苯丙氨酸脱氨酶在乳酸乳球菌NICE系统的高效表达及其实验动物研究

The Progressive Study on Gene Therapy for Hyperphenylalaninemia Rats

为构建苯丙氨酸脱氨酶的一种更新更高表达的基因工程菌 ,取得治疗高苯丙氨酸血症大鼠的更佳疗效。将欧芹PALcDNA重组到质粒pNZ80 48中 ,构建翻译融合载体和转录融合载体 ,分别转化乳酸乳球菌NZ90 0 0。对两种高效表达菌株的酶活性差异进行比较 ,观察Nisin诱导的动态进程 ,并利用新鲜培养的p(NZ80 48 PAL) 1 NZ90 0 0工程菌进行了高苯丙氨酸血症大鼠 (称PKU动物模型 )的治疗实验。结果表明 :(1)翻译融合型菌株具有更高的酶活性 ;(2 )当Nisin的诱导时间达 6h时 ,产物肉桂酸生成量基本达到峰值 ;(3)给高苯丙氨酸血症大鼠灌喂新鲜培养的p(NZ80 48 PAL) 1 NZ90 0 0工程菌 (翻译融合型 ) ,观察到该工程菌能显著降低模型动物血中Phe浓度 。

To construct a new high effective genetic engineering strain which can express active PAL enzyme in Lactococcus lactis(L.L) , and acquire better effect on curing hyperphenylalaninemia rats, Firstly translational fusion vector and transcriptional fusion vector were constructed in E.coli MC1061, and then PAL cDNA was transformed into L.L. Two kinds of high effect strain were compared with their enzyme activity and animal experiment was carried out. The results showed: (1) Two kinds of engineering L.L. were obtained and translational fusion strain has higher level enzyme activity. (2) The amount of transcinnamic aicd reach peak when induced for 6 hours. (3) The blood phe level of the treated rats was significantly reduced compared with non-treated rats when receiving fresh p(NZ8048-PAL) 1/NZ9000. The engineering L.L(translational fusion strain) can significantly reduce the blood phe level of the hyperphenylalaninemia rats, which has more superiority than pMG36e-PAL/L.L.