重组人硫氧还蛋白工程菌生物学特性稳定性的检测

Stability of biological characteristics of recombinant Escherichia coli expressing human thioredoxin

摘要:

【目的】观察和检测重组人硫氧还蛋白工程菌BL21/pET-22b(+)-rhTrx生物学特性的稳定性。【方法】工程菌连续传代50代, 通过对每10代进行质粒性状、蛋白表达水平、透射电镜观察、革兰氏染色及各项生化检查, 全面检测工程菌可能影响生产性能的生物学特性稳定性。【结果】各代工程菌提取的质粒经双酶切后均可见315 bp的目的基因片段; 各代工程菌中Trx蛋白的表达量均为菌体总蛋白的20%左右; 透射电镜观察呈现典型的大肠杆菌特性; 革兰染色显示为阴性杆菌; 各项生化检测结果与原始菌种无显著差异。【结论】该菌种生物学特性稳定, 可作为生产用菌种。

Abstract:

[Objective] This study was to detect the stability of biological characteristics of recombinant E. coli BL21/pET-22b(+)-rhTrx expressing human thioredoxin. [Methods] Recombinant E. coli BL21/pET-22b(+)-rhTrx was subcultured for 50 passages, and subjected to transmission electron microscopy, Gram staining and biochemical examination as well as test for expression level of target protein and property of plasmid every 10 passages. [Results] The results of restriction analysis and sequencing of various passages were in agreement. All the expression levels of target protein in recombinant E. coli BL21/pET-22b(+)-rhTrx of passages 0, 10, 20, 30, 40 and 50 passages were about 20%, which showed no significant difference. All the passages were negative in Gram staining and showed typical characteristics of E. coli under transmission electron microscope and in biochemical examination. [Conclusion] The recombinant E. coli BL21/pET-22b(+)-rhTrx showed stable biological characteristics and might be used as a bacterial seed for production.