斑马鱼GAPDH蛋白片段的原核表达差异分析

为了分析斑马鱼GAPDH基因不同区域原核表达构建的诱导效应差异,应用生物信息技术分析斑马鱼GAPDH分子结构,通过定向删除技术,构建表达部分斑马鱼GAPDH蛋白片段的质粒。通过IPTG诱导,SDS-PAG检测,分析GAPDH基因不同区域构建的蛋白表达差异。结果:引物对ZGCKF/ZGCXR构建的GAPDH重组表达质粒具有IPTG诱导表达效应,而引物对ZGNKF/ZGNXR构建的质粒则不具有IPTG诱导表达效应。因此,为了在原核表达真核蛋白时应考虑过表达的真核蛋白是否会影响宿主的代谢以及是否会导致宿主的抵抗,设计引物时应当选择合适的区域构建重组质粒才能获表达产物。

VARIATION ANALYSIS OF PROKARYOTIC EXPRESSION OF THE PROTEIN FRAGMENTS FROM ZEBRA FISH GAPDH

To analyze the variation of induction effect of the prokaryotic expressed Zebra fish GAPDH protein fragments which were constructed by different primers, the molecular structure of Zebra fish GAPDH was analyzed by bioinformatics approaches, and the targeting deletion technique was applied to construct the recombinant plasmids for partially expressed GAPDH. Then the variation of protein expression by the recombinant plasmids were induced with IPTG and detected by SDS-PAGE. And the primers had the efficiency of IPTG induced expression to the recombinant plasmids constructed by ZGCKF/ZGCXR, but not for ZGNKF/ZGNXR. The results indicated that it should be considered if the eukaryotic proteins will affect the metabolism of the prokaryotic host when the eukaryotic proteins over-expressed in prokaryotic organism, and result in the resistance of the host bacteria. Hence, proper region of the protein should be considered to express efficiently when the primers were designed.