TAT转导肽作为细胞穿膜载体的体内和体外实验分析
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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。
Objective
Most of the biopharmaceutical drugs, such as peptides. proteins, enzymes and oligonucleotides, have very limited effectiveness in clinical applications since they lack the ability to penetrate into cells and reach their pharmacological targets. Extensive studies have been conducted in an attempt to develop suitable delivery systems which can carry biopharmaceutical drugs across the impermeable plasma membrane for therapeutic purpose. HIV-1 transactivator of transcription (TAT) has demonstrated ability to traverse the cellular membrane in many studies. Recently, we conducted a systematic study to explore the mechanism of the TAT-mediated protein transduction process.Giant molecular substance go straight through cellular membrane difficultly as the selective permeability of the cellular membrane. So the cellular membrane becomes the main reason of the giant molecular drug producing a marked effect to human body. Many kinds of protein、polypeptide and oligonucleotide that has the effect of curing to human body go through the cellular hardly to produce marked effect. We are due to reserch protein transductive domain that can take along giant molecular drug to go through the cellular membrane.
Methods
We constructed an expression vector pET-28a-TAT-EGFP and expressed a quantity of His-TAT-EGFP fusion protein in E. coli. cells, purified the fusion protein using affinity chromatography. The fusion protein His-TAT-EGFP was confirmed with DNA sequence and protein sequence. With the same procedure, we also obtained His-EGFP as the negative control.
The fusion peptide His-TAT-EGFP was incubated with HeLa cells and PC 12 cells at 37℃temperature for 60 minutes. After rinsed three times. these cells were examined under fluorescence microscope. The fluorescence microscopic images showed that the fusion protein His-TAT-EGFP is able to traverse the cellular membrane in 20 minutes, whereas the protein His-EGFP cannot penetrate into the cells. In vivo experiments were also conducted where fusion protein His-TAT-EGFP and His-EGFP were injected intraperitoneally into Kunming mice, respectively. Two hours later, these mice were sacrificed and their organs (liver, brain, heart and kidney) were obtained and made frozen slides. All the slides showed fluorescent His-TAT-EGFP under the fluorescence microscope, of these mice which were injected with protein His-EGFP did not show any observable fluorescence signals. The data also showed that His-TAT-EGFP can reach and penetrate into these organs within 2 hours. However, the data showed that TAT-mediated protein transduction did not show any tissue specificity.
Through plasmid construction we construct plasmid that conect protein trunsductive domain and enhance green fluorescent protein gene order. Then through BL21 bacterium we expesses fusion protein and purificate fusion protein. Through fluorescent microscope overview to test the effect of going through cellular membrane in vitro and the effects of the protein going through the cellular membrane of live animal in vivo. Furthermore, we investigated the effect of the orientation and position of the TAT peptide on the protein transduction capability. The amino acid sequence of TAT was reversed (named as TAT(-)), and then fused with His-EGFP protein. The TAT and TAT(-) positions relevant to the His-EGFP protein were also changed. Thus, four fusion proteins were obtained, that is His-TAT-EGFP, His-TAT(-)-EGFP, His-EGFP-TAT and His-EGFP-TAT(-), respectively. Comparison of the results did not exhibit significant difference in terms of the transducing capability. The current study provides a reliable experimental foundation on which we established a technical platform for constructing more flexible fusion proteins for therapeutical applications.
Results
Result displays:the four fusion proteins His-TAT-EGFP, His-TAT(-)-EGFP, His-EGFP-TAT and His-EGFP-TAT(-) of expression have the effect of going through cellular membrane in vivo and in vitro, while His-EGFP doesn't have.
