TAT轉(zhuǎn)導(dǎo)肽作為細(xì)胞穿膜載體的體內(nèi)和體外實驗分析
發(fā)布時間：2018-08-15 17:25
【摘要】： 目的 由于細(xì)胞膜的選擇通透性,大分子物質(zhì)很難穿過細(xì)胞膜進(jìn)入細(xì)胞內(nèi)部,因此生物膜成了大分子物質(zhì)作用于人體發(fā)揮作用的主要障礙,從而限制了大分子物質(zhì)如大分子藥物的廣泛應(yīng)用。例如多種具有治療功效的蛋白質(zhì)、多肽和寡聚核苷酸等難以進(jìn)入細(xì)胞內(nèi)部發(fā)揮藥效,達(dá)不到臨床應(yīng)用目的。此外,很多具有應(yīng)用前景的藥物受到人體內(nèi)各種天然屏障(如血腦屏障)的限制也難以到達(dá)靶器官或靶細(xì)胞而發(fā)揮藥效。因此,本實驗致力于研究一種蛋白轉(zhuǎn)導(dǎo)域攜帶工具,它可以攜帶大分子蛋白藥物穿過細(xì)胞膜而發(fā)揮藥效。為進(jìn)一步解決大分子藥物的臨床應(yīng)用奠定基礎(chǔ)。 方法 通過生物信息學(xué)軟件分析His-EGFP和His-TAT-EGFP的理化性質(zhì)和二級結(jié)構(gòu)的變化與不同之處,分析得到反式激活蛋白TAT (transactivator protein)并不改變?nèi)诤系鞍譎is-TAT-EGFP中EGFP的二級結(jié)構(gòu)及生物學(xué)活性,并且與His-EGFP相比,His-TAT-EGFP的親水性和極性增加,等電點提高。通過質(zhì)粒構(gòu)建重組成融合質(zhì)粒載體,首先把HIV-1 TAT 47-57氨基酸基因序列連接到pET-28a質(zhì)粒上,將pET-28a-TAT轉(zhuǎn)化到DH5a菌中進(jìn)行擴(kuò)增,提取質(zhì)粒pET-28a-TAT;然后再把綠色熒光蛋白基因序列EGFP連接到構(gòu)建好的質(zhì)粒pET-28a-TAT上。將其轉(zhuǎn)化到DH5a菌中進(jìn)行擴(kuò)增,提取質(zhì)粒pET-28a-TAT-EGFP,然后進(jìn)行酶切鑒定及測序。用同樣的方法構(gòu)建質(zhì)粒pET-28a-EGFP,作為對照。分別把兩種質(zhì)粒轉(zhuǎn)化到BL21(DE3)菌中,進(jìn)行表達(dá),當(dāng)菌液OD為0.6時,加入IPTG,使IPTG終濃度為0.5mM,在37℃、200rpm條件下誘導(dǎo)表達(dá)四小時。大量表達(dá)出蛋白質(zhì)后,用His標(biāo)簽的親和層析柱進(jìn)行蛋白純化。得到純化的蛋白質(zhì)后,進(jìn)行體內(nèi)蛋白質(zhì)的細(xì)胞穿膜實驗,兩種蛋白His-TAT-EGFP和His-EGFP分別與HeLa細(xì)胞和PC12細(xì)胞共孵育,經(jīng)過PBS清洗過后,用熒光顯微鏡觀察兩種細(xì)胞分別被兩種蛋白孵育后的熒光情況。做蛋白濃度梯度與細(xì)胞穿膜功能之間量效關(guān)系實驗,利用不同濃度融合蛋白His-TAT-EGFP與HeLa細(xì)胞分別孵育90min,經(jīng)過PBS清洗和胰酶消化后,用PBS重懸細(xì)胞,用流式細(xì)胞儀檢測細(xì)胞穿膜效果。檢測融合蛋白穿活體動物細(xì)胞膜的活性,給體重為22g左右的健康成年昆明小鼠分別腹腔注射兩種蛋白His-TAT-EGFP和His-EGFP,檢測融合蛋白His-TAT-EGFP在活體動物體內(nèi)的細(xì)胞穿膜功能,做動物組織心、腦、肝和腎的冰凍切片,然后用熒光顯微鏡觀察切片。利用同樣方法構(gòu)建質(zhì)粒pET-28a-TAT(-)-EGFP, pET-28a-EGFP-TAT和pET-28a-EGFP-TAT(-),然后經(jīng)過表達(dá)和純化后,得到一系列融合蛋白,His-TAT(-)-EGFP、His-EGFP-TAT(+)和His-EGFP-TAT(-),利用這些融合蛋白進(jìn)行體外細(xì)胞穿膜實驗,比較各種不同序列和TAT在融合蛋白中N端或C端位置不同的TAT融合蛋白的細(xì)胞穿膜能力差異,從而在融合蛋白中找出不同TAT序列和在融合蛋白中位置不同的TAT融合蛋白的細(xì)胞穿膜功能的差異。 