小鼠S100A9蛋白在枯草芽孢杆菌中的表达及生物学活性分析  

Expression and biological activity analysis of recombinant mouse S100A9 in Bacillus subtilis

作　　者：李婷 李甜甜[1,2,3] 丁明玲 彭勇波[1,2,3] LI Ting;LI Tiantian;DING Minling;PENG Yongbo(South-Central Minzu University,College of Life Sciences,Wuhan 430074,China;South-Central Minzu University,Institute for Medical Biology,Wuhan 430074,China;South-Central Minzu University,Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China,Wuhan 430074,China)

机构地区：[1]中南民族大学生命科学学院,武汉430074 [2]中南民族大学生物医学研究所,武汉430074 [3]中南民族大学武陵山区特色资源植物种质保护与利用湖北省重点实验室,武汉430074

出　　处：《中南民族大学学报（自然科学版）》2025年第1期36-42,共7页Journal of South-Central Minzu University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(31371307);中央高校基本科研业务费专项资金资助项目(CZZ21005)。

摘　　要：钙结合蛋白S100A9与细胞炎症应答及肿瘤生长等密切相关,但对其生物学功能还不十分清楚.本研究构建了小鼠S100A9基因的枯草芽孢杆菌诱导表达载体,以期获得具有生物活性的S100A9蛋白并探讨其功能.采用无缝克隆将小鼠S100A9基因编码区序列克隆至pHT43载体的Pgrac启动子区,并将重组载体pHT43-S100A9转化至芽孢杆菌WB800N菌株,经IPTG诱导S100A9蛋白表达.通过LPS诱导的RAW264.7细胞炎症模型检测了重组蛋白S100A9对炎症应答的调控作用.Western Blot证实了S100A9重组蛋白分泌表达,且在0.2 mmol/L的IPTG、30℃的发酵条件下其表达水平最佳.ELISA检测表明S100A9重组蛋白分泌表达量达到了15.47 ng/mL.分别利用含有1.54、3.09、6.59 ng/mL三种不同浓度S100A9重组蛋白的发酵液上清作用于LPS诱导的RAW264.7细胞,与空载组比较,1.54 ng/mL的S100A9重组蛋白能显著抑制LPS诱导的RAW264.7细胞促炎因子TNF-α及IL-6等的表达(P<0.05).Calcium-binding protein S100A9 was associated with inflammatory response and growth of tumors,and the underlying functions remains further defined.To gain recombinant S100A9 protein with biological activity and explore its function,the mouse S100A9 coding sequence was amplified by PCR and cloned into the expression vector of pHT43 and fusion with Pgrac promoter.The recombinant plasmid pHT43-S100A9 was transferred to Bacillus subtilis WB800N and induced expression with IPTG.The expressed products were identified by Western Blot;the biological activity of recombined products were analyzed using LPS stimulated RAW264.7 cell.The recombinant bacteria WB800N/pHT43-S100A9 was induced expression by IPTG.The optimized induction temperature was 30℃with 0.2 mmol/L IPTG,and the secretory expression level of S100A9 is up to 15.47 ng/mL.To further analyzed the function of recombined protein,the LPS stimulated RAW264.7 cell was treated by the culture supernatants of the recombined Bacillus subtili with different concentration recombiner S100A9 protein(1.54,3.09,6.59 ng/mL),and the results showed that the pro-inflammatory factors,including TNF-αand IL-6 were significantly inhibited comparing with the empty vector group.

关 键 词：S100A9蛋白 枯草芽孢杆菌 异源表达 RAW264.7细胞 炎症因子 

分 类 号：Q785[生物学—分子生物学]