Conclusions
The plasmid construction can express the proteins His-TAT-EGFP, His-TAT(-)-EGFP, His-EGFP-TAT and His-EGFP-TAT(-), which named protein trunsductive domain can take along giant molecular protein to go through cellular membrane in vivo an in vitro. It shows that different fusion protein has different transmembrane capacity, the TAT(-) sequence and in C terminal has stronger transmembrane capacity. It constructs a technique flat-top for exploitation new drug
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。
Objective
Most of the biopharmaceutical drugs, such as peptides. proteins, enzymes and oligonucleotides, have very limited effectiveness in clinical applications since they lack the ability to penetrate into cells and reach their pharmacological targets. Extensive studies have been conducted in an attempt to develop suitable delivery systems which can carry biopharmaceutical drugs across the impermeable plasma membrane for therapeutic purpose. HIV-1 transactivator of transcription (TAT) has demonstrated ability to traverse the cellular membrane in many studies. Recently, we conducted a systematic study to explore the mechanism of the TAT-mediated protein transduction process.Giant molecular substance go straight through cellular membrane difficultly as the selective permeability of the cellular membrane. So the cellular membrane becomes the main reason of the giant molecular drug producing a marked effect to human body. Many kinds of protein、polypeptide and oligonucleotide that has the effect of curing to human body go through the cellular hardly to produce marked effect. We are due to reserch protein transductive domain that can take along giant molecular drug to go through the cellular membrane.
Methods
We constructed an expression vector pET-28a-TAT-EGFP and expressed a quantity of His-TAT-EGFP fusion protein in E. coli. cells, purified the fusion protein using affinity chromatography. The fusion protein His-TAT-EGFP was confirmed with DNA sequence and protein sequence. With the same procedure, we also obtained His-EGFP as the negative control.
The fusion peptide His-TAT-EGFP was incubated with HeLa cells and PC 12 cells at 37℃temperature for 60 minutes. After rinsed three times. these cells were examined under fluorescence microscope. The fluorescence microscopic images showed that the fusion protein His-TAT-EGFP is able to traverse the cellular membrane in 20 minutes, whereas the protein His-EGFP cannot penetrate into the cells. In vivo experiments were also conducted where fusion protein His-TAT-EGFP and His-EGFP were injected intraperitoneally into Kunming mice, respectively. Two hours later, these mice were sacrificed and their organs (liver, brain, heart and kidney) were obtained and made frozen slides. All the slides showed fluorescent His-TAT-EGFP under the fluorescence microscope, of these mice which were injected with protein His-EGFP did not show any observable fluorescence signals. The data also showed that His-TAT-EGFP can reach and penetrate into these organs within 2 hours. However, the data showed that TAT-mediated protein transduction did not show any tissue specificity.
Through plasmid construction we construct plasmid that conect protein trunsductive domain and enhance green fluorescent protein gene order. Then through BL21 bacterium we expesses fusion protein and purificate fusion protein. Through fluorescent microscope overview to test the effect of going through cellular membrane in vitro and the effects of the protein going through the cellular membrane of live animal in vivo. Furthermore, we investigated the effect of the orientation and position of the TAT peptide on the protein transduction capability. The amino acid sequence of TAT was reversed (named as TAT(-)), and then fused with His-EGFP protein. The TAT and TAT(-) positions relevant to the His-EGFP protein were also changed. Thus, four fusion proteins were obtained, that is His-TAT-EGFP, His-TAT(-)-EGFP, His-EGFP-TAT and His-EGFP-TAT(-), respectively. Comparison of the results did not exhibit significant difference in terms of the transducing capability. The current study provides a reliable experimental foundation on which we established a technical platform for constructing more flexible fusion proteins for therapeutical applications.
Results
Result displays:the four fusion proteins His-TAT-EGFP, His-TAT(-)-EGFP, His-EGFP-TAT and His-EGFP-TAT(-) of expression have the effect of going through cellular membrane in vivo and in vitro, while His-EGFP doesn't have.
