目的：构建穿膜肽TAT与EGFP融合蛋白的原核表达系统，研究该融合蛋白对人成熟精子的穿膜作用和对精子运动参数的影响。方法以pEGFP-N1载体为模板，设计N端和C端含有TAT序列的引物，用PCR技术扩增TAT-EGFP和EGFP-TAT基因，扩增产物插入载体 pET28a，构建成重组质粒pET28a-TAT-EGFP和pET28a-EGFP-TAT。将重组质粒转入大肠杆菌DE3中，IPTG诱导TAT-EGFP和EGFP-TAT融合蛋白表达。表达产物经Ni-NTA亲和层析柱纯化后SDS-PAGE电泳鉴定。将融合蛋白TAT-EGFP和EGFP-TAT分别加入正常人精子中孵育，荧光显微镜观察融合蛋白TAT-EGFP和EGFP-TAT穿膜情况。结果成功构建了高表达重组质粒pET28a-TAT-EGFP和pET28a-EGFP-TAT，纯化了分子质量约为32 kD的融合蛋白 TAT-EGFP和EGFP-TAT。融合蛋白TAT-EGFP和EGFP-TAT在正常人成熟精子中有穿膜作用。不同浓度的融合蛋白TAT-EGFP和EGFP-TAT对正常人成熟精子存活率及运动参数无明显影响。结论通过对融合蛋白TAT-EGFP和EGFP-TAT表达纯化及活性分析，证实穿膜肽TAT在精子中的跨膜转运作用，为将来精子研究提供了实验基础。

Objective To construct an prokaryotic expression system of cell-penptrating peptides(TAT)-enhanced green fluorescent protein fusion protein,and investigate its transmembrane effect on human mature sperm and the influence of sperm motility parametersin vitro.Methods Using pEGFP-N1 carrier as a template, the primers that contained TAT sequence of N-terminal and C-terminal respectively were designed. TAT-EGFP and EGFP-TAT gene were amplified by PCR,and its products were inserted into pET28a vector to construct recombinant plasmids pET28a-TAT-EGFP and pET28a-EGFP-TAT. The recombinant vectors were transformed into E.coli DE3.The fusion protein TAT-EGFP and EGFP-TAT were induced by IPTG. The expressed fusion proteins were purified by Ni-NTA affinity chromatography column and identified by SDS-PAGE electrophoresis. fusion proteins were added into human sperm in vitro toobserve transmembrane effect on human mature sperm.Results The high expression pET28a-TAT-EGFP and pET28a-EGFP-TAT

目的制备双功能RGD-TAT肽修饰的脂质体(RGD-TAT peptide modified liposomes,RGD-TAT-LPs),并对其脑胶质瘤靶向性进行研究。方法采用薄膜分散法制备双功能RGD-TAT-LPs并进行表征;细胞摄取实验研究脑胶质瘤C6细胞对普通脂质体(liposomes,LPs)、RGD修饰脂质体(RGD-LPs)、TAT修饰脂质体(TAT-LPs)和RGD-TAT-LPs的摄取效率。构建脑胶质瘤原位肿瘤模型,研究不同脂质体在荷瘤裸鼠的体内分布。结果RGD-TAT-LPs的粒径为(116.5±11.3)nm,电位为(23.2±3.5)mV。细胞摄取实验结果显示:C6细胞对RGD-TAT-LPs的摄取效率分别是LPs、TAT-LPs和RGD-LPs的4.7倍、2.3倍和2.9倍,差异有统计学意义(P0.01)。脂质体体内分布实验结果显示RGD-TAT修饰脂质体组荷瘤小鼠脑部荧光最强。结论RGD-TAT-LPs是一种潜在高效的肿瘤靶向给药系统。

Objective To construct RGD-TAT modified liposomes(RGD-TAT-LPs)and evaluate its glioma targeting efficiency.Methods RGD-TAT-LPs was constructed by film-ultrasonic method,its appearance,particle size and Zeta potential were mearsured. Cellular uptake of LPs,TAT-LPs, RGD-LPs and RGD-TAT-LPs was used to evaluate the affinity to C6 cells.C6 cells were xenografted in athymic mice to establish the animal model,which were used to evaluate the distribution of liposomes in vivo. Results The particle diameter of RGD-TAT-LPs was (1 16.5 ±1 1.3 )nm,and its Zeta potential was (23.2 ±3.5 )mV. Cellular uptake experiments demonstrated the cell uptake efficiency of RGD-TAT-LPs by C6 cells were 2.9-fold,2.3-fold and 4.7-fold than that of RGD-LPs,TAT-LPs and LPs respectively. The in vivo imaging showed that RGD-TAT-LPs had the strongest fluorescence intensity in brain. Conclusion The RGD-TAT-LPs might serve as a promising delivery system of antitumor drugs.

