HIV融膜蛋白抑制剂C34的红光调控研究
发布时间：2018-08-09 19:06
【摘要】：艾滋病(acquired immune deficiency syndrome,AIDS)全称获得性免疫缺陷综合征,是由人类免疫缺陷病毒(Human Immunodeficiency Virus,HIV)引起的一种严重传染病。HIV的生命周期一般分为几个不同的阶段:病毒吸附、结合和融合,病毒脱壳、转录、翻译、整合、组装、成熟和出芽。了解艾滋病病毒感染的过程细节对于我们开发药物是非常重要的。目的:开发艾滋病病毒感染机制研究工具,实现定性或定量控制病毒感染过程。方法:Gp41为HIV-I的包膜蛋白复合体,其控制着病毒侵入靶细胞的早期关键过程,主要功能是介导病毒与靶细胞膜的融合。C34为模拟核心CHR区的合成肽,可以通过抑制HIV-I gp41自身的C34肽与NHR区的N36肽结合而抑制HIV-I的融膜过程。现在市场上的很多抗HIV药物应用此原理。在这里,我们开发了一个打开和关闭的系统,它可以使用光来控制HIV进入靶细胞的融膜过程。偶氮苯(azobenzene)结构和功能的光致异构化为我们提供了一个重要的分子手段。偶氮苯具有较强的光响应特性,这一特性使其在许多领域表现出巨大的开发潜力。通常偶氮苯存在顺式、反式两种异构体。反式构型偶氮苯转变为顺式构型,需要在特定波长的紫外光照射下才能发生;在可见光或热的作用下,顺式构型可还原为反式构型。偶氮苯分子的两种构型的紫外-可见吸收光谱有明显不同。同时,两者在立体结构、偶极矩等物理和化学性质方面也存在明显差异。目前,偶氮苯的顺反异构体的不同特点,顺反异构化和各种诱导的光响应的现象,引起了社会各界的广泛关注。由于紫外对细胞和组织具有强烈的杀伤作用,所以想在体内应用偶氮苯的关键是需要为其提供一个发生异构化反应合适的照射波长。我们对偶氮苯进行修饰,使其调控波长处于红光区(630-660 nm)。这个开关系统的关键特征是修改C34肽,通过设计C34使红移的光控偶氮苯与设计位置的半胱氨酸突变体交联。在这里我们设计了6条突变的多肽和两个光控开关。多肽的合成采用固相合成法;多肽的纯化采用半制备型高压液相色谱仪进行;多肽结构鉴定采用MALDI-TOF法进行,纯度采用分析型高压液相色谱仪测定。光控开关采用化学合成法,纯化采用硅胶层析柱法进行纯化,结构采用MALDI-TOF及核磁共振氢谱进行分析,纯度采用HPLC测定;使用紫外分光光度计扫描光控开关的紫外-可见光光谱。开关系统的活性通过细胞融合实验的方法,其结构采用圆二色谱光谱扫描法进行鉴定。结果:1、多肽的合成和纯化实验较为关键,是整个实验成败的基础。本实验完成的较成功,经质谱分析和HPLC分析多肽结构准确、纯度较好,符合实验要求。2、开关的合成较为复杂,经过反复实验及优化,最终得到产品产率较高、纯度高。经质谱分析和HPLC分析所得产物结构准确、纯度高,符合实验要求。3、开关系统的组装相对较简单,所得产物经质谱分析和HPLC分析所得产物结构准确、纯度高,符合实验要求。4、圆二色谱法(CD)扫描可以看出开关系统均有两个比较明显的α-螺旋缝,这也符合C肽和N肽结合后形成的螺旋六聚体结构。通过C34和其突变体CD谱的比较可以看出经过氨基酸的突变对螺旋的形成存在一定的干扰作用,但峰型与C34对照组基本一致。5、活性测定结果表明其突变体的活性普遍降低,C34-M3基本丧失活性。通过红蓝光对比可以看出与CD相同的结果。C34-M4红光较蓝光活性降低一倍左右,C34-a红光较蓝光活性降低一倍左右,其他红蓝光照射不存在明显差异。结论:开关系统的异构化反应波长向长波长(红光)移动,这也符合最初开关设计的目的。红光可以穿透生物体细胞及各个组织和器官,对组织和器官没有危害。这就使得这个开关系统更适用于在生物体内的研究。开关系统我们进行了光控功能测试:它们中的一些在接受蓝光照射后表现出艾滋病毒的抑制活性,在红光照射后抑制功能减弱。
[Abstract]:Acquired immune deficiency syndrome (AIDS) is fully known as acquired immunodeficiency syndrome. The life cycle of a serious infectious disease caused by the human immunodeficiency virus (Human Immunodeficiency Virus, HIV) is generally divided into several different stages: disease adsorption, binding and fusion, viral dehulling, transcription, translation, and integration. Assemble, mature and bud. Understanding the details of the process of HIV infection is very important for us to develop drugs. Objective: to develop the AIDS virus infection mechanism research tool to realize the qualitative or quantitative control of the virus infection process. Method: Gp41 is a HIV-I envelope protein complex, which controls the early key of the virus invasion of the target cells. Process, the main function is to mediate the fusion of the virus and the target cell membrane.C34 as the synthetic peptide in the core CHR region, which can inhibit the melting of HIV-I by inhibiting the C34 peptide of the HIV-I gp41 itself and the N36 peptide in the NHR region. It can use light to control the melting process of HIV into the target cells. The photoisomerization of the structure and function of azobenzene (azobenzene) provides us with an important molecular means. Azobenzene has a strong light response characteristic, which shows great potential for development in many fields. Usually azobenzene exists CIS. Trans two isomers. The trans configuration azobenzene changes to a cis configuration, which needs to occur at a specific wavelength of ultraviolet light. Under visible light or heat, the CIS configuration can be reduced to a trans configuration. The UV visible absorption spectra of the two configurations of azobenzene molecules are distinctly different. At the same time, the two are in the stereostructure and dipole. There are obvious differences in physical and chemical properties. At present, the characteristics of the CIS and trans isomerization of azobenzene, CIS trans isomerization and various induced light responses have aroused widespread concern in the community. The key to the application of azobenzene in the body is the strong killing effect of ultraviolet on cells and tissues. We need to provide a suitable irradiation wavelength for the isomerization reaction. We modify the azobenzene to control the wavelength in the red region (630-660 nm). The key feature of the switching system is to modify the C34 peptide and cross link the red shift azobenzene with the designed cysteine mutant by designing C34. 