Conclusions
The plasmid construction can express the proteins His-TAT-EGFP, His-TAT(-)-EGFP, His-EGFP-TAT and His-EGFP-TAT(-), which named protein trunsductive domain can take along giant molecular protein to go through cellular membrane in vivo an in vitro. It shows that different fusion protein has different transmembrane capacity, the TAT(-) sequence and in C terminal has stronger transmembrane capacity. It constructs a technique flat-top for exploitation new drug
引文
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a) (ZHOU Guo-Yu, ZHOU Shcng-Nian, LOU Zhi-Yin,etal Prokaryotic Expression, Purification of Ngb Fusion Protein Containing Protein Transduction Domain and Its Biologic Activity. [J] Progress in Biochemistry and Biophysics.2007, (34)6:634-641).
19严世荣等,闫莹,孙军,等。TAT-EGFP融合蛋白对小鼠生物膜穿透性的研究。[J]药物生物技术,2007,14(5)：324-327。
a) (YAN Shi-rong, YAN Ying, SUN Jun, etal. Study on the TAT-EGFP Fusion Protein Penetrability into Biomem-branes in Mice. [J] Pharmaceutical Biotechnology 2007,
14(5):324-327.
20 Popiel H A, Nagai Y, Fujikake N, et al. Protein transduction domain-mediated delivery of QBP1 supresses poleglutamine-induced neurodegeneration in vivo.[J] Mol Ther,2007, 15(2):303-309.
21 Vives E, Granier C, Prevot P and Lebleu B Structure-activity relationship study of the plasma membrane translocating potential of a short peptide from HIV-1 Tat protein. [J] Lett in Pept Sci 1997,4:429-436.
22 Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. [J] Proc Natl Acad Sci USA,2000,97:13003-13008.
1 Green M,Loewenstein PM.Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein.[J] Cell,1988 Dec 23; 55 (6): 1179-1188.
2 王怡,蔡邵晖等。一种新穿膜肽的构建、表达及活性检测[J]药物生物技术2007,14(2)：079-084。
3 宋金丹,杨恬,李丰。细胞膜的结构。[B]医学细胞生物学2004,051-063。
4 Schwarze SR, Hruska KA, Dowdy SF.Protein transduction:unrestricted delivery into all cells? [J] Trends Cell Biol.2000 Jul;10(7):290-295.
5 Frankel AD, Pabo CO. Cellular uptake of the TAT protein from immunodeficiency virus. [J] Cell,1998,55(8):1189.
6 Vives E, Brodin P, Lebleu B.A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.[J] J Biol Chem.1997 Jun 20;272(25):16010-16017.
7 杨永臣,袁崇刚。Tat蛋白及其内化作用。[J]生命的化学,2001,21(4)：265-268.
8 Derosai D, Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent.[J] J Biol Chem,1996,271(30):18188-18193
9 Suzuki T, Futaki S, Niwa M,et al.Possible existence of common internalization mechanis-m among arginine-rich peptides.[J] J Biol Chem,2002,277(4):2473-2483.
10 Consde S, Marty C, Garcia-Echeveria C,et al.Antenapedia and HIV ransactivator of transcription(TAT)"protein transductiondomain"promote endocytosis of high mdecular weight cargo upon binding to cell surafce glycosaminoglycans.[J] J Biol Cehm.2003,278 (37):35109-35114.
11 Schwarze SR, Ho A, Vocero2 Akbani A, et al. In vivo protein transduction:delivery of a biologically active protein into the mouse.[J] Science,1999,285(5433):1569-1572.
12梁英民,孙强。TAT蛋白转导结构域介导的BCR/ABL融合蛋白在小鼠体内的跨膜转运。[J]第三军医大学学报,2002,24(4)：421-424。
13 Dietz G P, Bahr M. Delivery of bioactive molecules into the cell:the Trojan horse approach.[J] Mol Cell Neurosi,2004,27(2):85-131.
14 Aarts M, Liu Y, Liu L, et al. Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions.[J] Science,2002,298(5594):846-850.
15 Gong B, Cao Z, Zheng P, et al.Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual momery.[J] Cell,2006,126(4):775-788.
16 Fu A L, Li Q, Dong Z H, et al. Alternative therapy of Alzheimer's disease via supplementation with choline acetyltransferase.[J] Neurosci Leu,2004,368(3):258-262.
