TAT转导肽作为细胞穿膜载体的体内和体外实验分析
发布时间：2018-08-15 17:25
【摘要】： 目的 由于细胞膜的选择通透性,大分子物质很难穿过细胞膜进入细胞内部,因此生物膜成了大分子物质作用于人体发挥作用的主要障碍,从而限制了大分子物质如大分子药物的广泛应用。例如多种具有治疗功效的蛋白质、多肽和寡聚核苷酸等难以进入细胞内部发挥药效,达不到临床应用目的。此外,很多具有应用前景的药物受到人体内各种天然屏障(如血脑屏障)的限制也难以到达靶器官或靶细胞而发挥药效。因此,本实验致力于研究一种蛋白转导域携带工具,它可以携带大分子蛋白药物穿过细胞膜而发挥药效。为进一步解决大分子药物的临床应用奠定基础。 方法 通过生物信息学软件分析His-EGFP和His-TAT-EGFP的理化性质和二级结构的变化与不同之处,分析得到反式激活蛋白TAT (transactivator protein)并不改变融合蛋白His-TAT-EGFP中EGFP的二级结构及生物学活性,并且与His-EGFP相比,His-TAT-EGFP的亲水性和极性增加,等电点提高。通过质粒构建重组成融合质粒载体,首先把HIV-1 TAT 47-57氨基酸基因序列连接到pET-28a质粒上,将pET-28a-TAT转化到DH5a菌中进行扩增,提取质粒pET-28a-TAT;然后再把绿色荧光蛋白基因序列EGFP连接到构建好的质粒pET-28a-TAT上。将其转化到DH5a菌中进行扩增,提取质粒pET-28a-TAT-EGFP,然后进行酶切鉴定及测序。用同样的方法构建质粒pET-28a-EGFP,作为对照。分别把两种质粒转化到BL21(DE3)菌中,进行表达,当菌液OD为0.6时,加入IPTG,使IPTG终浓度为0.5mM,在37℃、200rpm条件下诱导表达四小时。大量表达出蛋白质后,用His标签的亲和层析柱进行蛋白纯化。得到纯化的蛋白质后,进行体内蛋白质的细胞穿膜实验,两种蛋白His-TAT-EGFP和His-EGFP分别与HeLa细胞和PC12细胞共孵育,经过PBS清洗过后,用荧光显微镜观察两种细胞分别被两种蛋白孵育后的荧光情况。做蛋白浓度梯度与细胞穿膜功能之间量效关系实验,利用不同浓度融合蛋白His-TAT-EGFP与HeLa细胞分别孵育90min,经过PBS清洗和胰酶消化后,用PBS重悬细胞,用流式细胞仪检测细胞穿膜效果。检测融合蛋白穿活体动物细胞膜的活性,给体重为22g左右的健康成年昆明小鼠分别腹腔注射两种蛋白His-TAT-EGFP和His-EGFP,检测融合蛋白His-TAT-EGFP在活体动物体内的细胞穿膜功能,做动物组织心、脑、肝和肾的冰冻切片,然后用荧光显微镜观察切片。利用同样方法构建质粒pET-28a-TAT(-)-EGFP, pET-28a-EGFP-TAT和pET-28a-EGFP-TAT(-),然后经过表达和纯化后,得到一系列融合蛋白,His-TAT(-)-EGFP、His-EGFP-TAT(+)和His-EGFP-TAT(-),利用这些融合蛋白进行体外细胞穿膜实验,比较各种不同序列和TAT在融合蛋白中N端或C端位置不同的TAT融合蛋白的细胞穿膜能力差异,从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融合蛋白的细胞穿膜功能的差异。 结果 成功地构建了pET-28a-TAT-EGFP和pET-28a-EGFP质粒,并在BL21(DE3)菌中得到了大量表达,经过His标签亲和层析柱进行蛋白纯化得到纯度比较高的两种融合蛋白。经过体外和体内细胞穿膜实验,结果显示表达出的融合蛋白His-TAT-EGFP具有体外和体内细胞穿膜功能,而单纯的His-EGFP蛋白是不具有这种细胞穿膜功能的。并且表明在同样时间的作用下,在一定浓度范围内,细胞穿膜能力与TAT融合蛋白的浓度成正比。成功地构建了融合质粒pET-28a-TAT(-)-EGFP, pET-28a-EGFP-TAT和pET-28a-EGFP-TAT(-),并表达纯化出几种蛋白His-TAT(-)-EGFP, His-EGFP-TAT(+)和His-EGFP-TAT(-),并且对几种融合蛋白分别进行了体外细胞穿膜实验,成功进行了TAT不同序列和在融合蛋白中不同位置的TAT融合蛋白的细胞穿膜功能比较,得到四种融合蛋白细胞穿膜能力不尽相同的结果,而His-EGFP-TAT(-)相对来说穿膜能力较强。 结论 蛋白转导域具有强大的携带大分子蛋白质细胞穿膜的功能。通过质粒构建可以表达出具有穿过生物膜功能的蛋白转导域和绿色荧光蛋白的融合蛋白,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融合蛋白细胞穿膜能力的差异,并且经过研究验证了不同序列TAT和在融合蛋白中位置不同的TAT穿膜能力不尽相同,在融合蛋白中处于C端的反向序列的融合蛋白细胞穿膜能力较强,并且在一定浓度范围内的穿膜能力会随浓度升高而穿膜能力增强,并且各种融合蛋白穿膜机制与膜受体无关,属于被动运输的一种,各种融合蛋白对细胞无创伤作用,为下一步开发大分子新药成功建立了技术平台。
[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.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
本文编号：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.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
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