【摘要】 目的　确定在一个表达质粒载体上串联目的操纵子以提高目的蛋白表达量的可行性 ,阐明宿主细胞对胞内质粒 DNA总量调控的可能机制。方法　亚克隆构建操纵子正向串联的表达载体 ;SDS凝胶电泳和激光扫描测定目的蛋白表达量 ;3H- Td R掺入法测定质粒拷贝数。结果　构建两组质粒 :CW11系列分别含 1～ 4个正向操纵子 ;CW12系列分别含 1～ 3个正向操纵子。在未诱导时 ,操纵子的串联不影响宿主大肠杆菌的生长 ;温度诱导表达后 ,CW11系列目的蛋白表达量分别为菌体总蛋白的 46 .0 %、5 4.8%、5 6 .1%和 6 0 .1% ,CW12系列目的蛋白表达量分别为菌体总蛋白的 33.5 %、44 .0 %和 47.1%。两组质粒的拷贝数均随操纵子串联个数的增加而减少 ,但目的基因的总剂量随之增加。实验数据同时表明 ,在相同的培养条件下 ,同一菌株每个宿主细胞内的质粒 DNA总量被限制在一定范围内。结论　操纵子串联增加了目的基因的剂量 ,从而提高了目的基因在大肠杆菌中的表达水平。另外 ,质粒大小和其拷贝数呈负相关 ,在同一培养条件下 ,对于特定的大肠杆菌菌株 ,宿主细胞内质粒 DNA总量在一定程度上是一个相对的被限定的常数。更多还原

【Abstract】 Objective To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli and to clarify the potential control mechanism of the total plasmid DNA amount in each host cell. Methods Two series of expression plasmids,CW11 and CW12,containing 1 to 4 and 1 to 3 heterologous gene operon(s),respectively,were constructed.The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments.The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments.Protein expression was analyzed via SDS PAGE and laser scanning.Plasmid copy number was determined by incorporation with 3H TdR([ 3H] thymidine). Results No influence of the tandem joined operons on host growth and plasmid stability was observed.Upon induction,the desired protein accumulated in the CW11 series were 46.0%,54.8%,56.1%,and 60.1% of total cell protein.In the CW12 series,the yields were 33.5%,44.0%,and 47.1% of total cell protein.With increasing size,however,plasmid copy number decreased.Further calculation showed that the total amount of plasmid DNA per cell is restricted to some extent.There seems exists a mechanism to control the maximum level of total plasmid DNA amount in each host cell. Conclusions Increasing the target gene dosage by tandem linking of operons enhanced the expression level of a desired protein.Although the size (kb) and the copy number of each plasmid are negatively interrelated,it seems that as for certain plasmids,their total DNA amount per cell is a restricted constant for a specific E.coli strain under identical incubation condition.更多还原