17 Fu A L, Huang S J, Sun M J. Complementary remedy of aged-related learning and memory deficits via exogenous choline acetyltransferase.[J] Biochem Biophys Res Common,2005,336(1):268-273.
18 Wu S P, Fu A L, Wang Y X, et al. A novel therapeutic approach to 6-OHDA-induced Parkinson's disease in rats via supplementation of PTD-conjugated tyrosine hydroxylase. [J] B iochem B iophys Res Common,2006,346(1):1-6.
19 Fu A L, Wu S P, Dong Z H, et al. A novel therapeutic approach to depression via supplement with tyrosine hydroxylase.[J] Biochem Biophys Res Common,2006, 351(1):140-145.
20 McConnell K W, Muenzer J T, Chang K C, et al. Anti-apoptotic peptides protect against radiation-induced cell death.[J] Biochem Biophys Res Common,2007,355(2):501-507.
21 Yin W, Cao G, Johnnides M J, et al. TAT-mediated delivery of Bcl-cl protein is neuroprotective against neonatal hypoxic-ischemic brain injury via inhibition of Caspases and AIF.[J] Neurobiol Dis,2006, (2):358-371.
22 Popiel H A, Nagai Y, Fujikake N, et al. Protein transduction domain-mediated delivery of QBP1 supresses poleglutamine-induced neurodegeneration in vivo.[J] Mol Ther,2007, 15(2):303-309.
23 Temsamani J, Bonnafous C, Rousselle C, et al. Improved brain uptake and pharmacological activity profile of morphine-6-glucuronide using a peptide vector-mediated strategy. [J] J pharmacol Exp Ther,2005,313(2):712-719.
24 Pizzi M, Sarnico I, Boroni F, et al, NF-kappaB factor c-Rel mediates neuroprotection elicited by mGlu5 receptor agonists against amyloid beta-peptide toxicity. [J] Cell Death Differ,2005,12(7):761-772.
25 Chang W P, Koelsch G, Wong S, et al. In vivo inhibition of Abeta production by memapsin 2(beta-secretase) inhibitors. [J] J Neurochem,2004,89(6):1409-1416.
26 Dietz G P, Valbuena P C, Dietz B. Application of a blood-brain-barrier-penetrating form of GDNF in a mouse model for Parkinson's disease. [J] Brain Res,2006,1082(1):61-66.
27 Kilic U, Kilic E, Dietz G P, et al. Intravenous TAT-GDNF is protective after focal cerebral ischemia in mice.[J] Stroke,2003,34(5):1304-1310.
28 Kim D W, Eurn W S, Jang S H, et al. Transduced Tat-SOD fusion protein protects against ischemic brain injury. [J] Mol Cells,2005,19(1):88-96.
29 Choi S H, Kim D W, Kim S Y, et al. Transduced human copper chaperone for Cu, Zn-SOD(PEP-1-CCS) protects against neuronal cell death.[J] Mol Cells,2005,20(3): 401-408.
30 Eum W S, Kim D W, Hwang I K, et al. In vivo protein transduction:biologically active intact pep-1-superoxide dismutase fusion protein efficiently protects against ischemic insult. [J] Free Radic Biol Med,2004,37(10):1656-1669.
31 Choi H S. An J J. Kim S Y. et al. PEP-1-SOD fusion protein efficiently protects against paraquat-induced dopaminergic neuron damage in a Parkinson disease mouse model.[J] Free Radic Biol Med,2006.41(7):1058-1068.
32 Choi H S. Lee S H. Kim S Y. et al. Transduced Tat-alpha-synuclein protects against oxidative stress in vitro and in vivo.[J] J Biochem Mol Biol.2006,39(3):253-262.
33 Rousselle C. Smirnova M. Clair P. et al. Enhanced delivery of doxorubicin into the brain via a peptide-vector-mediated strategy:saturation kinetics and specificity.[J] J Pharmacol Exp Ther.2001,296(1):124-131.
