组蛋白H1和TAT融合蛋白向细胞内转运DNA能力的研究
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摘要
组蛋白H1包括了多个亚型,但这些亚型有相类似的结构域:中心球形结构域、N末端区和C末端区,该家族成员都是富含碱性氨基酸的蛋白质,因而能够与带负电荷的DNA通过静电引力相结合,参与细胞内染色体高级结构的形成。Thomas等长期研究发现,C末端区在染色质凝聚方面比另两个区域更为重要,即,就与DNA作用而言,该区的氨基酸构成非常重要。研究证实,C末端区氨基酸对DNA凝聚起了决定作用。而且经Schwamborn等的研究发现,H1组蛋白的C末端序列(sequence of H1 C terminatio以下统称H1ct)可能含有多个核定位信号。
蛋白转导(protein transduction)是指蛋白具有自主跨过细胞膜转运蛋白的功能,即蛋白自身及和其携带的一些生物大分子(蛋白质、多肽、DNA、寡核苷酸等)组成的融合蛋白均能在无任何辅助条件介导下(如脂质体、磷酸钙、电转化、病毒载体等)快速、高效、无障碍的穿过多种哺乳动物细胞膜进入细胞内,并可在周围细胞间传递,而且被转入细胞内的外源蛋白保留其原有的生物学活性与功能。这种独特的蛋白转导功能对于高效新型疫苗和药物的开发及基因治疗具有重要意义。最早被发现能跨过细胞膜进入细胞的蛋白是人免疫缺陷病毒(HIV-1)的转录激活蛋白(TAT),即HIV-TAT。研究表明,完整的TAT和截短的TAT蛋白(47～57位氨基酸)均具有蛋白转导功能,从而确定了TAT的蛋白转导结构域。由于TAT为穿膜肽,目前的研究表明穿膜肽可携带多种物质,包括亲水性蛋白、多肽、DNA甚至颗粒性物质等进行细胞间或细胞内的传输,并且不受细胞类型的限制。为了证明TAT在没有靶向的情况下可以携带蛋白进入细胞,我们做了第一个实验作为第二个实验的阳性对照。我们通过PCR重叠延伸技术将HIV-ⅠTat蛋白基因与绿色荧光蛋白(GFP)基因重组获得TAT-GFP融合基因,并通过原核表达系统实现融合基因TAT-GFP(TG)的表达和纯化。通过重组融合蛋白TG作用Hela细胞后荧光强度检测显示TAT有很高的转膜效率。
由于TAT的转膜效率高,我们将TAT基因和HIct蛋白基因融合,构建了基因转运载体。利用大肠杆菌通过基因工程技术表达TAT-H1ct融合蛋白,获得了TAT-H1ct蛋白的高效制备,通过融合表达His-Tag抗原表位标签对TAT-H1ct重组蛋白进行检测和纯化。用已纯化的TAT-H1ct融合蛋白作为DNA转运载体,对Hela细胞和鼠成纤维细胞进行绿色荧光蛋白质粒的转导实验并检测其表达情况。融合蛋白与绿色荧光光蛋白质粒以最佳配比形成复合物后,将其加入培养在24孔板的Hela细胞和鼠成纤维细胞,48小时后在激光共聚焦荧光显微镜下观察到Hela细胞和鼠成纤维细胞内有较强的绿色荧光,表明绿色荧光蛋白基因得到了表达。而对照组Hela细胞和鼠成纤维细胞中没有绿色荧光。这一结果显示TAT-H1ct融合蛋白可以介导DNA进入细胞,有较强的转运DNA的能力,为其发展成为基因导入系统奠定了基础。
Gene Therapy is a new treatment model developed in nineties of the twentieth century. It replaced the mutation pathogenic gene, also by changing the genetic structure of disease cells, or by importing genes of enhance immunity to human body, to control the disease and achieve the purpose of treatment. And compared to traditional drug therapy, if gene therapy as a fundamental method to control disease, the vector which can be injected into the body must be the developed. At present, the gene delivery system is divided into virus carriers and non-viral vector. Virus carriers: Non-targeting, a strong immunogenicity, prone to wild-type virus. Because of the low immunogenicity and the relatively safety of Non-viral vector, it is concerned increaslly.
