摘要
衣康酸是由巨噬细胞通过炎症刺激促使顺乌头酸脱羧酶生成,衣康酸具有免疫调节、抗氧化、抗炎和杀菌作用,开展衣康酸检测方法研究,是研究其调控作用的关键。沙门菌转录因子RipR(STM3121基因编码)可通过结合STM3121启动子抑制其转录活性,而RipR与衣康酸结合后可解除其抑制作用。基于此调控方式,可将表达绿色荧光蛋白的报告基因AmCyan、STM3121及其启动子3个元件串联后构建衣康酸检测质粒,通过检测AmCyan的转录活性,可实现对衣康酸的定性检测。因此,本研究首先通过重叠PCR技术,将AmCyan、STM3121及其启动子序列克隆至pACYC184质粒中,成功构建衣康酸检测质粒pACYC184-STM3121-AmCyan。其次,为了评价该质粒对衣康酸的检测作用,将其转化DH5α,成功获得DH5α-pACYC184-STM3121-AmCyan衣康酸检测模型菌株。最后,向该模型菌株分别添加外源和内源性(大肠杆菌感染RAW264.7巨噬细胞后的细胞裂解液)2种方式产生的衣康酸,验证该质粒对衣康酸的检测作用。结果表明,二者均可成功诱导DH5α-pACYC184-STM3121-AmCyan模型菌株产生绿色荧光,而对照组不产生。本研究成功建立衣康酸检测方法,为后续开展衣康酸生物学功能研究提供技术支撑。
Itaconate is produced by macrophages through inflammatory stimulation to generate cis-aconite decarboxylase.Itaconate has immunomodulatory,antioxidant,anti-inflammatory and bactericidal effects.The research on detection methods of itaconate is the key to study its regulatory effects.The Salmonella transcription factor RipR(encoding STM3121 gene)inhibits its transcriptional activity by binding to theSTM3121promoter,and the inhibition effect can be removed by binding RipR to itaconate.Based on this regulation mode,the reporter gene AmCyan,STM3121 and its promoter,which express greenfluorescent protein,can be connected in series to construct the itaconate detection plasmid.By detecting the transcriptional activity of AmCyan,qualitative detection of itaconate can be realized.Therefore,AmCyan,STM3121 and their promoter sequences werefirstly cloned into pACYC184 plasmid by overlapping PCR technique,and the itaconate detection plasmid pACYC184-STM3121-amcyan was successfully constructed.Secondly,in order to evaluate the detection effect of the plasmid on itaconate,it was transformed into DH5αand the DH5α-pACYC184-STM3121-Amcyan detection model strain was successfully obtained.Finally,exogenous and endogenous itaconate(cell lysate after Escherichiacoli infected RAW264.7 macrophages)were added to the model strain respectively to verify the detection effect of the plasmid on itaconate.The results showed that,both strains induced green fluorescence in DH5α-pACYC184-STM3121-AmCyan model but not in control group.This study developed a method for the detection of itaconate,which provided technical support for the subsequent research on the biological function of itaconate.
作者
范修齐
王志豪
孔兰芳
鲁壮壮
蒋蔚
陈兆国
韩先干
FAN Xiuqi;WANG Zhihao;KONG Lanfang;LU Zhuangzhuang;JIANG Wei;CHEN Zhaoguo;HAN Xiangan(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Anhui Agricultural University,Hefei 230036,China;Tarim University,Alaer 843300,China)
出处
《中国动物传染病学报》
北大核心
2025年第4期104-110,共7页
Chinese Journal of Animal Infectious Diseases
基金
政府间国际科技创新合作重点项目(2018YFE0102200)
国家自然科学基金(32072829)。
作者简介
第一作者:范修齐,女,硕士研究生,预防兽医学专业;通信作者:韩先干,E-mail:hanxgan@163.com。