6 mutant polypeptides and two light control switches were taken into account. The synthesis of polypeptides was synthesized by solid phase synthesis, and the purification of polypeptides was carried out by semi preparative high pressure liquid chromatograph. The polypeptide structure identification was carried out by MALDI-TOF method and the purity was determined by analytical high pressure liquid chromatograph. The optical control switch was chemically synthesized and purified by silica gel column. The method was purified, the structure was analyzed by MALDI-TOF and NMR, the purity was determined by HPLC, UV spectrophotometer was used to scan the UV visible light spectrum of the optical switch. The activity of the switch system was identified by the method of cell fusion. The structure was identified by the circular two chromatography scanning method. Results: 1, the synthesis of the polypeptide. The experiment is the basis of the experiment, which is the basis of the success and failure of the whole experiment. The results of this experiment are more successful. The mass spectrometry analysis and HPLC analysis of the polypeptide structure is accurate and the purity is good. It meets the requirements of the experiment.2. The synthesis of the switch is more complex. After repeated experiments and optimization, the yield of the product is high and the purity is high. The results of mass spectrometry analysis and HPLC analysis are obtained. The product has accurate structure and high purity, which meets the requirements of the experiment.3. The assembly of the switch system is relatively simple. The products obtained by mass spectrometry analysis and HPLC analysis are accurate and high in purity, which conform to the requirements of the experiment.4. The circular two chromatography (CD) scanning shows that there are two obvious alpha helix seams in the switch system, which also conforms to the C peptide and the N peptide junction. The spiral six polymer structure formed after the combination. Through the comparison of C34 and its mutant CD spectrum, it can be seen that there is a certain interference effect on the formation of the helix through the mutation of amino acids, but the peak type is basically the same as that of the C34 control group. The activity determination results show that the activity of the mutant is generally lower and the C34-M3 basic loss activity. Through the red and blue light, It can be seen that the same results as CD can be found that.C34-M4 Hong Guang is more than doubling the blue light activity, C34-a Hong Guang is more than twice the blue light activity, and other red and blue light does not exist. Conclusion: the wavelength of the isomerization reaction of the switch system moves to the long wavelength (Hong Guang), which is also in line with the original switch design. Hong Guang can penetrate. Biological cells and various tissues and organs do not harm tissues and organs. This makes the switch system more suitable for research in living organisms. The switch system has tested the light control function: some of them show the inhibitory activity of HIV after exposure to blue light, and the inhibition function is weakened after red light irradiation.