Non-viral vectors for potential gene replacement and therapy have been developed in order to overcome the drawbacks of viral vectors. The diversity of non-viral vector resulted in a number of non-viral vector products, they have a flexibility of application, ease of use, low costs of production, and enhanced the safety of gene. Use the strategy of non-viral vector, oligonucleotide (ODNS) can be delivered naked (less efficient) or embedded cationic liposomes, polymers or peptide formed low buffer delivery systems, to achieve the purpose of therapy according to the target of cells. Tissues and cells internalization has been further strengthened by changing of physical or chemical method . There are many kinds of non-viral gene transfer vector, histone belongs to this family, because of their positively charge, DNA with a negative charge, they can be closely integrated through the charge, so that the structure of DNA by the extension is reduced to a relatively small volume of DNA particles.
The first protein across the cell membrane to enter the cells is of human immunodeficiency virus ,HIV. Studies have shown that exogenous protein conjugated with TAT can be transported to the cells, confirmed the ability of TAT transgenic membrane. N-(47-57) of 11 amino acids of TAT protein is the domain of transduction, sequence is YGRKKRRQRRR.
Search for efficient gene transfer vector, we constructed histone H1 as the skeleton of the gene delivery system, recombinant histone H1 gene, applied overlap extension PCR technology to recombinant histone gene and TAT trarsmembrane systems, build a fusion protein gene TAT-H1ct and expressed in prokaryotic cells. combinate fusion protein after purificate with pEGFP/C1 plasmid and then transfect Hela cells and fibroblast of mouse , detecte the intensity of green fluorescence to determine the ability to transfer DNA.
1. Expression and purification of TG fusion gene and detection efficiency of TAT trarsmembrane
Applied overlap extension PCR technique to recombinant green fluorescent protein gene and TAT gene, acquired recombinant plasmid of the fusion gene: pMD 18-T- TAT-GFP and prokaryotic expression plasmid pET-28a-TAT-GFP, the recombinant plasmid was transformed into receptor BL21 (DE3) to express, and fusion protein was detected by 12% SDS-PAGE electrophoresis, The specific TG fusion protein appeared at 31.5 kDa, conformity with the expected molecular weight, induced conditions is 1 mmol / L of IPTG final concentration, and the recombinant protein is highest expression when induced time is 6h. Fusion protein TG is purified, fusion protein is role in Hela cells. The results showed that compared with the control group, after 6 h , tumor cells with TG fusion protein can be observated high green fluorescence intensity by the laser scanning confocal fluorescence microscope, in the control group did not have green fluorescence.
2 Expression of prokaryotic expression vector of TAT-H1ct fusion gene and study the ability of transfectedr DNA to the cells
Applied PCR technology to recombinant H1ct gene and TAT gene, TAT-H1ct recombinant fusion protein gene is acquired, based on the recombinant plasmid pMD18-T-TAT-H1ct of fusion gene, we constructe the recombinant expression plasmid pET-28a-TAT-H1ct successfully, the recombinant plasmid was transformed into receptor BL21 (DE3) to express, and the fusion protein was detected by 12% SDS-PAGE electrophoresis, TAT-H1ct fusion protein is appered at around 32 kDa, conformity with the expected molecular weight. Induced conditions is 1 mmol / L of IPTG final concentration, and the fusion protein is highest expression when induced time is 5h. Fusion protein TAT-H1ct is purified,. After purification, the matching conditions of fusion protein and pEGFP/C1 plasmid is optimized, detecte the ability of the fusion protein binding DNA by observate the electrophores and then transfect Hela cells and fibroblast of mouse, after 48 hours high green fluorescence is observated by laser scanning confocal fluorescence microscopy. However the control group Hela cells and mouse fibroblasts is no green fluorescence. The results show that the fusion protein can carry DNA into the cell membrane with high efficiency, have a stronger ability to transfer DNA, is expected to become a gene transfer vector.