34 Rousselle C. Clair P. Temsamani J, et al. Improved brain delivery of benzylpenicillin with a peptide-vector-mediated strategy. [J] J Drug Target,2002,10(4):309-315.
35 Rousselle C. Clair P. Smirnova M, et al. Improved brain uptake and pharmacological activity of dalargin using a peptide-vector-mediated strategy. [J] J Pharmacol Exp Ther, 2003.306(1):371-376.
36 Lewin M et al. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. [J] Nat Biotechnol,2000,18(4):410-414.
37 Torchilin VP et al. TAT-liposomes:a novel intracellular drug carrier. [J] Curr Protein Pept Sci,2003.4(2):133-134.
38 Jin LH et al. Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells. [J] Free Radic Biol Med,2001, 31(11):1509-1519.
39 Park J et al.9-polylysine protein transduction domain:enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin [J] Mol Cells.2002.13(2):202-208.
40 Lim JM et al. Penetration enhancement in mouse skin and lipolysis in adipocytes by TAT-GKH. a new cosmetic ingredient [J] J Cosmet Sei.2003.54(5):483-491.
41 Angdisen J et al. Mitochondrial trifunctional protein defects:molecular basis and novel therapeutic approaches.[J] Curr Drug Targets Immune Endocr Metabol Disord,2005.5(1): 27-40.
42 Alexeyev MF et al. alpha-complementation-enabled T7 expression vectors and their use for the expression of recombinant polypeptides for protein transduction experiments.[J] Methods Mol Biol 2004.267:91-100.
43 Shokolenko IN, Alexeyev MF. LeDoux SP, Wilson GL. TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells. [J] DNA Repair (Amst).2005 Apr 4:4(4):511-8. Epub 2005 Jan 13.
44 Hashida H et al. Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool. [J] Br J Cancer.2004,90(6):1252-1258.
45梁英民等。带有蛋白转导结构域的Bcr/Abl癌蛋白片段的表达及其跨膜转运。[J]中国生物化学与分生物学报,2001,17(5)：574-578。
46严世荣等。pET28a-TAT-LacZ重组子的构建。[J]遗传,2003,25(2)：141-144。
2 Frankel AD, Pabo CO.Cellular uptake of the tat protein from human immunodeficiency virus.[J] Cell.1988 Dec 23;55(6):1189-1193.
3 Fawell S, Seery J, Daikh Y, Moore C, Chen LL, Pepinsky B, Barsoum J.Tat-mediated delivery of heterologous proteins into cells.[J] Proc Natl Acad Sci U S A.1994 Jan 18;91(2):664-668.
4 Vives E, Brodin P, Lebleu B.A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. [J] J Biol Chem.1997 Jun 20;272(25):16010-16017.
5 Derossi D, Joliot AH, Chassaing G, Prochiantz A.The third helix of the Antennapedia homeodomain translocates through biological membranes.[J] J Biol Chem.1994 Apr 8;269(14):10444-10450.
6 Elliott G, O'Hare P. Intercellular trafficking and protein delivery by a herpesvirus structural protein.[J] Cell.1997 Jan 24;88(2):223-233.
7 Schwarze SR, Hruska KA, Dowdy SF.Protein transduction:unrestricted delivery into all cells?[J] Trends Cell Biol.2000 Jul;10(7):290-295.
8 McConnell K W, Muenzer J T, Chang K C, et al. Anti-apoptotic peptides protect against radiation-induced cell death[J] Biochem Biophys Res Common,2007,355(2):501-507.
9 Le Roux I, Joliot AH, Bloch-Gallego E, Prochiantz A, Volovitch M.Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties. [J] Proc Natl Acad Sci U S A.1993 Oct 1;90(19):9120-9124.
10 Eguchi A, Akuta T, Okuyama H, Senda T, Yokoi H, Inokuchi H, Fujita S, Hayakawa T, Takeda K, Hasegawa M, Nakanishi M Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells.[J] J Biol Chem.2001 Jul 13;276(28):26204-26210.
