嗜粘蛋白阿克曼菌Amuc1100蛋白对高脂饲料联合链脲佐菌素干预大鼠的预防保护作用  

Prokaryotic expression of Amuc100 protein and its effects on high-fat diet rats combined streptozotocin injection

作　　者：董新燕 刘曼妮 刘雪 刘敏[1] 张翔凌 王国庆[1] Dong Xinyan;Liu Manni;Liu Xue;Liu Min;Zhang Xiangling;Wang Guoqing(West China School of Public Health and West China Fourth Hospital,Sichuan University,Chengdu 610041,China)

机构地区：[1]四川大学华西公共卫生学院/四川大学华西第四医院,成都610041

出　　处：《卫生研究》2020年第5期785-789,822,共6页Journal of Hygiene Research

基　　金：四川省科技厅重点研发项目(No.2019YFS0304)。

摘　　要：目的构建嗜粘蛋白阿克曼菌Amuc1100蛋白的原核表达体系,分析该蛋白对高脂饲料联合链脲佐菌素(streptozotocin,STZ)干预大鼠体重、血糖、肠道屏障功能及嗜粘蛋白阿克曼菌丰度的影响。方法将Amuc1100基因(ID:34174504)的扩增产物与双酶切后的pET-26b质粒载体连接。重组的表达质粒pET-26b-Amuc1100转化大肠杆菌BL21感受态细胞。选取测序正确的阳性克隆菌株,异丙基-β-D-硫代半乳糖苷(isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导表达。纯化后的蛋白以高低两个剂量(6和3μg/d)灌胃高脂饲料联合30 mg/kg STZ干预的大鼠8周,分析大鼠体重、血糖、肠道屏障功能相关指标(脂多糖、肿瘤坏死因子-α、二胺氧化酶、D-乳酸)及粪便中嗜粘蛋白阿克曼菌含量的变化。结果成功构建pET-26b-Amuc1100重组表达质粒,构建的克隆质粒测序结果与GenBank中Amuc1100基因的同源性为100%,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)和蛋白质印迹法(Western blot)结果显示获得的蛋白分子质量约为42 kDa。Amuc1100蛋白对高脂饲料联合STZ干预大鼠的灌胃可以在一定程度上减轻大鼠体重(P<0.05和P<0.01),改善其肠道屏障功能(P<0.01)及增加肠道中嗜粘蛋白阿克曼菌含量(P<0.01)。结论成功构建Amuc1100蛋白的原核表达系统,该蛋白对高脂饲料联合STZ干预大鼠的代谢状况有一定的调节作用。OBJECTIVE Establish the prokaryotic expression system of Amuc1100 protein from Akkermansia muciniphila, and analyze the effects of this protein on the body weight, blood glucose,intestinal barrier function and Akkermansia muciniphila abundance in rats fed with high-fat diet combined streptozotocin(STZ)injection. METHODS PCR product of Amuc1100 Gene(Gene ID: 34174504) was linked to the double enzyme-digested pET-26 b plasmid vector. The recombinant expression plasmid pET-26 b-Amuc1100 then transformed into E.Coli BL21. The verified clones through sequence analysis were induced by the addition of IPTG. High-fat diet rats were interfered with the purified protein at high and low doses. The changes of body weight, blood glucose, intestinal barrier function and Akkermansia muciniphila abundance were analyzed. RESULTS The recombinant expression plasmid pET-26 b-Amuc1100 was successfully constructed. The sequencing result showed 100% similarity to the Amuc1100 gene in GenBank. The molecular weight of the protein obtained was 42 kDa. The intervention of Amuc1100 protein can reduce the weight of rats fed with high-fat diet combined STZ injection to some extent, improve barrier function and increase the abundance of Akkermansia muciniphila in intestine. CONCLUSION The prokaryotic expression system of Amuc1100 protein was successfully constructed, which has a certain regulatory effect on rats fed with high-fat diet combined STZ injection.

关 键 词：嗜粘蛋白阿克曼菌 Amuc1100蛋白 高脂饲料 

分 类 号：Q591.2[生物学—生物化学] Q51