保加利亚乳杆菌分解多种尿毒素的基因定向诱变及其功能研究

Lactobacillus bulgaricus is one kind of human probiotics, which colonizes in the intestinal mucosa for long-term。It can enhance immunity、inhibit pathogens, Synthesize vitamins, decrease cholesterol etc. If the genes of the bacteria is modified, it is likely to continuously secrete urotoxin clastic enzyme and efficiently decompose urine toxins。This Study will use a culture medium with serum of uremic patients to induce the Lactobacillus bulgaricus , and select the lactobacilli with strongest degradation capacity of urine toxins as an original strain，then the genes of lactobacillus strains is modified by using physical and chemistry mutagenesis technique, to obtain an gene engineering bacterium with the ability of urine toxins degradation, and the gene sequences of the gene engineering bacterium are analyzed and identified, in order to construct a related mutant database。The manipulated gene engineering L.lactis is made into biological agents to feed the rats with Chronic renal failure. Then a variety of effects of this gene engineering bacterium are investigated, including the ability of decomposing urine toxins, the intestinal mucosal adhesion of colonization and viability, genetic stability of the plasmid, plasmid duplication and inter-bacterial transmission functions. In addition, the damage of urine toxins on residual kidney function is explored via measuring cytokines related with kidney damage and observing renal pathological changes of rats withc hronic renal failure If this mutant gene engineering bacterium is constructed successfully，it will provide a candidate bacteria as a great biological agents with comprehensive therapeutic effect on chronic renal failure 。It can not only reduce various urotoxins ，but also improve the immunocompromised patients with chronic renal failure , as well as compensate the defect of vitamin deficiency and fat metabolism disorders。

保加利亚乳酸杆菌是人体益生菌，长期定植于肠粘膜，有增强免疫、抑制病原菌、合成多种维生素及降胆固醇等功能。如改变该菌的基因有可能持续分泌尿毒素分解酶，高效分解尿毒素。本课题将保加利亚乳酸杆菌置于含尿毒症患者血清的培养基作定向诱导培养，选出尿毒症毒素分解能力最强的菌株作出发菌株，用物理化学双重多次诱变改变乳酸杆菌的基因，获取一种能够分解多种尿毒素的乳酸工程菌并进行基因序列分析鉴定，构建突变体库。后将乳酸工程菌制成生物制剂喂饲慢性肾衰大鼠，观察其分解尿毒素的能力；研究其肠粘膜粘附定植及生存能力、质粒的遗传稳定性、质粒复制及细菌间传递的功能；通过测定与肾损害相关的细胞因子和观察慢性肾衰大鼠肾脏病理改变，探讨尿毒素对残余肾功能的损害作用。如本工程菌诱变成功，将为慢性肾衰研制具有综合治疗作用的生物制剂提供一种候选菌，使其在降低多种尿毒素基础上，弥补慢性肾衰患者免疫力低下、维生素缺乏和脂肪代谢紊乱等缺限

摘要：本课题已按年度计划完成全部研究内容：首先筛选具有较高分解尿毒素能力的野生保加利亚乳酸杆菌为出发菌株，用尿毒症患者血清为定向诱变剂将筛选的乳酸杆菌进行化学和物理双重6次诱变，选育了能分解多种尿毒素的保加利亚乳杆菌突变株L.b-DUC-17和L.b-DUU-22。并对诱变乳杆菌在消化道的生存能力进行监测，发现该菌具有抗酸、抗碱、抗胃蛋白酶、胆盐和胰蛋白酶作用，并有良好的肠粘膜粘附功能；本菌能抑制致病性大肠杆菌（EPEC、）粪肠球菌（E.faecalis）及金黄色葡萄球菌（SA）的生长。为在肠道寄居持续分解尿毒素提供了生物学基础。体外研究发现该菌能分解尿素、肌酐、尿酸、硫酸吲哚酚、甲状旁腺素等多种尿毒素。用诱变乳杆菌治疗慢性肾衰SD大鼠，结果显示该菌在体内能分解多种尿毒素，与体外研究结果吻合；诱变乳杆菌能抑制炎性细胞因子TGF-β1和FN在肾脏的表达，减轻肾间质纤维化对肾脏病理有明显修复作用。其降解尿毒素功能的细菌间传递实验结果表明诱变乳杆菌不能传递给大肠埃希菌、肠球菌.诱变乳酸杆菌的全基因测序结果如下：基因长度为3.1mp，基因个数2768，GC含量47.4%+-0.03，SNP变异数在56,389-56,575之间，变异率53%。插入Insertion20-27，缺失10-15.其基因突变范围广，证明物理和化学复合诱变易于得到饱和突变。与本课题研究目标一致，即多种尿毒素分解酶基因可以同时一次性突变。对以往一种诱导剂只能诱导一种酶基因突变的方法作了根本性的改革。本课题还构建了第一个分解尿毒素乳酸杆菌的突变体库。本突变体库包括6组突变基因群体：野生菌、诱导菌、物理诱变菌、化学诱变菌、复合诱变菌和多次复合诱变菌的突变体。为探明不同诱变方法的基因改变和研究多种尿毒素分解酶基因定位提供了实验基础。乳酸菌的研究国内外均集中在农业和食品工业。医药卫生领域的研究刚起步。本课题开启了乳酸杆菌特殊功能的开发研究。为尿毒症毒素的生物分解提供了经济环保的候选菌，为非透析疗法治疗慢性肾衰提供了新的理论和方法.