HIV融膜蛋白抑制劑C34的紅光調(diào)控研究
發(fā)布時(shí)間：2018-08-09 19:06
【摘要】：艾滋病(acquired immune deficiency syndrome,AIDS)全稱獲得性免疫缺陷綜合征,是由人類免疫缺陷病毒(Human Immunodeficiency Virus,HIV)引起的一種嚴(yán)重傳染病。HIV的生命周期一般分為幾個(gè)不同的階段:病毒吸附、結(jié)合和融合,病毒脫殼、轉(zhuǎn)錄、翻譯、整合、組裝、成熟和出芽。了解艾滋病病毒感染的過程細(xì)節(jié)對于我們開發(fā)藥物是非常重要的。目的:開發(fā)艾滋病病毒感染機(jī)制研究工具,實(shí)現(xiàn)定性或定量控制病毒感染過程。方法:Gp41為HIV-I的包膜蛋白復(fù)合體,其控制著病毒侵入靶細(xì)胞的早期關(guān)鍵過程,主要功能是介導(dǎo)病毒與靶細(xì)胞膜的融合。C34為模擬核心CHR區(qū)的合成肽,可以通過抑制HIV-I gp41自身的C34肽與NHR區(qū)的N36肽結(jié)合而抑制HIV-I的融膜過程�，F(xiàn)在市場上的很多抗HIV藥物應(yīng)用此原理。在這里,我們開發(fā)了一個(gè)打開和關(guān)閉的系統(tǒng),它可以使用光來控制HIV進(jìn)入靶細(xì)胞的融膜過程。偶氮苯(azobenzene)結(jié)構(gòu)和功能的光致異構(gòu)化為我們提供了一個(gè)重要的分子手段。偶氮苯具有較強(qiáng)的光響應(yīng)特性,這一特性使其在許多領(lǐng)域表現(xiàn)出巨大的開發(fā)潛力。通常偶氮苯存在順式、反式兩種異構(gòu)體。反式構(gòu)型偶氮苯轉(zhuǎn)變?yōu)轫樖綐?gòu)型,需要在特定波長的紫外光照射下才能發(fā)生;在可見光或熱的作用下,順式構(gòu)型可還原為反式構(gòu)型。偶氮苯分子的兩種構(gòu)型的紫外-可見吸收光譜有明顯不同。同時(shí),兩者在立體結(jié)構(gòu)、偶極矩等物理和化學(xué)性質(zhì)方面也存在明顯差異。目前,偶氮苯的順反異構(gòu)體的不同特點(diǎn),順反異構(gòu)化和各種誘導(dǎo)的光響應(yīng)的現(xiàn)象,引起了社會(huì)各界的廣泛關(guān)注。由于紫外對細(xì)胞和組織具有強(qiáng)烈的殺傷作用,所以想在體內(nèi)應(yīng)用偶氮苯的關(guān)鍵是需要為其提供一個(gè)發(fā)生異構(gòu)化反應(yīng)合適的照射波長。我們對偶氮苯進(jìn)行修飾,使其調(diào)控波長處于紅光區(qū)(630-660 nm)。這個(gè)開關(guān)系統(tǒng)的關(guān)鍵特征是修改C34肽,通過設(shè)計(jì)C34使紅移的光控偶氮苯與設(shè)計(jì)位置的半胱氨酸突變體交聯(lián)。在這里我們設(shè)計(jì)了6條突變的多肽和兩個(gè)光控開關(guān)。多肽的合成采用固相合成法;多肽的純化采用半制備型高壓液相色譜儀進(jìn)行;多肽結(jié)構(gòu)鑒定采用MALDI-TOF法進(jìn)行,純度采用分析型高壓液相色譜儀測定。光控開關(guān)采用化學(xué)合成法,純化采用硅膠層析柱法進(jìn)行純化,結(jié)構(gòu)采用MALDI-TOF及核磁共振氫譜進(jìn)行分析,純度采用HPLC測定;使用紫外分光光度計(jì)掃描光控開關(guān)的紫外-可見光光譜。開關(guān)系統(tǒng)的活性通過細(xì)胞融合實(shí)驗(yàn)的方法,其結(jié)構(gòu)采用圓二色譜光譜掃描法進(jìn)行鑒定。結(jié)果:1、多肽的合成和純化實(shí)驗(yàn)較為關(guān)鍵,是整個(gè)實(shí)驗(yàn)成敗的基礎(chǔ)。本實(shí)驗(yàn)完成的較成功,經(jīng)質(zhì)譜分析和HPLC分析多肽結(jié)構(gòu)準(zhǔn)確、純度較好,符合實(shí)驗(yàn)要求。2、開關(guān)的合成較為復(fù)雜,經(jīng)過反復(fù)實(shí)驗(yàn)及優(yōu)化,最終得到產(chǎn)品產(chǎn)率較高、純度高。經(jīng)質(zhì)譜分析和HPLC分析所得產(chǎn)物結(jié)構(gòu)準(zhǔn)確、純度高,符合實(shí)驗(yàn)要求。3、開關(guān)系統(tǒng)的組裝相對較簡單,所得產(chǎn)物經(jīng)質(zhì)譜分析和HPLC分析所得產(chǎn)物結(jié)構(gòu)準(zhǔn)確、純度高,符合實(shí)驗(yàn)要求。4、圓二色譜法(CD)掃描可以看出開關(guān)系統(tǒng)均有兩個(gè)比較明顯的α-螺旋縫,這也符合C肽和N肽結(jié)合后形成的螺旋六聚體結(jié)構(gòu)。通過C34和其突變體CD譜的比較可以看出經(jīng)過氨基酸的突變對螺旋的形成存在一定的干擾作用,但峰型與C34對照組基本一致。5、活性測定結(jié)果表明其突變體的活性普遍降低,C34-M3基本喪失活性。通過紅藍(lán)光對比可以看出與CD相同的結(jié)果。C34-M4紅光較藍(lán)光活性降低一倍左右,C34-a紅光較藍(lán)光活性降低一倍左右,其他紅藍(lán)光照射不存在明顯差異。結(jié)論:開關(guān)系統(tǒng)的異構(gòu)化反應(yīng)波長向長波長(紅光)移動(dòng),這也符合最初開關(guān)設(shè)計(jì)的目的。紅光可以穿透生物體細(xì)胞及各個(gè)組織和器官,對組織和器官?zèng)]有危害。這就使得這個(gè)開關(guān)系統(tǒng)更適用于在生物體內(nèi)的研究。開關(guān)系統(tǒng)我們進(jìn)行了光控功能測試:它們中的一些在接受藍(lán)光照射后表現(xiàn)出艾滋病毒的抑制活性,在紅光照射后抑制功能減弱。
[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.
【學(xué)位授予單位】：河北師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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.
【學(xué)位授予單位】：河北師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.91
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