11 Victoria Del Gaizo Moore and R. Mark Payne.Transactivator of Transcription Fusion Protein Transduction Causes Membrane Inversion. [J] The Journal of Biological Chemistry,279,32541-32544.
12 Del Gaizo V, MacKenzie JA, Payne RM.Targeting proteins to mitochondria using TAT.[J] Mol Genet Metab.2003 Sep-Oct;80(1-2):170-180.
13 Eum W S, Choung 1 S, Li M Z, et al. HIV-1 Tat-mediated protein transduction of Cu, Zn-superoxide dismutase into pancreatic β-cells in vitro and in vivo[J] Free Radic Viol Med,2004,37(3):339.
14 Park J, Ryu J, Kim K A, et al. Mutational analysis of a human immunodeficiency virus type 1 Tat protein transduction domain which is required for delivery of an exogenous protein into mammalian cells. [J] J Gen Virol,2002,83(Pt 5):1173～1181.
15 Dodd C H, Hsu H C, Chu WJ, et al. Normal T-cell response and in vivo magnetic resonance imaging of T-cells loaded with HIV transactivator-peptide-derived superparamagnetic nanoparticles.[J] Immunol Methods,2001,256(1-2):89～105.
16 Rothbard J B,Jessop T C, Lewis R S, et al. Role of membrane potential and hydrogen binding in the mechanism of translocation of guanidinium-rich peptides into cells. [J] J Am Chem Soc,2004,126(31):9506-9507.
17 Shen H, Mai J C, Qiu L, et al. Evaluation of peptide-mediated transduction in human CD34+cell. [J] Hum Gcne Ther,2004,15(4):415～419.
18 周国钰,周盛年,楼之茵,等,含蛋白质转导域的Ngb融合蛋白的原核表达、纯化及其生物学活性的初步研究。[J]生物化学与生物物理学进展,2007,(34)6：634-641。
a) (ZHOU Guo-Yu, ZHOU Shcng-Nian, LOU Zhi-Yin,etal Prokaryotic Expression, Purification of Ngb Fusion Protein Containing Protein Transduction Domain and Its Biologic Activity. [J] Progress in Biochemistry and Biophysics.2007, (34)6:634-641).
19严世荣等,闫莹,孙军,等。TAT-EGFP融合蛋白对小鼠生物膜穿透性的研究。[J]药物生物技术,2007,14(5)：324-327。
a) (YAN Shi-rong, YAN Ying, SUN Jun, etal. Study on the TAT-EGFP Fusion Protein Penetrability into Biomem-branes in Mice. [J] Pharmaceutical Biotechnology 2007,
14(5):324-327.
20 Popiel H A, Nagai Y, Fujikake N, et al. Protein transduction domain-mediated delivery of QBP1 supresses poleglutamine-induced neurodegeneration in vivo.[J] Mol Ther,2007, 15(2):303-309.
21 Vives E, Granier C, Prevot P and Lebleu B Structure-activity relationship study of the plasma membrane translocating potential of a short peptide from HIV-1 Tat protein. [J] Lett in Pept Sci 1997,4:429-436.
22 Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. [J] Proc Natl Acad Sci USA,2000,97:13003-13008.
1 Green M,Loewenstein PM.Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein.[J] Cell,1988 Dec 23; 55 (6): 1179-1188.
2 王怡,蔡邵晖等。一种新穿膜肽的构建、表达及活性检测[J]药物生物技术2007,14(2)：079-084。
3 宋金丹,杨恬,李丰。细胞膜的结构。[B]医学细胞生物学2004,051-063。
4 Schwarze SR, Hruska KA, Dowdy SF.Protein transduction:unrestricted delivery into all cells? [J] Trends Cell Biol.2000 Jul;10(7):290-295.
5 Frankel AD, Pabo CO. Cellular uptake of the TAT protein from immunodeficiency virus. [J] Cell,1998,55(8):1189.
6 Vives E, Brodin P, Lebleu B.A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.[J] J Biol Chem.1997 Jun 20;272(25):16010-16017.
