Mohseni Amir Hossein, Razavilar Vadood, Keyvani Hossein, Razavi Mohammad Reza, Khavari-Nejad Ramazan Ali
Department of Microbiology, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Islamic Republic of Iran.
Department of Food Hygiene, Faculty of Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tehran, Islamic Republic of Iran.
Microb Pathog. 2017 Sep;110:554-560. doi: 10.1016/j.micpath.2017.07.039. Epub 2017 Jul 25.
The present work was aimed at investigating the expression and optimization of a human papillomavirus (HPV) type 16 gene encoding oncoprotein E7 in Lactococcus lactis. We genetically engineered Lactococcus lactis using nisin-controlled gene expression (NICE) system pNZ8148 to express the native and codon optimized E7 oncogenes isolated from Iranian HPV-16. The results of optimizing fermentation showed, the concentration of produced protein was expressively improved by 10 ng/mL nisin after 3.5, and 4 h induction for NZ9000 harboring the codon-optimized, and native E7 respectively. Furthermore the recombinant NZ9000 strains expressed rE7 by maximum value of 4.7 (Codon-optimized), and 1.82 μg/mL (Native) in static flask experiments at initial glucose concentrations of 50 and 75 g/L respectively. The rE7 yield was further enriched in batch fermenter experiments using controlled pH. Thus, the overall production of rE7 under optimized conditions accumulated in the cytoplasm to nearly 33.25 μg/mL by L. lactis NZ9000 containing codon-optimized E7, which was over ∼2.7-fold higher compared to the NZ9000 having native E7 strain (12.01 μg/mL). Accordingly, the maximum biomass production was calculated 4.87, and 1.51 g/L respectively.
本研究旨在调查人乳头瘤病毒16型（HPV-16）编码致癌蛋白E7的基因在乳酸乳球菌中的表达及优化情况。我们利用乳链菌肽控制基因表达（NICE）系统pNZ8148对乳酸乳球菌进行基因工程改造，以表达从伊朗HPV-16分离出的天然及密码子优化的E7致癌基因。优化发酵结果显示，对于分别携带密码子优化型和天然型E7的NZ9000，在诱导3.5小时和4小时后，添加10 ng/mL乳链菌肽可显著提高蛋白产量。此外，在初始葡萄糖浓度分别为50 g/L和75 g/L的摇瓶实验中，重组NZ9000菌株表达的重组E7（rE7）最大值分别为4.7 μg/mL（密码子优化型）和1.82 μg/mL（天然型）。在使用pH控制的分批发酵实验中，rE7产量进一步提高。因此，在优化条件下，含有密码子优化型E7的乳酸乳球菌NZ9000在细胞质中积累的rE7总产量接近33.25 μg/mL，比含有天然型E7菌株的NZ9000（12.01 μg/mL）高出约2.7倍。相应地，最大生物量产量分别计算为4.87 g/L和1.51 g/L。