結(jié)果 成功地構(gòu)建了pET-28a-TAT-EGFP和pET-28a-EGFP質(zhì)粒,并在BL21(DE3)菌中得到了大量表達(dá),經(jīng)過His標(biāo)簽親和層析柱進(jìn)行蛋白純化得到純度比較高的兩種融合蛋白。經(jīng)過體外和體內(nèi)細(xì)胞穿膜實驗,結(jié)果顯示表達(dá)出的融合蛋白His-TAT-EGFP具有體外和體內(nèi)細(xì)胞穿膜功能,而單純的His-EGFP蛋白是不具有這種細(xì)胞穿膜功能的。并且表明在同樣時間的作用下,在一定濃度范圍內(nèi),細(xì)胞穿膜能力與TAT融合蛋白的濃度成正比。成功地構(gòu)建了融合質(zhì)粒pET-28a-TAT(-)-EGFP, pET-28a-EGFP-TAT和pET-28a-EGFP-TAT(-),并表達(dá)純化出幾種蛋白His-TAT(-)-EGFP, His-EGFP-TAT(+)和His-EGFP-TAT(-),并且對幾種融合蛋白分別進(jìn)行了體外細(xì)胞穿膜實驗,成功進(jìn)行了TAT不同序列和在融合蛋白中不同位置的TAT融合蛋白的細(xì)胞穿膜功能比較,得到四種融合蛋白細(xì)胞穿膜能力不盡相同的結(jié)果,而His-EGFP-TAT(-)相對來說穿膜能力較強。 結(jié)論 蛋白轉(zhuǎn)導(dǎo)域具有強大的攜帶大分子蛋白質(zhì)細(xì)胞穿膜的功能。通過質(zhì)粒構(gòu)建可以表達(dá)出具有穿過生物膜功能的蛋白轉(zhuǎn)導(dǎo)域和綠色熒光蛋白的融合蛋白,并且找出不同序列和在融合蛋白中N或C端的不同位置的TAT融合蛋白細(xì)胞穿膜能力的差異,并且經(jīng)過研究驗證了不同序列TAT和在融合蛋白中位置不同的TAT穿膜能力不盡相同,在融合蛋白中處于C端的反向序列的融合蛋白細(xì)胞穿膜能力較強,并且在一定濃度范圍內(nèi)的穿膜能力會隨濃度升高而穿膜能力增強,并且各種融合蛋白穿膜機制與膜受體無關(guān),屬于被動運輸?shù)囊环N,各種融合蛋白對細(xì)胞無創(chuàng)傷作用,為下一步開發(fā)大分子新藥成功建立了技術(shù)平臺。
[Abstract]:objective
Because of the selective permeability of cell membranes, it is difficult for macromolecules to penetrate the cell membranes into the cell, so biofilms become the main obstacle for macromolecules to act on the human body, thus limiting the wide application of macromolecules such as macromolecular drugs, such as a variety of therapeutic proteins, peptides and oligonucleotides. In addition, many promising drugs are limited by various natural barriers (such as blood-brain barrier) in the human body and are difficult to reach target organs or target cells. Therefore, this experiment is devoted to the study of a protein transduction domain carrier tool, which can be carried. Macromolecular protein drugs penetrate the cell membrane to exert their efficacy, which lays a foundation for further solving the clinical application of macromolecular drugs.