7 杨永臣,袁崇刚。Tat蛋白及其内化作用。[J]生命的化学,2001,21(4)：265-268.
8 Derosai D, Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent.[J] J Biol Chem,1996,271(30):18188-18193
9 Suzuki T, Futaki S, Niwa M,et al.Possible existence of common internalization mechanis-m among arginine-rich peptides.[J] J Biol Chem,2002,277(4):2473-2483.
10 Consde S, Marty C, Garcia-Echeveria C,et al.Antenapedia and HIV ransactivator of transcription(TAT)"protein transductiondomain"promote endocytosis of high mdecular weight cargo upon binding to cell surafce glycosaminoglycans.[J] J Biol Cehm.2003,278 (37):35109-35114.
11 Schwarze SR, Ho A, Vocero2 Akbani A, et al. In vivo protein transduction:delivery of a biologically active protein into the mouse.[J] Science,1999,285(5433):1569-1572.
12梁英民,孙强。TAT蛋白转导结构域介导的BCR/ABL融合蛋白在小鼠体内的跨膜转运。[J]第三军医大学学报,2002,24(4)：421-424。
13 Dietz G P, Bahr M. Delivery of bioactive molecules into the cell:the Trojan horse approach.[J] Mol Cell Neurosi,2004,27(2):85-131.
14 Aarts M, Liu Y, Liu L, et al. Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions.[J] Science,2002,298(5594):846-850.
15 Gong B, Cao Z, Zheng P, et al.Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual momery.[J] Cell,2006,126(4):775-788.
16 Fu A L, Li Q, Dong Z H, et al. Alternative therapy of Alzheimer's disease via supplementation with choline acetyltransferase.[J] Neurosci Leu,2004,368(3):258-262.
17 Fu A L, Huang S J, Sun M J. Complementary remedy of aged-related learning and memory deficits via exogenous choline acetyltransferase.[J] Biochem Biophys Res Common,2005,336(1):268-273.
18 Wu S P, Fu A L, Wang Y X, et al. A novel therapeutic approach to 6-OHDA-induced Parkinson's disease in rats via supplementation of PTD-conjugated tyrosine hydroxylase. [J] B iochem B iophys Res Common,2006,346(1):1-6.
19 Fu A L, Wu S P, Dong Z H, et al. A novel therapeutic approach to depression via supplement with tyrosine hydroxylase.[J] Biochem Biophys Res Common,2006, 351(1):140-145.
20 McConnell K W, Muenzer J T, Chang K C, et al. Anti-apoptotic peptides protect against radiation-induced cell death.[J] Biochem Biophys Res Common,2007,355(2):501-507.
21 Yin W, Cao G, Johnnides M J, et al. TAT-mediated delivery of Bcl-cl protein is neuroprotective against neonatal hypoxic-ischemic brain injury via inhibition of Caspases and AIF.[J] Neurobiol Dis,2006, (2):358-371.
22 Popiel H A, Nagai Y, Fujikake N, et al. Protein transduction domain-mediated delivery of QBP1 supresses poleglutamine-induced neurodegeneration in vivo.[J] Mol Ther,2007, 15(2):303-309.
23 Temsamani J, Bonnafous C, Rousselle C, et al. Improved brain uptake and pharmacological activity profile of morphine-6-glucuronide using a peptide vector-mediated strategy. [J] J pharmacol Exp Ther,2005,313(2):712-719.
24 Pizzi M, Sarnico I, Boroni F, et al, NF-kappaB factor c-Rel mediates neuroprotection elicited by mGlu5 receptor agonists against amyloid beta-peptide toxicity. [J] Cell Death Differ,2005,12(7):761-772.
25 Chang W P, Koelsch G, Wong S, et al. In vivo inhibition of Abeta production by memapsin 2(beta-secretase) inhibitors. [J] J Neurochem,2004,89(6):1409-1416.
