高苯丙氨酸血症大鼠基因疗法的实验研究

A new strategeutics of gene therapy for hyperphenylalaninemia rats

目的 构建能表达活性苯丙氨酸脱氨酶 (PAL)的重组质粒 ,使其在乳酸乳球菌 (L .L)中表达 ,将苯丙氨酸 (phe)脱氨 ,从而减少外周血中phe的水平 ,达到治疗苯丙酮尿症 (PKU)或大鼠高苯丙氨酸血症的目的。方法 将欧芹PALcDNA重组到质粒pMG36e中 ,转化乳酸乳球菌 ,用聚合酶链反应 (PCR)及高效液相色谱法 (HPLC)等技术筛选阳性表达株 ,将工程菌pMG36ePAL/L .L .制成不同剂型 ,“治疗”高苯丙氨酸血症模型大鼠。结果 获得了能表达PAL的乳酸乳球菌基因工程菌株。灌喂pMG36ePAL/L .L .制成的肠溶微囊及抗酸缓冲液体制剂 ,观察到高苯丙氨酸血症大鼠外周血phe水平明显降低 (2组治疗后 5d为 1 2 5mg % ,第 12d为 1 75mg% ) ,与对照组相比 (治疗后 5d为 3 75mg % ,第 12d为 2 9mg% )有显著性差异。不同剂型显示出的效果有差别。结论 能表达活性PAL的基因工程菌 (乳酸乳球菌 )的适当口服剂型 ,能够显著降低高苯丙氨酸血症大鼠外周血phe水平 。

Objective To construct a recombinant vector which expresses active phenylalanine amonia lyase(PAL) in Lactococcus lactis (L.L.), and to convert phe into cinnamic acid in small intestine by the engineering L.L. to decrease the phe level in the peripheral blood, and to cure hyperphenylalaninemia rats. Methods PAL cDNA from Petroselinum crispum was subcloned into expression vector pMG36e and transformed L.L.. The pMG36ePAL/L.L. was screened and characterized by using PCR and HPLC,and prepared as enteric coated microcapsules and oral liquid type preparation that were given orally to hyperphenylalaninemia rats. Results Engineering L.L. expressing PAL activity was obtained. The phe levels plasma of in the rats receiving preparations made from the engineering L.L. were significantly reduced compared with non treated hyperphenylalaninemia rats. And the effects of different preparations were different from each other. Conclusion The engineering L.L. expressing PAL activity can reduce the blood phe level of the hyperphenylalaninemia rats. This may be a potential way for PKU gene therapeutics.