高表达尿酸氧化酶乳酸杆菌的制备与应用研究.docVIP

高表达尿酸氧化酶乳酸杆菌的制备与应用研究 木研究日的是制备一种可高效表达尿酸氧化酶的乳酸杆菌，并探索通过口服该菌以 降低机体血尿酸含量的有效性。首先，通过体外检测发现，0%。二1时的正常乳酸杆菌，40°C静止 孵育3小时，可引发尿酸含量从0增加到881.3±12.3umol/Lo体内试验表明，分别灌胃1天和 2天正常乳酸杆菌(菌体浓度为0. lg/ml,0.2ml/d)的小鼠，可导致血清中的尿酸水平从179. 7± 1.3 u mol/ml分别增加到了 239?8土19?8 u mol/L 285. 6±23. 8 y mol/L为了降低乳酸杆菌的尿酸 产生量，木研究根据猪尿酸氧化酶基因序列，人工合成了猪尿酸氧化酶的全基因，以此构建了乳 酸菌内可组成性表达猪尿酸氧化酶基因的表达载体pMG36-puOo通过电穿孔方法最终获得了可组 成型的表达重组猪尿酸氧化酶的干酪乳酸杆菌，质粒可稳定传代。体外实验表明，该基因丁稈菌 株的体外尿酸产量与不携带尿酸氧化酶的菌株相比，尿酸水平下降了 39.9%。体内试验证实，连 续灌胃两天后(浓度为0. lg/ml,剂量为0.2ml/d),食用表达尿酸氧化酶乳酸菌的小鼠与对照组 相比，血清尿酸含量降低了 19.2%,差异显著。同时，木研究还探索了口服这种乳酸杆菌对于预 防高尿酸血症的情况，结果发现：低剂量灌胃乳酸杆菌(0.01g/ml,0.2nil/d) 7天后，腹腔注射 尿酸( 250mg/kg) ,2小时后取血，最后检测血清屮的尿酸水平较对照组和生理盐水组低 (305. 9±56. 5 vs 341.5±52. 3 vs 310.0±13.3)。由上述研究可见，乳酸类杆菌具有尿酸分泌 功能，大剂最食用会增加体内血尿酸的负担；在乳酸杆菌屮纽?成性的高效表达尿酸氧化酶，可显 著降低菌体自身的尿酸产量，口服具有高效尿酸分解能力的乳酸杆菌能够显著降低血尿酸含量， 同时对高尿酸血症具有一定的预防效果。 【关键词】 尿酸氧化酶；高尿酸血症；乳酸杆菌 The Preparation and Application of a New Type of Lactobacilli with High Expression of Urate Oxidase ZHANG Ze~ren, Shanghai Gezhi High School, Shanghai 200003, China [Abstract] The purpose of this study is to prepare a new type of lactobacilli with high expression of urate oxidase, and explore its effectiveness in reducing the content of blood uric acid by oral administration. First of all, in this study we found that the content of blood uric acid was increased from 0 to 881.3±12.3pmol/L in normal lactobacilli(OD600=l) by incubation at 40 °C for 3 hours. We also found that the blood uric acid content increased from 179.7±1.3pmol/L to 239.8±19.8pmol/L and 285.6±23.8pmol/L separately in the mice which were intragastric administrated with normal lactobacilli(0.1 g/ml,0.2ml/d) for one day and two days. Secondly, wc synthesized the entire gene of pig urate oxidase optimized by codon usage of predicted highly expressed genes in lactobacilli,and constructed a expression vector pMG36-puo, which could induce the constitutive expression of pig urate oxidase in lactobacilli, then we got the fondue lactobacilli, which could constitutively express