Method
The physicochemical properties and secondary structure of his-EGFP and his-TAT-EGFP were analyzed by bioinformatics software. The results showed that transactivator protein did not change the secondary structure and biological activity of EGFP in his-TAT-EGFP, and compared with his-EGFP, his-TAT-EGFP was more hydrophilic and biologically active. The fusion plasmid was constructed by recombining the HIV-1 TAT 47-57 amino acid gene sequence into the pET-28a plasmid, transforming the pET-28a-TAT into DH5a strain for amplification, extracting the plasmid pET-28a-TAT, and then connecting the green fluorescent protein gene sequence EGFP to the constructed plasmid pET-28a-TA. The plasmid pET-28a-TAT-EGFP was constructed by the same method as control. The two plasmids were transformed into BL21 (DE3) bacteria and expressed. When OD was 0.6, IPTG was added to make the final concentration of IPTG 0.5mM, at 37 C and 200 rpm. The protein was purified by His-labeled affinity chromatography column. After the purified protein was obtained, the in vivo protein penetration test was carried out. The two proteins, His-TAT-EGFP and His-EGFP, were incubated with HeLa cells and PC12 cells respectively. After being washed by PBS, the two proteins were observed by fluorescence microscope. The fluorescence of HeLa cells incubated with different concentrations of fusion protein His-TAT-EGFP was measured by flow cytometry after being washed with PBS and digested with trypsin for 90 minutes. The activity of the fusion protein penetrating the cell membrane of living animals was studied by intraperitoneal injection of his-TAT-EGFP and his-EGFP into healthy adult Kunming mice weighing about 22g. The function of the fusion protein His-TAT-EGFP penetrating the cell membrane in vivo was detected. The frozen sections of heart, brain, liver and kidney were made and observed by fluorescence microscope. The plasmids pET-28a-TAT (-) -EGFP, pET-28a-EGFP-TAT and pET-28a-EGFP-TAT (-) were constructed by the same method. After expression and purification, a series of fusion proteins, His-TAT (-) -EGFP, His-EGFP-TAT (+) and His-EGFP-TAT (-), were obtained. These fusion proteins were used for cell penetration test in vitro to compare the different sequences and TAT in vitro. The cell membrane penetrating ability of TAT fusion proteins with different N-terminal or C-terminal positions in the fusion proteins was different, so the cell membrane penetrating function of TAT fusion proteins with different TAT sequences and different TAT fusion proteins in the fusion proteins was found.
Result
The plasmids of pET-28a-TAT-EGFP and pET-28a-EGFP were successfully constructed and expressed in BL21 (DE3) strain. Two fusion proteins with high purity were purified by His-labeled affinity chromatography column. The results showed that the fusion protein His-TAT-EGFP was expressed in vitro and in vivo. Cell membrane penetrating function was not found in the pure H-EGFP protein, and the cell membrane penetrating ability was proportional to the concentration of TAT fusion protein at the same time. Fusion plasmids pET-28a-TAT (-) -EGFP, pET-28a-EGFP-TAT and pET-28a-EGFP-TAT (-) were successfully constructed. He-TAT (-) - EGFP, His-EGFP-TAT (+) and His-EGFP-TAT (-) were expressed and purified. The cell penetration ability of the four fusion proteins was compared between different TAT sequences and different TAT fusion proteins in different positions. The results are the same, while His-EGFP-TAT (-) is relatively strong in penetrating.
conclusion
Protein transduction domain has a powerful function of carrying macromolecule protein through cell membrane. Fusion proteins of protein transduction domain and green fluorescent protein can be expressed through plasmid construction, and the ability of TAT fusion protein cells to penetrate membrane can be found in different sequences and in different positions at N or C ends of fusion protein. Different TAT sequences and different TAT locations in the fusion proteins have different membrane penetrating abilities. The reverse sequence of TAT in the fusion proteins has stronger membrane penetrating ability, and the membrane penetrating ability will increase with the concentration of TAT in a certain range of concentration, and all kinds of fusion proteins have different membrane penetrating abilities. The mechanism of protein penetration has nothing to do with membrane receptor, and belongs to passive transport. All kinds of fusion proteins have no invasive effect on cells, which provides a technical platform for the further development of new macromolecular drugs.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346
本文編號：2184903
[Abstract]:objective
Because of the selective permeability of cell membranes, it is difficult for macromolecules to penetrate the cell membranes into the cell, so biofilms become the main obstacle for macromolecules to act on the human body, thus limiting the wide application of macromolecules such as macromolecular drugs, such as a variety of therapeutic proteins, peptides and oligonucleotides. In addition, many promising drugs are limited by various natural barriers (such as blood-brain barrier) in the human body and are difficult to reach target organs or target cells. Therefore, this experiment is devoted to the study of a protein transduction domain carrier tool, which can be carried. Macromolecular protein drugs penetrate the cell membrane to exert their efficacy, which lays a foundation for further solving the clinical application of macromolecular drugs.
