本文選題：心肌型脂肪酸結(jié)合蛋白 + 表達(dá)與純化��； 參考：《吉林大學(xué)》2005年碩士論文

【摘要】：信號序列可使新合成的蛋白質(zhì)結(jié)合到真核細(xì)胞的內(nèi)質(zhì)網(wǎng)上或者細(xì)菌的質(zhì)膜。所有的信號序列都有一個疏水的核心區(qū),但除此之外,它們的長度和氨基酸的序列都有很大的差異。這些差異使得信號序列在引導(dǎo)蛋白及本身的膜插入時都存在特異性,甚至即使在與蛋白裂解后還行使一定的功能。 信號肽對蛋白質(zhì)的轉(zhuǎn)移和膜插入具有重要作用。不同的信號肽引導(dǎo)蛋白轉(zhuǎn)移和膜插入的途徑是相同的,因此不同蛋白的信號肽間可互換,甚至不同有機(jī)體的蛋白信號肽間也可互換研究表明,信號肽具有多種功能,在引導(dǎo)蛋白轉(zhuǎn)移和膜插入時,它們有各自不同的作用。較為明確的是:信號肽可選擇不同的轉(zhuǎn)移途徑;調(diào)解N-端或C-端跨膜的方向;甚至改變跨膜的方式,使得蛋白或留在胞漿,或插入膜間,或位于細(xì)胞的囊腔內(nèi)。甚至,在信號肽與蛋白裂解后,仍具有進(jìn)一步的作用。 信號肽一般由三部分構(gòu)成,即中間的疏水核心區(qū)(h 區(qū),hydropHobic coreregion),疏水核心區(qū)碳端(C-端)的極性區(qū)(c-區(qū)),疏水核心區(qū)氮端(N-端)的強(qiáng)極性區(qū)(n 區(qū))。信號肽的疏水核(h-區(qū))對于蛋白質(zhì)的引導(dǎo)和膜插入有重要意義。信號肽c-區(qū)大多含有解螺旋的脯氨酸和甘氨酸,在c-區(qū)-3 和-1 的位置上含有小的不帶電殘基,為信號肽裂解的位點(diǎn)。信號肽的強(qiáng)極性區(qū)帶有純正電。比較分析大量的信號序列表明:信號肽長度變化范圍在15～50個氨基酸殘基之間,其中,強(qiáng)極性區(qū)(n-區(qū))對信號肽長度影響最大。并且n-區(qū)的陽性電荷可影響信號肽分泌蛋白的量。 GFP 因其獨(dú)特的發(fā)光特性引起人們極大的興趣;它能在長波紫外光或Ca~(2+)激發(fā)下發(fā)出特有的綠色熒光,更為奇特的是它發(fā)光時不需要任何其它外源底物或輔助因子參與,且沒有任何種屬特異性,只要有GFP 存在均能發(fā)出特有的綠色熒光。目前研究未發(fā)現(xiàn)GFP 對細(xì)胞本身的生理狀態(tài)有何干擾,也未發(fā)現(xiàn)其毒性作用。GFP 的發(fā)光特性能長期保持,甚至福爾馬林固定亦不能改變其發(fā)光強(qiáng)度,這一特性使其成為一種新的基因表達(dá)標(biāo)記物,而且它的靈敏度和分辨率均高于現(xiàn)有的免疫組織化學(xué)法,所以是一類能在遺傳工程研究中發(fā)揮重要作用的較理想的基因表達(dá)標(biāo)記物。 綜上所述,本次研究采用重疊延伸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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：Q782

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本文編號：1819119