In this study, through the detection of fluorescence intensity show that TAT-H1ct has high trarsmembrane efficiency, is expected to become a gene transfer vector, and establish foundation for further study of tumor treatment.
蛋白转导(protein transduction)是指蛋白具有自主跨过细胞膜转运蛋白的功能,即蛋白自身及和其携带的一些生物大分子(蛋白质、多肽、DNA、寡核苷酸等)组成的融合蛋白均能在无任何辅助条件介导下(如脂质体、磷酸钙、电转化、病毒载体等)快速、高效、无障碍的穿过多种哺乳动物细胞膜进入细胞内,并可在周围细胞间传递,而且被转入细胞内的外源蛋白保留其原有的生物学活性与功能。这种独特的蛋白转导功能对于高效新型疫苗和药物的开发及基因治疗具有重要意义。最早被发现能跨过细胞膜进入细胞的蛋白是人免疫缺陷病毒(HIV-1)的转录激活蛋白(TAT),即HIV-TAT。研究表明,完整的TAT和截短的TAT蛋白(47～57位氨基酸)均具有蛋白转导功能,从而确定了TAT的蛋白转导结构域。由于TAT为穿膜肽,目前的研究表明穿膜肽可携带多种物质,包括亲水性蛋白、多肽、DNA甚至颗粒性物质等进行细胞间或细胞内的传输,并且不受细胞类型的限制。为了证明TAT在没有靶向的情况下可以携带蛋白进入细胞,我们做了第一个实验作为第二个实验的阳性对照。我们通过PCR重叠延伸技术将HIV-ⅠTat蛋白基因与绿色荧光蛋白(GFP)基因重组获得TAT-GFP融合基因,并通过原核表达系统实现融合基因TAT-GFP(TG)的表达和纯化。通过重组融合蛋白TG作用Hela细胞后荧光强度检测显示TAT有很高的转膜效率。
由于TAT的转膜效率高,我们将TAT基因和HIct蛋白基因融合,构建了基因转运载体。利用大肠杆菌通过基因工程技术表达TAT-H1ct融合蛋白,获得了TAT-H1ct蛋白的高效制备,通过融合表达His-Tag抗原表位标签对TAT-H1ct重组蛋白进行检测和纯化。用已纯化的TAT-H1ct融合蛋白作为DNA转运载体,对Hela细胞和鼠成纤维细胞进行绿色荧光蛋白质粒的转导实验并检测其表达情况。融合蛋白与绿色荧光光蛋白质粒以最佳配比形成复合物后,将其加入培养在24孔板的Hela细胞和鼠成纤维细胞,48小时后在激光共聚焦荧光显微镜下观察到Hela细胞和鼠成纤维细胞内有较强的绿色荧光,表明绿色荧光蛋白基因得到了表达。而对照组Hela细胞和鼠成纤维细胞中没有绿色荧光。这一结果显示TAT-H1ct融合蛋白可以介导DNA进入细胞,有较强的转运DNA的能力,为其发展成为基因导入系统奠定了基础。
Gene Therapy is a new treatment model developed in nineties of the twentieth century. It replaced the mutation pathogenic gene, also by changing the genetic structure of disease cells, or by importing genes of enhance immunity to human body, to control the disease and achieve the purpose of treatment. And compared to traditional drug therapy, if gene therapy as a fundamental method to control disease, the vector which can be injected into the body must be the developed. At present, the gene delivery system is divided into virus carriers and non-viral vector. Virus carriers: Non-targeting, a strong immunogenicity, prone to wild-type virus. Because of the low immunogenicity and the relatively safety of Non-viral vector, it is concerned increaslly.