【学位授予单位】：河北师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91
[Abstract]:Acquired immune deficiency syndrome (AIDS) is fully known as acquired immunodeficiency syndrome. The life cycle of a serious infectious disease caused by the human immunodeficiency virus (Human Immunodeficiency Virus, HIV) is generally divided into several different stages: disease adsorption, binding and fusion, viral dehulling, transcription, translation, and integration. Assemble, mature and bud. Understanding the details of the process of HIV infection is very important for us to develop drugs. Objective: to develop the AIDS virus infection mechanism research tool to realize the qualitative or quantitative control of the virus infection process. Method: Gp41 is a HIV-I envelope protein complex, which controls the early key of the virus invasion of the target cells. Process, the main function is to mediate the fusion of the virus and the target cell membrane.C34 as the synthetic peptide in the core CHR region, which can inhibit the melting of HIV-I by inhibiting the C34 peptide of the HIV-I gp41 itself and the N36 peptide in the NHR region. It can use light to control the melting process of HIV into the target cells. The photoisomerization of the structure and function of azobenzene (azobenzene) provides us with an important molecular means. Azobenzene has a strong light response characteristic, which shows great potential for development in many fields. Usually azobenzene exists CIS. Trans two isomers. The trans configuration azobenzene changes to a cis configuration, which needs to occur at a specific wavelength of ultraviolet light. Under visible light or heat, the CIS configuration can be reduced to a trans configuration. The UV visible absorption spectra of the two configurations of azobenzene molecules are distinctly different. At the same time, the two are in the stereostructure and dipole. There are obvious differences in physical and chemical properties. At present, the characteristics of the CIS and trans isomerization of azobenzene, CIS trans isomerization and various induced light responses have aroused widespread concern in the community. The key to the application of azobenzene in the body is the strong killing effect of ultraviolet on cells and tissues. We need to provide a suitable irradiation wavelength for the isomerization reaction. We modify the azobenzene to control the wavelength in the red region (630-660 nm). The key feature of the switching system is to modify the C34 peptide and cross link the red shift azobenzene with the designed cysteine mutant by designing C34. 6 mutant polypeptides and two light control switches were taken into account. The synthesis of polypeptides was synthesized by solid phase synthesis, and the purification of polypeptides was carried out by semi preparative high pressure liquid chromatograph. The polypeptide structure identification was carried out by MALDI-TOF method and the purity was determined by analytical high pressure liquid chromatograph. The optical control switch was chemically synthesized and purified by silica gel column. The method was purified, the structure was analyzed by MALDI-TOF and NMR, the purity was determined by HPLC, UV spectrophotometer was used to scan the UV visible light spectrum of the optical switch. The activity of the switch system was identified by the method of cell fusion. The structure was identified by the circular two chromatography scanning method. Results: 1, the synthesis of the polypeptide. The experiment is the basis of the experiment, which is the basis of the success and failure of the whole experiment. The results of this experiment are more successful. The mass spectrometry analysis and HPLC analysis of the polypeptide structure is accurate and the purity is good. It meets the requirements of the experiment.2. The synthesis of the switch is more complex. After repeated experiments and optimization, the yield of the product is high and the purity is high. The results of mass spectrometry analysis and HPLC analysis are obtained. The product has accurate structure and high purity, which meets the requirements of the experiment.3. The assembly of the switch system is relatively simple. The products obtained by mass spectrometry analysis and HPLC analysis are accurate and high in purity, which conform to the requirements of the experiment.4. The circular two chromatography (CD) scanning shows that there are two obvious alpha helix seams in the switch system, which also conforms to the C peptide and the N peptide junction. The spiral six polymer structure formed after the combination. Through the comparison of C34 and its mutant CD spectrum, it can be seen that there is a certain interference effect on the formation of the helix through the mutation of amino acids, but the peak type is basically the same as that of the C34 control group. The activity determination results show that the activity of the mutant is generally lower and the C34-M3 basic loss activity. Through the red and blue light, It can be seen that the same results as CD can be found that.C34-M4 Hong Guang is more than doubling the blue light activity, C34-a Hong Guang is more than twice the blue light activity, and other red and blue light does not exist. Conclusion: the wavelength of the isomerization reaction of the switch system moves to the long wavelength (Hong Guang), which is also in line with the original switch design. Hong Guang can penetrate. Biological cells and various tissues and organs do not harm tissues and organs. This makes the switch system more suitable for research in living organisms. The switch system has tested the light control function: some of them show the inhibitory activity of HIV after exposure to blue light, and the inhibition function is weakened after red light irradiation.
【学位授予单位】：河北师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91
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