Lactobacillus kefiranofaciens 乳糖酶基因克隆及在毕赤酵母中表达

以马乳酒样乳杆菌ZW3基因组为模板，PCR扩增得到乳糖酶中LacL、LacM两个大小亚基片段，经EcoRI和SnaBI双酶切后，分别连接pPIC9K质粒，转化大肠杆菌感受态细胞DH5α，并验证其核苷酸序列正确。将重组质粒pPIC9K-LacL、pPIC9K-LacM分别电转化毕赤酵母GS115，构建pPIC9K-LacL-GS115重组子和pPIC9K-LacM-GS115重组子，通过筛选得到抗G418浓度达到3.0 mg/mL，具有多拷贝基因的重组子。经反转录PCR验证，cDNA上存在LacL片段，并可检测到乳糖酶酶活达到1.17 U/mL；pPIC9K-LacM-GS115重组子诱导表达72 h后的发酵液上清经SDS蛋白电泳检测到目的条带。

Cloning of Lactobacillus kefiranofaciens β-galactosidase genes and expression in Pichia pastoris

Using kumiss Lactobacillus kefiranofaciens ZW3 genome as template, the β-galactosidase genes(LacL and LacM) were amplified from L. kefiranofaciens by PCR, and it was recombined into vector pPIC9K after digestion by EcoRI and SnaBI. Then two plasmids of pPIC9K-LacL and pPIC9K-LacM were constructed through transformation into Escherichia coli DH5α and the nucleic acid sequence was verified by sequencing results. The recombinations of pPIC9K-LacL-GS115 and pPIC9K-LacM-GS115 were constructed by electroporating the vectors into Pichia pastoris GS115. Through screening, the multi-copy recombinations pPIC9K-LacL-GS115 and pPIC9K-LacM-GS115 were obtained resistant to 3.0 mg/ml G418. Through reverse PCR verification, LacL genes in recombination pPIC9K-LacL-GS115 were detected, in which fermented for 72 h induced by methanol. Moreover, the activity of β-galactosidase from recombination pPIC9K-LacL-GS115 reached to 1.17 U/ml. Also, the target protein band of recombination pPIC9K-LacM-GS115 was observed by SDS-PAGE.