression level of TNF by TNFbiologic activity measurement, we find that the biologic activity of TNF induced by IPTGis 5 ×105u / cm3, while that of TNF induced at 42t is 1 ×106u / cm3. If both Ptac andpRPL work, TNF activity is 5 x 106u/ cm3. SDS-PAGE result also shows that when induced by IPTG at 42C, the expression level of TNF is about 15% of total cellular proteins,when done by either IPTG or at 42C, the level is about 5% or 11 % repectively. It indicatesthat the amout of TNF promoted by two promoters is twice as much as that done by Ptaconly. These two results imply that only part of expressed TNF is soluble, others is inclusionho,dies. Prpl, Ptac in PNJ may promote foreign to express individually or simultaneously.更多还原