木聚糖酶-甘露聚糖酶融合酶基因Linker优化及其在猪肾pK15细胞中共表达

【目的】构建木聚糖酶（XynB）与甘露聚糖酶（ManA）的融合酶基因，使其能在哺乳动物表达系统中表达并分泌兼有木聚糖酶和甘露聚糖酶活性的双功能融合酶。【方法】利用基因融合技术（SOE-PCR），把 11条Linker融合到木聚糖酶（XynB）和甘露聚糖酶（ManA）基因间，构建真核表达载体，经转染猪肾细胞（pK15）收集细胞培养液，用DNS法测定其酶活并进行酶学分析。【结果】经表达分析12条不同Linker构建的融合酶与亲本酶酶活，发现用Linker a3、pS3和具有“自我剪切”能力T2A构建的融合酶在木聚糖酶和甘露聚糖酶活性都显著高于两亲本酶。融合酶XynB-a3-ManA的木聚糖酶和甘露聚糖酶酶活比亲本酶XynB和ManA分别提高了54.06%和104.40%；该融合酶在酸性环境3.0—7.0起作用，对pH3.0~8.0具有一定的耐受力。【结论】首次获得可在哺乳动物系统表达分泌的增强型双功能XynB-ManA融合酶。

Peptide Linkers Optimized Recombinant Enzyme Gene of XynB-ManA and Its Co-expression in pK15 Cells

【Objective】This study was aimed at constructing a fusion enzyme of xylanase and mannase which functioned well in mammalian expression system for the research of transgenic animals and potential utilization in the industry of feed enzyme.【Method】Eleven chimeric genes of xylanase and mannase differed in linkers were constructed by SOE-PCR, and then subcloned into pcDNA3.1 vector. The cell free culture supernatants were analyzed by the dinitrosalicylic (DNS) acid method after the recombinant plasmids were transiently transformed into PK15 cells .【Result】Compared with the parental enzymes，the fusion enzymes with Linkers a3, pS3, and “self-cleavage”T2A displayed significantly higher catalytic efficiency. Specifically, the XynB-a3-ManA was increased by 54.06% for the xylanase and of 104.40% for the mannase in enzyme activity, and worked well at pH3.0-7.0 and was partially resistant in pH3.0-8.0.【Conclusion】The results demonstrated that the firstly obtained enhanced bifunctional fusion XynB-ManA enzyme which could be expressed and secreted in mammalian expression system by optimizing the peptide Linkers between two parental genes.