Method
The physicochemical properties and secondary structure of his-EGFP and his-TAT-EGFP were analyzed by bioinformatics software. The results showed that transactivator protein did not change the secondary structure and biological activity of EGFP in his-TAT-EGFP, and compared with his-EGFP, his-TAT-EGFP was more hydrophilic and biologically active. The fusion plasmid was constructed by recombining the HIV-1 TAT 47-57 amino acid gene sequence into the pET-28a plasmid, transforming the pET-28a-TAT into DH5a strain for amplification, extracting the plasmid pET-28a-TAT, and then connecting the green fluorescent protein gene sequence EGFP to the constructed plasmid pET-28a-TA. The plasmid pET-28a-TAT-EGFP was constructed by the same method as control. The two plasmids were transformed into BL21 (DE3) bacteria and expressed. When OD was 0.6, IPTG was added to make the final concentration of IPTG 0.5mM, at 37 C and 200 rpm. The protein was purified by His-labeled affinity chromatography column. After the purified protein was obtained, the in vivo protein penetration test was carried out. The two proteins, His-TAT-EGFP and His-EGFP, were incubated with HeLa cells and PC12 cells respectively. After being washed by PBS, the two proteins were observed by fluorescence microscope. The fluorescence of HeLa cells incubated with different concentrations of fusion protein His-TAT-EGFP was measured by flow cytometry after being washed with PBS and digested with trypsin for 90 minutes. The activity of the fusion protein penetrating the cell membrane of living animals was studied by intraperitoneal injection of his-TAT-EGFP and his-EGFP into healthy adult Kunming mice weighing about 22g. The function of the fusion protein His-TAT-EGFP penetrating the cell membrane in vivo was detected. The frozen sections of heart, brain, liver and kidney were made and observed by fluorescence microscope. The plasmids pET-28a-TAT (-) -EGFP, pET-28a-EGFP-TAT and pET-28a-EGFP-TAT (-) were constructed by the same method. After expression and purification, a series of fusion proteins, His-TAT (-) -EGFP, His-EGFP-TAT (+) and His-EGFP-TAT (-), were obtained. These fusion proteins were used for cell penetration test in vitro to compare the different sequences and TAT in vitro. The cell membrane penetrating ability of TAT fusion proteins with different N-terminal or C-terminal positions in the fusion proteins was different, so the cell membrane penetrating function of TAT fusion proteins with different TAT sequences and different TAT fusion proteins in the fusion proteins was found.
Result
The plasmids of pET-28a-TAT-EGFP and pET-28a-EGFP were successfully constructed and expressed in BL21 (DE3) strain. Two fusion proteins with high purity were purified by His-labeled affinity chromatography column. The results showed that the fusion protein His-TAT-EGFP was expressed in vitro and in vivo. Cell membrane penetrating function was not found in the pure H-EGFP protein, and the cell membrane penetrating ability was proportional to the concentration of TAT fusion protein at the same time. Fusion plasmids pET-28a-TAT (-) -EGFP, pET-28a-EGFP-TAT and pET-28a-EGFP-TAT (-) were successfully constructed. He-TAT (-) - EGFP, His-EGFP-TAT (+) and His-EGFP-TAT (-) were expressed and purified. The cell penetration ability of the four fusion proteins was compared between different TAT sequences and different TAT fusion proteins in different positions. The results are the same, while His-EGFP-TAT (-) is relatively strong in penetrating.
conclusion
Protein transduction domain has a powerful function of carrying macromolecule protein through cell membrane. Fusion proteins of protein transduction domain and green fluorescent protein can be expressed through plasmid construction, and the ability of TAT fusion protein cells to penetrate membrane can be found in different sequences and in different positions at N or C ends of fusion protein. Different TAT sequences and different TAT locations in the fusion proteins have different membrane penetrating abilities. The reverse sequence of TAT in the fusion proteins has stronger membrane penetrating ability, and the membrane penetrating ability will increase with the concentration of TAT in a certain range of concentration, and all kinds of fusion proteins have different membrane penetrating abilities. The mechanism of protein penetration has nothing to do with membrane receptor, and belongs to passive transport. All kinds of fusion proteins have no invasive effect on cells, which provides a technical platform for the further development of new macromolecular drugs.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346
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