重组草酸降解酶基因的乳酸菌治疗高草酸尿症的实验研究
发布时间：2019-04-12 06:22
【摘要】：目的:草酸钙结石是泌尿外科最常见的疾病之一,高草酸尿症是其主要致病因素,目前尚无有效的防治方法。肠道吸收的食源性草酸是尿草酸的重要来源,减少食物中草酸含量可有效降低尿草酸排泄量。草酸脱羧酶(oxalate decarboxylase/ODC)和草酸氧化酶(oxalate oxidase/Ox O)是自然界中存在的两种特异性草酸降解酶,在预防高草酸尿方面具有广泛的应用前景。本研究将ODC和Ox O基因转入乳酸菌(lactic acid bacteria/Lab)中,使其表达草酸降解酶,达到持续有效降解草酸的效果,通过口服途径降解食物中的草酸,减少肠道草酸的吸收,降低尿草酸排泄量,从而预防草酸钙结石的形成。方法:提取ODC及Ox O编码基因,将基因片段与Lab表达质粒p MG36e相连接,为了提高表达效率,在编码基因前插入p H诱导的强启动子p170;将重组表达质粒通过电转化转入Lab菌株MG1363中。在含有100mmol/L草酸的MRS培养基中于37℃条件下静置培养转基因乳酸菌48h,利用分光光度计和高效液相色谱仪测量不同时间的培养基的吸光度和草酸浓度,检测Lab的生长情况及降解草酸效率;用含5%草酸食物喂养SD大鼠,构建高草酸尿模型,同时转基因乳酸菌灌胃治疗1个月,分别在第0、7、14、21、28天收集24h尿液,测量尿液中草酸含量,并在第30天处死大鼠,取出肾脏,切片固定,利用硝酸银染色,观察肾脏中草酸钙结晶形成情况。结果:转ODC基因乳酸菌(ODC-Lab)和转Ox O基因乳酸菌(Ox O-Lab)可在高草酸浓度培养基中生长,ODC-Lab可有效降解培养基中草酸,插入强启动子p170(p170-ODC-Lab)可提高草酸降解效率;而Ox O-Lab和p170-Ox O-Lab并无有效降解草酸的功能;在动物实验中,ODC-Lab和p170-ODC-Lab灌胃治疗可明显缓解高草酸尿并预防草酸钙结石形成,其中p170-ODC-Lab效果更佳,相反Ox O-Lab和p170-Ox O-Lab无防治高草酸尿症和草酸钙结石的功效。结论:重组ODC乳酸菌可有效降解草酸,p170可以增强目的基因的生物学效应,ODC-Lab和p170-ODC-Lab都可通过口服途径有效减少食源性草酸的吸收,并对预防草酸钙结石具有显著作用。
[Abstract]:Aim: calcium oxalate stone is one of the most common diseases in urology, hyperoxaluria is the main cause of disease, and there is no effective method to prevent and cure it. Food-derived oxalic acid absorbed by intestinal tract is an important source of urinary oxalic acid. Reducing oxalic acid content in food can effectively reduce urinary oxalic acid excretion. Oxalic acid decarboxylase (oxalate decarboxylase/ODC) and oxalate oxidase (oxalate oxidase/Ox O) are two kinds of specific oxalate degrading enzymes in nature. In this study, ODC and Ox O genes were transferred into Lactobacillus (lactic acid bacteria/Lab to express oxalic acid degrading enzyme, which could degrade oxalic acid continuously and effectively. The oxalic acid in food was degraded by oral administration, and the absorption of oxalic acid in intestinal tract was reduced. Reduce urinary oxalic acid excretion, thereby preventing the formation of calcium oxalate stones. Methods: ODC and Ox O genes were extracted and ligated with the Lab expression plasmid p MG36e. In order to improve the expression efficiency, the strong promoter p170 induced by pH was inserted before the coding gene. The recombinant expression plasmid was transformed into Lab strain MG1363 by electroporation. Transgenic lactic acid bacteria were statically cultured in MRS medium containing 100mmol/L oxalic acid at 37 鈩，
本文编号：2456768
[Abstract]:Aim: calcium oxalate stone is one of the most common diseases in urology, hyperoxaluria is the main cause of disease, and there is no effective method to prevent and cure it. Food-derived oxalic acid absorbed by intestinal tract is an important source of urinary oxalic acid. Reducing oxalic acid content in food can effectively reduce urinary oxalic acid excretion. Oxalic acid decarboxylase (oxalate decarboxylase/ODC) and oxalate oxidase (oxalate oxidase/Ox O) are two kinds of specific oxalate degrading enzymes in nature. In this study, ODC and Ox O genes were transferred into Lactobacillus (lactic acid bacteria/Lab to express oxalic acid degrading enzyme, which could degrade oxalic acid continuously and effectively. The oxalic acid in food was degraded by oral administration, and the absorption of oxalic acid in intestinal tract was reduced. Reduce urinary oxalic acid excretion, thereby preventing the formation of calcium oxalate stones. Methods: ODC and Ox O genes were extracted and ligated with the Lab expression plasmid p MG36e. In order to improve the expression efficiency, the strong promoter p170 induced by pH was inserted before the coding gene. The recombinant expression plasmid was transformed into Lab strain MG1363 by electroporation. Transgenic lactic acid bacteria were statically cultured in MRS medium containing 100mmol/L oxalic acid at 37 鈩，
本文编号：2456768
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