微管蛋白β-2b链突变体基因真核重组表达质粒的构建及其对α微管蛋白聚合的影响

Construction of tubulin β-2b chain mutant gene eukaryotic recombinant expression plasmid and its effect onα-tubulin polymerization

为了获得小鼠微管蛋白β-2b链突变体基因TUBB2B(N247S),并分析其对α微管蛋白聚合的影响,试验采用RT-PCR法扩增TUBB2B基因的全长cDNA序列,通过重叠延伸PCR点突变技术获得TUBB2B(N247S)突变基因,并采用分子克隆技术构建真核重组表达质粒TUBB2B(N247S)-p3-flag-cmv-10,用真核重组表达质粒转染小鼠成纤维细胞(L929细胞),并从细胞中提取α微管蛋白,采用Western-blot技术分析游离态及聚合态α微管蛋白的表达量。结果表明:通过重叠延伸PCR点突变技术成功扩增出大小为1362 bp的TUBB2B(N247S)突变基因,并成功构建出真核重组表达质粒TUBB2B(N247S)-p3-flag-cmv-10,分子质量为55 ku的突变蛋白在L929细胞中成功表达,在转染真核重组表达质粒的L929细胞中游离态α微管蛋白含量显著高于空载体转染组及空白对照组(P<0.05),而聚合态α微管蛋白含量显著低于空载体转染组及空白对照组(P<0.05)。说明TUBB2B(N247S)基因突变蛋白可抑制α微管蛋白的聚合。

In order to obtain the mouse tubulinβ-2b chain mutant gene TUBB2B(N247S)and analyze its effect on the polymerization ofα-tubulin,the full-length cDNA sequence of TUBB2B was amplified by RT-PCR,and the TUBB2B(N247S)gene was obtained by point mutation technique.The TUBB2B(N247S)-p3-flag-cmv-10 eukaryotic recombinant expression plasmid was constructed by molecular cloning technique.The eukaryotic recombinant expression plasmid was transfected into mouse fibroblast L929.Theα-tubulin protein was extracted from the cells and the expression of free and polymerizedα-tubulin was analyzed by Western-blottechnique.The results showed that the 1362 bp TUBB2B(N247S)mutant gene was successfully amplified by overlapping extension PCR point mutation technology,and the TUBB2B-p3-flag-cmv-10 eukaryotic recombinant expression plasmid was successfully constructed,and the mutant protein with a relative molecular weight of 55 ku was successfully expressed in L929 cells.The content of freeα-tubulin in L929 cells transfected with the eukaryotic recombinant expression plasmid was significantly higher than that in the empty vector transfection group and the blank control group(P<0.05),while the content of polymerizedα-tubulin was significantly lower than that in the empty vector transfection groupand theblank control group(P<0.05).It indicated that TUBB2B(N247S)gene mutant protein could inhibit the polymerization of α-tubulin.