TAT蛋白转导域介导BCR/ABL融合蛋白通过小鼠血脑屏障的作用

目的　探讨HIV - 1反式激活蛋白 (TAT)的蛋白转导结构域 (PTD)介导的BCR/ABL(TATPTD -BCR/ABL)融合蛋白通过血脑屏障的作用。方法　在大肠杆菌中表达PTD -BCR/ABL融合蛋白。将经Ni-NTA树脂亲和层析纯化的融合蛋白 ,经尾静脉注射小鼠体内 ,用免疫组织化学染色法 ,对小鼠脑组织细胞内的PTD -BCR/ABL融合蛋白进行检测。结果　①表达和纯化了相对分子质量 (Mr)为 2 3× 10 3 的PTD -BCR/ABL融合蛋白 ;②将其经静脉注射到小鼠体内 ,在小鼠脑组织细胞中可检测到PTD -BCR/ABL融合蛋白。结论　PTD可在体内介导较大Mr的融合蛋白通过血脑屏障 ,有望为治疗性的药物通过血脑屏障进入中枢神经系统提供了一种新的方法     

PTD—bcr／abl融合癌蛋白片段的表达及其跨膜转运

目的 探讨蛋白转导结构域（PTD）介导的转导bcr/abl癌蛋白片段的方法,为进行免疫治疗提供实验依据。方法 合成编码PTD的基因片段,并与PCR扩增的慢性粒细胞白血病癌蛋bcr/abl基因片段融合,在胸有成竹肠杆菌中表达,将纯化的融合蛋白PTD-bcr/abl与HL-60细胞共孵育,用Western blot分析融合蛋白的跨膜转运。结果 PTD基序可介导bcr/abl蛋白由细胞外跨膜转导进行细胞内。结论 这一结果可能为应用外源蛋白负载（loading)抗原提呈细胞（APC）提供新的途径。     

蛋白转导域介导BCR/ABL抗原对CML患者T细胞的活化作用

目的：研究蛋白转导域(PTD)介导的BCR／ABL抗原对慢性髓细胞白血病(CML)患者T细胞的特异性活化作用。方法：利用基因工程技术，将PTD基因与CML b3a2 bcr／abl基因融合并原核表达。将纯化的PTD—BCR／ABL融合蛋白与CML患者外周血单个核细胞(PBMC)体外共孵育，用流式细胞仪分别检测CD4^ 、CD8^ T细胞上活化抗原CD25的表达。结果：终浓度为100mg／L的PTD—BCR／ABL抗原体外刺激4d后，10例CML患者中，5例表现为CD8^ T细胞活化，2例表现为CD4^ T细胞活化，其中有1例CD8^ 和CD4^ T细胞同时活化；而作为对照的BCR／ABL抗原刺激组无一例表现为CD8^ 或CD4^ T细胞活化。结论：PTD能将外源性BCR／ABL抗原转导入抗原呈递细胞内，加工呈递后激活抗原特异性CD8^ 及CD4^ T细胞，为CML特异性CD8^ 、CD4^ T细胞的体外活化及细胞免疫治疗开辟一条新的途径。     

分泌型TAT蛋白转导结构域基因通用真核表达载体的构建及表达

目的：构建一个全新的分泌型TAT蛋白转导结构域基因哺乳动物细胞融合表达载体并对融合蛋白的表达进行分析。方法：依照编码TAT—PTD11肽及所引入的BamHⅠ酶识别序列和增强性绿色荧光蛋白N端7个氨基酸的碱基序列合成了3条引物，以egfp基因为模板通过依次3轮PCR扩增得到tat-egfp融合基因，该基因片段以sfiⅠ和HindⅢ双酶切后插入哺乳动物细胞表达载体pSectagA，构建成重组载体pSecTAT-m-egfp，BamHⅠ单酶切后该载体自连，即得到可通用的真核表达载体pSecTAT—m，重组质粒psecTAT-m-egfp转染HEK293细胞，融合蛋白的表达用Western blot分析。结果：酶切及测序证实表达载体pSecTAT—m构建成功，Western blot表明TAT—EGFP融合蛋白在HEK293细胞中获得表达并分泌到细胞培养液中。结论：成功地构建了分泌型TAT蛋白转导结构域基因的哺乳动物细胞融合表达载体，为更充分发挥TAT蛋白转导结构域在蛋白质功能及疾病治疗研究中的应用打下了基础。     

基于支持向量机预测蛋白质转导域的研究

目的 蛋白转导域（PTD）是一类能携带分子穿越细胞膜的短肽,利用支持向量机对多肽片段PTD进行预测.方法 对来源于SwissProt数据库的多肽序列用68个特征值描述其整体和局部的理化特性以及空间结构特征,利用支持向量机（SVM）和直推式支持向量机（TSVM）并结合聚类的方法进行PTD的预测.结果 5次交叉验证的结果显示,TSVM的预测准确率达到（94±4）%,SVM预测准确率达到（94±5）%.2种预测方法共同预测了1210个可能的PTD片段.结论 TSVM和SVM均显示了很好的预测性能,预测的PTD为实验方法有目的 地发现、确认PT提供了基础.     

