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目的 确定在一个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 亚克隆构建了两组操纵子正向串联的表达质粒 :CWll系列分别含 1- 4个正向操纵子 ,质粒大小以 2 2 5kb的增加量从 5 4 7kb增加至 12 2 6kb ;CW12系列分别含 1- 3个正向操纵子 ,质粒大小以 2 16kb的增加量从 5 4 0kb增加至 9 72kb。SDS凝胶电泳和激光密度扫描测定目的蛋白表达量 ;3 H TdR掺入法测定质粒拷贝数。结果 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 4 4 9%± 3 9%、5 1 3%± 4 1%、5 4 8%± 3 3%和 5 8 2 %± 3 4 % ,CW12系列目的蛋白表达量分别为菌体总蛋白的 32 2 %± 5 0 %、4 2 8%± 4 1%和 4 6 9%± 4 0 %。两组质粒的拷贝数均随操纵子串联个数的增加而显著减少 (P <0 0 1) ,但目的基因的总剂量随之显著增加 (P <0 0 1) ,而同一系列的质粒在每个宿主细胞内的质粒DNA总量没有显著的变化 (P >0 0 5 )。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。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 ,同一系列的质粒在宿主细
OBJECTIVE: To determine the feasibility of tandemly connecting the target operon on a plasmid vector to increase the expression of the target protein, and to elucidate the possible mechanism by which host cells regulate the total amount of intracellular plasmid DNA. METHODS: Two pairs of expression plasmids were constructed by subcloning: the CWll series contains 1 to 4 forward operons, respectively, and the size of the plasmids increased from 574kb to 1226kb with an increase of 225kb. CW12 series Containing 1 to 3 forward operons, respectively, the plasmid size increased from 5400kb to 972kb with an increase of 216kb. The SDS-PAGE and laser densitometry were used to determine the expression level of the target protein. The plasmid copy number was determined by 3 H TdR incorporation method. The results showed that the cascade of operon did not affect the growth of E.coli. The expression of target protein of CW11 series after temperature induction were 44.9% ± 39%, 51.3% ± 41.1%, 54 8% ± 3 3% and 5 8 2% ± 34% respectively. The expression of target proteins in CW12 series were 32 2% ± 5 0%, 42 8% ± 4 1% and 4 6 9% ± 40%. The copy numbers of plasmids of both groups were significantly decreased with the increase of the number of the intron (P <0.01), but the total dose of the target gene was significantly increased (P <0.01), while the same series of plasmids The total amount of plasmid DNA in each host cell did not change significantly (P> 0.05). Conclusion Operators in series increase the dose of the target gene and increase the expression level of the target gene in E. coli. Plasmid size and its copy number was negatively correlated, under the same culture conditions, for a specific strain of E. coli, the same series of plasmids in the host fine