三种带有不同信号肽的GFP真核表达载体和H-FABP原核表达载体的构建

发布时间：2018-04-29 08:12

本文选题：心肌型脂肪酸结合蛋白 + 表达与纯化　；参考：《吉林大学》2005年硕士论文

【摘要】：信号序列可使新合成的蛋白质结合到真核细胞的内质网上或者细菌的质膜。所有的信号序列都有一个疏水的核心区,但除此之外,它们的长度和氨基酸的序列都有很大的差异。这些差异使得信号序列在引导蛋白及本身的膜插入时都存在特异性,甚至即使在与蛋白裂解后还行使一定的功能。信号肽对蛋白质的转移和膜插入具有重要作用。不同的信号肽引导蛋白转移和膜插入的途径是相同的,因此不同蛋白的信号肽间可互换,甚至不同有机体的蛋白信号肽间也可互换研究表明,信号肽具有多种功能,在引导蛋白转移和膜插入时,它们有各自不同的作用。较为明确的是:信号肽可选择不同的转移途径;调解N-端或C-端跨膜的方向;甚至改变跨膜的方式,使得蛋白或留在胞浆,或插入膜间,或位于细胞的囊腔内。甚至,在信号肽与蛋白裂解后,仍具有进一步的作用。信号肽一般由三部分构成,即中间的疏水核心区(h 区,hydropHobic coreregion),疏水核心区碳端(C-端)的极性区(c-区),疏水核心区氮端(N-端)的强极性区(n 区)。信号肽的疏水核(h-区)对于蛋白质的引导和膜插入有重要意义。信号肽c-区大多含有解螺旋的脯氨酸和甘氨酸,在c-区-3 和-1 的位置上含有小的不带电残基,为信号肽裂解的位点。信号肽的强极性区带有纯正电。比较分析大量的信号序列表明:信号肽长度变化范围在15～50个氨基酸残基之间,其中,强极性区(n-区)对信号肽长度影响最大。并且n-区的阳性电荷可影响信号肽分泌蛋白的量。 GFP 因其独特的发光特性引起人们极大的兴趣;它能在长波紫外光或Ca~(2+)激发下发出特有的绿色荧光,更为奇特的是它发光时不需要任何其它外源底物或辅助因子参与,且没有任何种属特异性,只要有GFP 存在均能发出特有的绿色荧光。目前研究未发现GFP 对细胞本身的生理状态有何干扰,也未发现其毒性作用。GFP 的发光特性能长期保持,甚至福尔马林固定亦不能改变其发光强度,这一特性使其成为一种新的基因表达标记物,而且它的灵敏度和分辨率均高于现有的免疫组织化学法,所以是一类能在遗传工程研究中发挥重要作用的较理想的基因表达标记物。综上所述,本次研究采用重叠延伸PCR 拼接方法(splicing by overlap
[Abstract]:Signal sequences enable newly synthesized proteins to bind to the endoplasmic reticulum of eukaryotic cells or to the plasma membrane of bacteria. All signal sequences have a hydrophobic core region, but their lengths and amino acid sequences differ greatly. These differences make the signal sequence specific to both the guiding protein and its own membrane insertion, and even perform certain functions even after cleavage with the protein. Signal peptides play an important role in protein transfer and membrane insertion. The pathway of different signal peptide guiding protein transfer and membrane insertion is the same, so the signal peptide of different protein can be interchangeable, and even the signal peptide of different organism can be interchangeable research shows that signal peptide has many functions. They play different roles in guiding protein transfer and membrane insertion. It is clear that the signal peptide can choose different transfer pathways; mediate the direction of the N-terminal or C-terminal transmembrane; or even change the transmembrane way so that the protein is left in the cytoplasm or inserted between the membranes or in the cytosolic cavity of the cell. Even after the cleavage of signal peptide and protein, it still has further effect. The signal peptide is generally composed of three parts, namely, the polar region of the middle hydrophobic core region, the central region of the hydrophobic core region, the carbon terminal of the hydrophobic core region, the polar region of the hydrophobic core region, and the strong polar region of the hydrophobic core region, the nitrogen terminal of the hydrophobic core region and the N-terminal of the hydrophobic core region. The hydrophobic nucleotide region of the signal peptide is important for protein guidance and membrane insertion. Most of the signal peptide c- region contains proline and glycine, and there are small uncharged residues in the c-region-3 and 1, which are the sites of signal peptide cleavage. The strong polar region of the signal peptide contains pure electricity. A large number of signal sequences show that the length of signal peptide varies from 15 to 50 amino acid residues, and the strong polar region (n-) has the greatest influence on the signal peptide length. And the positive charge of n- region can affect the amount of signal peptide secreted protein. GFP is of great interest because of its unique luminescent properties; it can emit unique green fluorescence under the excitation of long wavelength ultraviolet light or Ca~(2. What is more peculiar is that it does not require any other external substrate or auxiliary factor to participate in the luminescence. And there is no species-specific, as long as the existence of GFP can emit a unique green fluorescence. At present, it has not been found that GFP interferes with the physiological state of the cell itself, nor does it find that the luminescence properties of GFP can be maintained for a long time, and even formalin fixation can not change its luminescence intensity. This feature makes it a new marker for gene expression, and its sensitivity and resolution are higher than those of existing immunohistochemical methods, Therefore, it is an ideal gene expression marker that can play an important role in genetic engineering. To sum up, the method of overlapping extended PCR splicing by overlap is used in this study.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q782

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