【摘要】 目的本基因工程大肠杆菌DH5α/pCW-PL-XE-TNFαm2所表达的靶向融合蛋白XE-TNFαm2已被初步证明具有用于清除艾滋病患者体内HIV病毒的前景。其目的蛋白表达水平为32%～36%细胞总蛋白。本研究旨在验证其遗传稳定性。方法工程菌株DH5α/pCW-PL-XE-TNFαm分别在LBAmp+与LBAmp-二种固体培养基上逐日单菌落划线传代,32℃培养过夜。每间隔十代运用一般温控表达技术,确定其XE-TNFαm2的蛋白含量,最后比较分析各代之间目的蛋白(20.3 kDa)表达水平的差异情况。结果该重组基因工程菌连续传100代后XE-TNFαm2的蛋白表达水平没有明显差异(P>0.05);只是在上述两种情况下传至100代后将其置于4℃保藏4、5、6个月,其目的蛋白表达水平有8%的下降。本载体质粒含有的CIts857序列、PL启动子与T1T2末端终止序列,是确保目的基因稳定高表达的3个关键元件。结论本研究结果证明该工程菌DH5α/pCW-PL-XE-TNFαm2具有良好的遗传稳定性。更多还原

【Abstract】 Objective The targeting fusion protein XE-TNFαm2 produced by the genetic engineered E.coli strain DH5α/pCW-PL-XE-TNFαm2 was shown a prospect to effectively eliminate the HIV in AIDS patients.Its expression level reached 32%～36% of total cell soluble proteins.The purpose of this study was to investigate the hereditary stability of this engineered strain.Methods This strain was transferred on LBAmp+ and LBAmp-solid culture media day by day as generation by generation for a total of 100 generations,and then stored at 4℃ for 4,5,6 months,respectively.Finally,to check the protein XE-TNFαm2(20.3 kDa) expression levels of each transferred generation strain on LBAmp+ and LBAmp-media cases,respectively,by general protocol for temperature control expression,so as to compare the expression levels in the above different cases.The samples were taken from generations transferred for 1,10,20,30,40,50,60,70,80,90,100 days on LBamp+ and LBAmp-media,respectively.Results The protein XE-TNFαm2 expression levels were not obviously changed from the 1st generation to 100th generation under LBamp+ and LBAmp-cases,respectively(P>0.05).Only in the case that after the strains were transferred for 100 generations on LBamp+ or LBAmp-media and then stored at 4℃ for 4,5 or 6 months,respectively,the protein XE-TNFαm2 expression levels were reduced by 8%.Conclusions CIts857 sequence,PL promoter and termination T1T2 sequence,existing in this plasmid vector,are three key elements to maintain such a high expression level.This genetic engineering E.coli strain DH5α/pCW-PL-XE-TNFαm2 has good hereditary stability.更多还原