26 Dietz G P, Valbuena P C, Dietz B. Application of a blood-brain-barrier-penetrating form of GDNF in a mouse model for Parkinson's disease. [J] Brain Res,2006,1082(1):61-66.
27 Kilic U, Kilic E, Dietz G P, et al. Intravenous TAT-GDNF is protective after focal cerebral ischemia in mice.[J] Stroke,2003,34(5):1304-1310.
28 Kim D W, Eurn W S, Jang S H, et al. Transduced Tat-SOD fusion protein protects against ischemic brain injury. [J] Mol Cells,2005,19(1):88-96.
29 Choi S H, Kim D W, Kim S Y, et al. Transduced human copper chaperone for Cu, Zn-SOD(PEP-1-CCS) protects against neuronal cell death.[J] Mol Cells,2005,20(3): 401-408.
30 Eum W S, Kim D W, Hwang I K, et al. In vivo protein transduction:biologically active intact pep-1-superoxide dismutase fusion protein efficiently protects against ischemic insult. [J] Free Radic Biol Med,2004,37(10):1656-1669.
31 Choi H S. An J J. Kim S Y. et al. PEP-1-SOD fusion protein efficiently protects against paraquat-induced dopaminergic neuron damage in a Parkinson disease mouse model.[J] Free Radic Biol Med,2006.41(7):1058-1068.
32 Choi H S. Lee S H. Kim S Y. et al. Transduced Tat-alpha-synuclein protects against oxidative stress in vitro and in vivo.[J] J Biochem Mol Biol.2006,39(3):253-262.
33 Rousselle C. Smirnova M. Clair P. et al. Enhanced delivery of doxorubicin into the brain via a peptide-vector-mediated strategy:saturation kinetics and specificity.[J] J Pharmacol Exp Ther.2001,296(1):124-131.
34 Rousselle C. Clair P. Temsamani J, et al. Improved brain delivery of benzylpenicillin with a peptide-vector-mediated strategy. [J] J Drug Target,2002,10(4):309-315.
35 Rousselle C. Clair P. Smirnova M, et al. Improved brain uptake and pharmacological activity of dalargin using a peptide-vector-mediated strategy. [J] J Pharmacol Exp Ther, 2003.306(1):371-376.
36 Lewin M et al. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. [J] Nat Biotechnol,2000,18(4):410-414.
37 Torchilin VP et al. TAT-liposomes:a novel intracellular drug carrier. [J] Curr Protein Pept Sci,2003.4(2):133-134.
38 Jin LH et al. Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells. [J] Free Radic Biol Med,2001, 31(11):1509-1519.
39 Park J et al.9-polylysine protein transduction domain:enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin [J] Mol Cells.2002.13(2):202-208.
40 Lim JM et al. Penetration enhancement in mouse skin and lipolysis in adipocytes by TAT-GKH. a new cosmetic ingredient [J] J Cosmet Sei.2003.54(5):483-491.
41 Angdisen J et al. Mitochondrial trifunctional protein defects:molecular basis and novel therapeutic approaches.[J] Curr Drug Targets Immune Endocr Metabol Disord,2005.5(1): 27-40.
42 Alexeyev MF et al. alpha-complementation-enabled T7 expression vectors and their use for the expression of recombinant polypeptides for protein transduction experiments.[J] Methods Mol Biol 2004.267:91-100.
43 Shokolenko IN, Alexeyev MF. LeDoux SP, Wilson GL. TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells. [J] DNA Repair (Amst).2005 Apr 4:4(4):511-8. Epub 2005 Jan 13.
44 Hashida H et al. Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool. [J] Br J Cancer.2004,90(6):1252-1258.
45梁英民等。带有蛋白转导结构域的Bcr/Abl癌蛋白片段的表达及其跨膜转运。[J]中国生物化学与分生物学报,2001,17(5)：574-578。
46严世荣等。pET28a-TAT-LacZ重组子的构建。[J]遗传,2003,25(2)：141-144。