欧芹苯丙氨酸脱氨酶cDNA在乳酸乳球菌中的表达研究

将欧芹（Ｐｅｔｒｏｓｅｌｉｎｕｍｃｒｉｓｐｕｍ）苯丙氨酸脱氨酶（ＰＡＬ）ｃＤＮＡ亚克隆到组成型表达载体ｐＭＧ３６ｅ启动子Ｐ３２下游，电穿孔法转化乳酸乳球菌，获得有ＰＡＬ表达活性的乳酸乳球菌工程菌（ｐＭＧ３６ｅＰＡＬ／Ｌ．ｌａｃｔｉｓＭＧ１３６３）。通过递归ＰＣＲ合成了一段１２０ｂｐ的调控片段，用以将ｐＭＧ３６ｅ改造为分泌型表达载体ｐＸＨＳ，以翻译偶联的方式表达ＰＡＬ，可使ＰＡＬ的Ｎ末端带上ｕｓｐ４５信号肽，结果亦检测到ＰＡＬ酶活性。自行分离克隆了乳酸乳球菌热休克蛋白基因ｄｎａＪ的启动子区域，构建了热诱导表达载体ｐＸＨＪ，获得ＰＡＬ热诱导表达工程菌（ｐＸＨＪＰＡＬ／Ｌ．ｌａｃｔｉｓＩＬ１４０３），经３０℃至３７℃热诱导，可使ＰＡＬ表达活性提高至２倍。本文还就乳酸乳球菌ＰＡＬ工程菌在经典型苯丙酮尿症防治中的应用进行了分析和讨论

Expression in Lactococcus lactis of catalytically active phenylalanine ammonia-lyase from parsley]

The phenylalanine ammonia-lyase (PAL) cDNA of parsley (Petroselinum crispum) was subcloned into constitutive expression vector pMG36e downstream of the P32 promoter. The resulting plasmid pMG36ePAL was introduced into Lactococcus lactis subsp. lactis MG1363 by electroporation. The recombinant strain showed its PAL activity conversing the L-phenylalanine in the culture medium into trans-cinnamic acid. A new secretory vector pXHS was constructed by recombination of pMG36e with a Lactococcal usp45 secretion leader coding sequence and a translational coupling sequence. Then the pXHSPAL was constructed and used for expression of PAL in L. lactis, the PAL activity was also detectable. The L. lactis dnaJ promoter sequence was cloned and used to construct a heat inducible vector pXHJ. PAL cDNA was cloned into pXHJ and the L. lactis IL1403 was transformed with the recombinant plasmid pXHJPAL. After a heat shock from 30 degrees C to 37 degrees C, the PAL activity of the pXHJPAL strain could increase approximately onefold. The prospect of using these engineering L. lactis strains for PKU therapy was also discussed.