蛋白质转导技术及其应用

蛋白质转导结构域(protein transduction domain,PTD)是指一类能将其他生物大分子导入多种哺乳动物细胞的小分子阳离子多肽,它可以用于研究细胞内某些蛋白质的功能,还可以与具有治疗作用的蛋白质、多肽等生物大分子偶联,在缺血、肿瘤等多种疾病的治疗中有潜在的应用价值。     

新型神经营养因子TAT-BDNF生物合成及生物活性研究

目的：合成新型神经营养因子TAT-BDNF并验证其生物活性，为进一步应用功能蛋白质治疗中枢神经损伤提供研究方法。方法：采用分子克隆方法构建带有TAT蛋白转导区序列及无信号肽序列人BDNF基因的原核表达载体pTAT—HA-DNF。经原核表达系统表达、纯化、复性获得纯净TAT—BDNF融合蛋白。利用体外培养的新生大鼠小脑颗粒细胞，通过双重免疫荧光细胞染色检测其蛋白转导活性；Hoechst33342染色观察TAT-BDNF对颗粒细胞谷氨酸兴奋性毒性损伤神经保护作用。结果：重组质粒能有效的通过原核表达系统表达并获得高纯度TAT-BDNF。免疫荧光细胞染色显示TAT-BDNF能快速转导入小脑颗粒细胞中，并能显著减少由谷氨酸诱导的细胞凋亡坏死的比例，改善神经元的存活状态。结论：合成的新型神经营养因子TAT-BDNF具有蛋白转导活性及神经保护作用。     

神经元保护蛋白TAT-Bcl-XL的体外高效表达及其功能的初步研究

目的：在原核表达系统中表达对凋亡神经元具有保护作用的重要蛋白Bcl-XL与蛋白质转导序列（PTD）的融合蛋白，并检测重组蛋白对重金属离子所诱导的细胞凋亡的保护作用。方法：通过RT-PCR的方法，用Bcl-XL特异引物从乳腺癌细胞系MCF-7细胞的总RNA中扩增出Bcl-XL基因，构建相应的原核表达载体，体外表达的TAT-Bcl-XL融合蛋白经镍亲和层析介质纯化后，通过免疫荧光方法检测其转导293T细胞的能力；并用流式细胞仪检测融合蛋白抑制细胞凋亡的能力。结果：经RT-PCR从MCF-7细胞总RNA中得到相应的TAT-Bcl-XL基因片段，并将其克隆入pCRT7/CT-TOPO载体中，重组质粒pTBTOPO转化大肠杆菌后，在SDS-PAGE和Western blot的结果中出现了与预期分子量相同的蛋白条带和阳性信号，纯化的TAT-Bcl-XL重组蛋白经免疫荧光检测，主要分布于细胞的细胞质中。流式细胞仪的检测结果显示融合表达蛋白可以有效地抑制Zn2＋离子所诱导的细胞凋亡，使细胞的存活率提高40%。结论：TAT-Bcl-XL融合蛋白在大肠杆菌中获得高效表达，初步的功能性检测表明融合蛋白具有抑制细胞凋亡的功能。     

重组人腺病毒表达的MDV-1 VP22蛋白的不同定位模式

目的:研究MDV-1 CVI988 VP22蛋白的转导机制及细胞定位机理, 分析该蛋白在表达过程中细胞定位的影响因素.方法:构建表达VP22蛋白的重组人腺病毒, 将重组病毒感染的AD-293细胞裂解产物加至正常的MDBK细胞上, 通过免疫荧光(IFA)及Western blot鉴定VP22的蛋白转导功能.观察感染后不同时间VP22在AD-293细胞上的定位, 并与瞬时表达的VP22蛋白定位比较.结果:重组人腺病毒表达的VP22在MDBK细胞上能够进入几乎所有的细胞, 说明VP22蛋白具有很强的蛋白转导功能.进一步鉴定VP22的细胞定位发现, 在重组病毒感染的AD-293细胞中, VP22首先聚集于细胞核周围, 随后以特殊的荧光粒子的形式散在于胞质中, 而AD-293细胞中瞬时表达的VP22及MDV感染的CEF中VP22均均匀分布于细胞核.结论:重组人腺病毒表达的VP22蛋白具有很强的蛋白转导功能, 不同重组病毒表达的VP22在细胞中的定位模式有所差别.     

细胞穿透肽PEP-1介导增强型绿色荧光蛋白在小鼠体内跨膜转导

目的 研究细胞穿透肽PEP-1介导的大分子物质在小鼠体内的跨膜转导能力.方法 用基因工程的方法制备并纯化增强型绿色荧光蛋白EGFP和PEP-1-EGFP融合蛋白,分别将500 μg的EGFP蛋白和PEP-1-EGFP融合蛋白通过尾静脉注射入昆明小鼠体内,2 h后麻醉小鼠,PBS充分灌流并取心、脑、肝、脾、肾快速冷冻切片后立即置荧光显微镜下观察.结果 2 h后PEP-1-EGFP融合蛋白处理的小鼠大脑、心肌、肝、脾和肾组织里出现均一的明亮绿色荧光,而EGFP蛋白处理的小鼠各脏器内均未见到绿色荧光.结论 细胞穿透肽PEP-1能携带增强型绿色荧光蛋白穿透小鼠细胞膜并分布于心、脑、肝、脾、肾组织内,为将来用PEP-1介导各种大分子药物跨膜转导进行各种疾病的蛋白治疗提供实验依据.     

Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68.     

细胞穿透肽转运机制、分子改造及相关免疫治疗

细胞穿透肽(cell penetrating peptides,CPPs)也称为蛋白转导结构域(protein transduction domain,PTD)是一类具有较强的穿膜活性的多肽,可以携带各种生物分子转运到细胞内,这一特点使其在药物转运系统及疾病治疗等领域有着广泛的研究,但是其穿膜转运机制尚不清楚。CPPs的穿膜过程可以分为3步,首先CPPs特殊的生化性质使其与细胞表面相互作用,形成空间优势,然后通过不同的方式进行跨膜转运,之后在CPPs在细胞内有不同的转运路径与定位。这种特性使其在免疫治疗方面有广阔的应用前景。本文就其运转过程的研究做一概括总结。随着研究不断深入,需要对传统的PTD进行改建、开发或者构建新的仿CPPs分子达到提高其运转效率、细胞内特异性定位及靶向不同种类细胞的目的,优化CPPs运转系统,为疫苗设计提供新的思路,并满足基础研究的需要。     

Dendritic cells transduced with TAT protein transduction domain-containing tyrosinase-related protein 2 vaccinate against murine melanoma

Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (TAT PTD) into proteins facilitates rapid, efficient entry into cells. We previously showed that rTAT-PTD-OVA-transduced dendritic cells (DC) stimulated antigen (Ag)-specific CTL and Th cells, and vaccinated against OVA-expressing tumors. Here we studied B16 melanoma in C57BL/6 mice, using murine tyrosinase-related protein 2 (Trp2) as a candidate tumor Ag. We produced a 472-amino acid N-terminal fragment of Trp2 protein (rTrp2Delta) with or without PTD. Although PTD-deficient rTrp2Delta was ineffective, mice given rTAT-PTD-Trp2Delta-transduced DC were efficiently primed for Trp2(180-188) peptide-specific and B16-reactive CTL. In 58% of such mice, growth of melanomas was prevented. Trp2(180-188) peptide-pulsed DC protected 35% of recipients, and irradiated GM-CSF-producing B16 cells protected 75%. rTAT-PTD-Trp2Delta-transduced DC induced a more vigorous memory response to B16 rechallenge than the other regimens, and protected 30% of recipients from progressive tumor development in treatment studies. In this setting, Trp2 peptide-treated DC protected 20% and irradiated GM-CSF-producing cells protected 0%. Both tumor prevention and tumor treatment were CD8(+) T cell dependent. Vaccination with rTAT-PTD-Trp2Delta-transduced DC induced a robust CTL response and durable anti-melanoma immunity. This approach should be clinically applicable, and offers theoretical and practical advantages to those that are in current use.     

Protein transduction domains and their utility in gene therapy

Protein transduction domains (PTDs, sometimes termed cell permeable proteins (CPP) or membrane translocating sequences (MTS)) are small peptides that are able to ferry much larger molecules into cells independent of classical endocytosis. This property makes PTDs ideal tools to transfer proteins and other molecules into living cells for research purposes. The mechanism by which this internalization takes place is poorly understood. It is evident, however, that many known PTDs bind to the same surface molecules (Heparan Sulphate Proteoglycans, HSPG) before internalization, and that internalization is dependent on these molecules. PTDs, although at this moment mainly used for the chemical or bacterial production of membrane permeable proteins can become powerful tools for gene therapy. By incorporating a PTD in the therapeutic gene product, the protein produced in the transfected cell might be enabled to spread to non-transfected cells, thereby creating an increased therapeutic effect. In this review, we give an overview of PTDs that may be useful for gene therapy applications, and discuss some of the problems that can be expected when incorporating PTDs in gene therapy approaches.     

Delivery of the aggregate inhibitor peptide QBP1 into the mouse brain using PTDs and its therapeutic effect on polyglutamine disease mice

The polyglutamine (polyQ) diseases are neurodegenerative diseases caused by proteins with an abnormally expanded polyQ stretch, which triggers abnormal aggregation of these proteins in the brain. We previously showed that the polyQ-binding peptide QBP1 inhibits polyQ aggregation, and further that administration of QBP1 fused with a protein transduction domain (PTD) suppresses polyQ-induced neurodegeneration in Drosophila. As the next step towards developing a therapy using QBP1, we investigated the delivery of PTD-QBP1 to the mouse brain upon its administration. Here we successfully detected delivery of PTD-QBP1 into mouse brain cells upon its single intracerebroventricular injection. In addition, long-term administration of PTD-QBP1 to polyQ disease mice improved their weight loss phenotype, suggesting a possible therapeutic effect. Our study indicates the potential of PTD-mediated delivery of QBP1 as a therapeutic strategy for the currently untreatable polyQ diseases.