Non-viral vectors for potential gene replacement and therapy have been developed in order to overcome the drawbacks of viral vectors. The diversity of non-viral vector resulted in a number of non-viral vector products, they have a flexibility of application, ease of use, low costs of production, and enhanced the safety of gene. Use the strategy of non-viral vector, oligonucleotide (ODNS) can be delivered naked (less efficient) or embedded cationic liposomes, polymers or peptide formed low buffer delivery systems, to achieve the purpose of therapy according to the target of cells. Tissues and cells internalization has been further strengthened by changing of physical or chemical method . There are many kinds of non-viral gene transfer vector, histone belongs to this family, because of their positively charge, DNA with a negative charge, they can be closely integrated through the charge, so that the structure of DNA by the extension is reduced to a relatively small volume of DNA particles.
The first protein across the cell membrane to enter the cells is of human immunodeficiency virus ,HIV. Studies have shown that exogenous protein conjugated with TAT can be transported to the cells, confirmed the ability of TAT transgenic membrane. N-(47-57) of 11 amino acids of TAT protein is the domain of transduction, sequence is YGRKKRRQRRR.
Search for efficient gene transfer vector, we constructed histone H1 as the skeleton of the gene delivery system, recombinant histone H1 gene, applied overlap extension PCR technology to recombinant histone gene and TAT trarsmembrane systems, build a fusion protein gene TAT-H1ct and expressed in prokaryotic cells. combinate fusion protein after purificate with pEGFP/C1 plasmid and then transfect Hela cells and fibroblast of mouse , detecte the intensity of green fluorescence to determine the ability to transfer DNA.
1. Expression and purification of TG fusion gene and detection efficiency of TAT trarsmembrane
Applied overlap extension PCR technique to recombinant green fluorescent protein gene and TAT gene, acquired recombinant plasmid of the fusion gene: pMD 18-T- TAT-GFP and prokaryotic expression plasmid pET-28a-TAT-GFP, the recombinant plasmid was transformed into receptor BL21 (DE3) to express, and fusion protein was detected by 12% SDS-PAGE electrophoresis, The specific TG fusion protein appeared at 31.5 kDa, conformity with the expected molecular weight, induced conditions is 1 mmol / L of IPTG final concentration, and the recombinant protein is highest expression when induced time is 6h. Fusion protein TG is purified, fusion protein is role in Hela cells. The results showed that compared with the control group, after 6 h , tumor cells with TG fusion protein can be observated high green fluorescence intensity by the laser scanning confocal fluorescence microscope, in the control group did not have green fluorescence.
2 Expression of prokaryotic expression vector of TAT-H1ct fusion gene and study the ability of transfectedr DNA to the cells
Applied PCR technology to recombinant H1ct gene and TAT gene, TAT-H1ct recombinant fusion protein gene is acquired, based on the recombinant plasmid pMD18-T-TAT-H1ct of fusion gene, we constructe the recombinant expression plasmid pET-28a-TAT-H1ct successfully, the recombinant plasmid was transformed into receptor BL21 (DE3) to express, and the fusion protein was detected by 12% SDS-PAGE electrophoresis, TAT-H1ct fusion protein is appered at around 32 kDa, conformity with the expected molecular weight. Induced conditions is 1 mmol / L of IPTG final concentration, and the fusion protein is highest expression when induced time is 5h. Fusion protein TAT-H1ct is purified,. After purification, the matching conditions of fusion protein and pEGFP/C1 plasmid is optimized, detecte the ability of the fusion protein binding DNA by observate the electrophores and then transfect Hela cells and fibroblast of mouse, after 48 hours high green fluorescence is observated by laser scanning confocal fluorescence microscopy. However the control group Hela cells and mouse fibroblasts is no green fluorescence. The results show that the fusion protein can carry DNA into the cell membrane with high efficiency, have a stronger ability to transfer DNA, is expected to become a gene transfer vector.
In this study, through the detection of fluorescence intensity show that TAT-H1ct has high trarsmembrane efficiency, is expected to become a gene transfer vector, and establish foundation for further study of tumor treatment.
引文
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