摘要
微管由交替出现的α微管蛋白和β微管蛋白组成，微管蛋白β-2b链(tubulin beta-2B chain,TUBB2B)是β微管蛋白的亚型基因，它是微管的主要成分。微管蛋白基因TUBB2B的突变会改变微管的正常功能和结构。研究发现TUBB2B蛋白第247位点天冬酰胺突变为丝氨酸会导致小鼠大脑皮质的损坏和异常发育，本文就TUBB2B突变基因TUBB2B（N247S）对微管聚合的影响展开分析。本实验以C57bl/6j小鼠总RNA为模板，反转录得到cDNA，通过与T载体连接构建了TUBB2B基因克隆载体，并通过点突变技术构建了TUBB2B(N247S)基因克隆载体。使用DNA限制性酶切技术构建了TUBB2B(N247S)-p3-flag-cmv-10真核表达载体，并在转染试剂的介导下，将真核重组表达载体及空载体转染至小鼠成纤维细胞L929中，设置空白组和对照组，对不同组的细胞进行微管蛋白的提取，通过Western-blot技术对α微管蛋白表达量进行分析，收集表达的突变蛋白并通过磁分离技术进行纯化，为了研究突变蛋白对微管骨架的影响，对纯化后的突变蛋白进行体外聚合实验分析，所得结果如下： 1.构建的TUBB2B以及TUBB2B(N247S)克隆载体经菌液PCR及DNA鉴定证明克隆载体构建正确。
2.目的片段与真核表达载体连接成功，并通过菌液PCR，双酶切及DNA 测序结果鉴定，成功构建TUBB2B(N247S)-p3-flag-cmv-10真核重组表达载体。
3.成功转染L929细胞，以及成功表达突变蛋白，经过优化诱导表达条件，确定了以6孔板为准，最佳目
的基因与转染试剂的比例为8：1，且实现了TUBB2B 基因在L929中的高效表达。
4.转染组与空白组及对照组进行比较，游离态的α微管蛋白显著高于对照组和空白组(P值分别为0.0001和0.0004)，聚合态的α微管蛋白显著低于对照组和空白组(P值分别为0.0004和0.0036)。
5.磁分离技术纯化可以得到较高浓度的目的蛋白。
6.TUBB2B(N247S)突变蛋白对微管蛋白体外聚合具有一定的抑制作用。
关键词：微管蛋白，微管蛋白β-2b链，真核表达，磁分离技术，体外聚合
I
Abstract
The microtubules are composed of alternating α-tubulin and β-tubulin. The tubulin beta-2B chain (TUBB2B) is a subtype of β-tubulin, which is the main component of microtubules. Mutations in the tubulin gene TUBB2B alter the normal function and structure of microtubules. The mutation of asparagine to serine was found at position 247 of TUBB2B caused damage and abnormal development of mouse cerebral cortex. This paper analyzes the effect of TUBB2B mutant on microtu
bule polymerization. In this experiment, the total RNA of C57bl/6j mouse was used as a template, and the cDNA was reverse transcribed. The TUBB2B cloning vector was constructed by ligation with T vector, and the TUBB2B(N247S) cloning vector was constructed by point mutation technique. The TUBB2B-p3-flag-cmv-10 eukaryotic expression vector was constructed by DNA restriction enzyme digestion, and the eukaryotic recombinant expression vector and empty vector were transfected into mouse fibroblasts under the guidance of transfection reagent. In the fibroblast L929, a blank group and a control group were set up.and tubulin was extracted from different groups of cells, and the expression of α-tubulin was analyzed by Western-blot. The mutant protein was expressed, collected and purified by magnetic separation techniques. In order to study the effect of the mutant protein on the microtubule skeleton, the purified mutant protein was analyzed in vitro, and the results were as follows:
1.The constructed TUBB2B and TUBB2B(N247S) cloning vectors were confirmed to be correctly constructed by PCR and DNA identification.
2.The target fragment was successfully ligated with the eukaryotic expression vector, and the TUBB2B(N247S)-p3-flag-cmv-10 eukaryotic recombinant expression vector was successfully constructed by PCR, double enzyme digestion and DNA sequencing.
3.Successfully transfected L929 cells and successfully expressed mutant proteins. After optimization and induction of expression conditions, it was determined that the ratio of the best target gene to transfection reagent was 8:1, and the TUBB2B gene was achieved. Efficient expression in L929.
4.The transfection group was compared with the blank group and the control group. The free α-tubulin was significantly higher than the control group and the
blank group (P values were 0.0001 and 0.0004, respectively), and the aggregated α-tubulin was significantly lower than the control group. In addition, blank groups (P values are 0.0004 and 0.0036, respectively).
5.Magnetic separation technology can obtain higher concentration of target protein.
6.TUBB2B(N247S) mutant protein has a certain inhibitory effect on the in vitro polymerization of tubulin.
Key words: Tubulin, tubulin beta-2b chain, eukaryotic expression, magnetic separation technique, in vitro polymerization
目 录
摘要 ................................................................................................................................ II 目录 ..................................................................................................................... I V 第一篇文献综述 (1)
1.1 微管蛋白研究现状 (1)
1.2 TUBB2B突变体与人类疾病的研究进展 (2)
1.3 TUBB2B突变体表型的致病机制 (5)
1.4 研究目的及意义 (7)
第二篇实验内容 (8)
第一章TUBB2B(N247S)重组表达载体的构建及鉴定 (8)
1 实验材料 (8)
1.1 实验动物、质粒、菌株、细胞 (8)
1.2 试剂 (8)
1.3 主要实验仪器 (9)
1.4 相关实验实验配方 (9)
2实验方法 (11)
2.1 TUBB2B基因克隆载体的构建 (11)
2.2 TUBB2B(N247S)突变基因克隆载体的构建 (14)
2.3 真核表达载体TUBB2B(N247S)-p3-flag-cmv-10的构建 (15)
2.4 成纤维细胞L929的培养及真核表达载体的转染 (16)
2.5 重组目的蛋白的提取及鉴定 (17)
3 结果与分析 (20)
3.1 TUBB2B克隆载体的构建 (20)
3.2 TUBB2B突变克隆载体的构建 (22)
3.3 TUBB2B(N247S)-p3-flag-cmv-10表达载体的构建 (25)
3.4 表达蛋白的浓度测定及重组目的蛋白的检测 (27)
4 讨论 (30)
5 小结 (31)
第二章突变蛋白TUBB2B(N247S)对微管聚合影响的研究 (32)
1实验材料 (32)
1.1 相关试剂 (32)
1.2 主要实验仪器 (32)
2实验方法 (33)
2.1 突变蛋白TUBB2B（N247S）细胞内对微管蛋白聚合的影响 (33)
2.2 重组目的蛋白的纯化及鉴定 (33)
2.3 重组蛋白体外聚合检验 (34)
3实验结果 (35)
3.1 TUBB2B（N247S）突变对α微管蛋白的聚合状态分析 (35)
3.2 重组蛋白纯化 (36)
3.2 重组蛋白体外聚合检验 (37)
4讨论 (38)
5小结 (40)
结论 (41)
作者简介 (47)
致谢 (48)
附录A：TUBB2B点突变致病情况 (49)
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