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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。
OBJECTIVE: The target fusion protein XE-TNFαm2 expressed in this genetically engineered E. coli DH5α / pCW-PL-XE-TNFαm2 has been initially demonstrated to have the potential to eradicate the HIV virus in AIDS patients. The target protein expression level of 32% to 36% of total cellular protein. The aim of this study was to verify the genetic stability. Methods The strain DH5α / pCW-PL-XE-TNFαm was subcultured by single colony on LBAmp + and LBAmp- two solid medium respectively and cultured overnight at 32 ℃. At intervals of ten generations, the expression of XE-TNFαm2 protein was determined by the general temperature-dependent expression technique. Finally, the differences of the expression level of the target protein (20.3 kDa) between the generations were compared and analyzed. Results The expression levels of XE-TNFαm2 in the recombinant genetically engineered bacteria after 100 generations were not significantly different (P> 0.05) except that in the above two cases, 6 months, the target protein expression level of 8% decline. The vector plasmid contains CIts857 sequence, PL promoter and T1T2 terminal termination sequence, is to ensure that the target gene stable high expression of the three key elements. Conclusion The results of this study demonstrate that the engineering strain DH5α / pCW-PL-XE-TNFαm2 has good genetic stability.