Yuan Ling-Zhi, Shi Xiao, Tang Dan, Zheng Shao-Peng, Xiao Zhi-Ming, Wang Fen
Department of Gastroenterology, the Third Xiangya Hospital, Central South University, Changsha, Hunan, 410013, China.
Hunan Key Laboratory of Non-resolving Inflammation and Cancer, Central South University, Changsha, Hunan, 410013, China.
BMC Microbiol. 2021 Aug 18;21(1):229. doi: 10.1186/s12866-021-02262-7.
H. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further exploration of the role of VacA in H. pylori -induced inflammation of cancer.
A 2502-bp fragment and VacA gene were identified. An 89.7-kDa VacA recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay.
A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).
幽门螺杆菌与各种消化性胃炎、消化性溃疡及黏膜相关淋巴组织（MALT）淋巴瘤的发生发展密切相关。幽门螺杆菌也是胃癌的Ⅰ类致癌物。空泡毒素（VacA）是幽门螺杆菌唯一的外分泌毒素，在幽门螺杆菌致病机制中发挥着非常重要的作用。在自然条件下VacA的产生过程复杂，工作量大且产量低。因此，获得稳定且具有生物活性的重组VacA蛋白非常重要。本研究旨在探索幽门螺杆菌VacA毒素在大肠杆菌中的表达、纯化及稳定保存方法，为进一步探究VacA在幽门螺杆菌诱导的炎症及致癌作用中的角色提供实验依据。
鉴定出一段2502 bp的片段及VacA基因。从重组工程菌中表达并纯化出了一个89.7 kDa的VacA重组蛋白，该蛋白在50 mM乙酸缓冲液（pH 2.9）中能稳定保存。重组蛋白在包涵体中的含量比在上清液中多。此外，用VacA重组蛋白培养GES-1细胞24小时后，在倒置显微镜和透射电镜下观察到细胞出现凋亡迹象，包括早期细胞核固缩和聚集。通过TUNEL检测发现VacA重组蛋白可诱导细胞凋亡。
成功构建了一种稳定高表达且具有促凋亡活性的VacA重组蛋白。该蛋白在50 mM乙酸缓冲液（pH 2.9）中能稳定保存。