借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体(LacI HPM)高亲和力结合DNA的特性,建立一种安全高效、无基因插入片段大小限制的基因转导系统。方法:在TAT-LacI HPM片段C端和N端分别添加GST标签,构建pET-28a(+)-TAT-LacI HPM-GST和pGEX-GST-TAT-LacI HPM重组表达载体,可溶性表达TAT-LacI HPM-GST及GST-TAT-LacI HPM融合蛋白并纯化,获得TAT-LacI HPM二聚体,免疫荧光检测TAT-LacI HPM融合蛋白穿过HeLa细胞膜的情况,观察EGFP的表达,用免疫印迹检测TAT-LacI HPM融合蛋白介导质粒DNA进入细胞的能力。结果:表达、纯化并获得二聚体融合蛋白,体内实验表明其具有跨膜能力,能介导带有LacI结合序列的DNA质粒进入细胞,并在转染细胞里检测到了目的蛋白。结论:初步证实TAT-LacI HPM融合蛋白作为一种新型通用性非病毒DNA转运载体的可行性,为评价这种新型DNA疫苗载体在提高免疫效果方面的可行性奠定了前期实验基础。

Objective: To establish a novel DNA delivery system which is safe, high efficient and no limitation of inserting DNA fragments, by using cell-penetrating peptides TAT with the ability of high efficient cellular pene-tration and LacI to specifically bind to its recognizing DNA sequence with high affinity. Methods: GST-tag was inserted into either C- or N-terminal of TAT-LacI HPM(LacI headpiece mutant) fragment to obtain the expres-sion plasmids. TAT-LacI HPM fusion proteins expressed in E.coli were purified by glutathione Sepharose 4B beads, and were dialyzed against buffer to form the TAT-LacI HPM dimers. Whether the transduced dimeric TAT-LacI HPM fusion proteins could penetrate in cells was tested by immunofluorescence, and the ability of TAT-LacI HPM dimers to deliver DNA into the cells were examined by observing the expression of the reporter EGFP gene and by Western blotting. Results: The expression plasmids of pET-28a(+)-TAT-LacI HPM-GST and pGEX-GST-TAT-LacI HPM were obtained, a

目的 观察Tat-Smac N7融合肽对人肺癌细胞系H460和人食管癌细胞系EC109的辐射增敏作用,探讨其辐射增敏机制.方法 取对数生长期H460和EC109细胞,分为DAPI对照组、FITC-Smac N7和FITC-Tat-Smac N7组,荧光显微镜观察两种细胞不同时间药物入核情况.取对数生长期H460和EC109细胞,分为单纯照射组、照射联合Tat-Smac N7组,单纯照射组给予0、2、4、6 Gy照射,照射联合Tat-Smac N7组中Tat-Smac N7的浓度为20 μmol/L,WST-1测定Tat-Smac N7的辐射增敏作用.取对数生长期H460和EC109细胞,分为对照组、Tat-Smac N7组、单纯照射组和照射联合Tat-Smac N7组,吸收剂量为4 Gy,Tat-Smac N7浓度为20 μmol/L,细胞流式分析仪测定细胞不同时间的细胞凋亡率.结果 Tat-Smac N7融合蛋白进入2种细胞系后2h可以有蓄积,且这种蓄积可延续到24 h,而Smac N7则不能进入细胞.Tat-Smac N7能够增强H460和EC109细胞的辐射敏感性(F=22.2、13.2,P＜0.05),照射联合Tat-Smac N7可明显增加辐射诱导的细胞凋亡率(24 h:F=9.32、5.86,P＜0.05;48 h:F =7.09、8.25,P＜0.05).Tat-Smac N7联合照射后凋亡诱导效应具有时间依赖性.结论 Tat-Smac N7融合肽可促进肿瘤细胞的辐射敏感性,作为一种新的Smac蛋白类似物,有望用于肿瘤的辐射增敏治疗.

Objective To observe the radiosensitization effect of Tat-Smac N7 fusion peptide in EC109 and H460 cell lines,and to explore the mechanism of Tat-Smac N7 in radio sensitization.Methods H460 and EC109 cells were divided into DAPI group,and FITC-Smac N7 group,FITC-Tat-Smac N7 group.Fluorescence microscope was used to detect if the peptide had been entered into tumor cells at deferent times.H460 and EC109 cells were divided into radiation group and Tat-Smac N7 combined with radiation group.The cells were irradiated with 4 Gy γ-ray and the concentration of Tat-Smac N7 was 20 μmol/L.The proliferation of tumor cells was detected by WST-1 assay.Among control group,radiation group,Tat-Smac N7 group and Tat-Smac N7 combined with radiation group,apoptosis was detected by flow cytometry.Results Tat-Smac N7 could enter cells for 2-24 h,but Smac N7 couldn''t.Tat-Smac N7 promoted the radio sensitization of H460 and EC109 cells obviously (F =22.2,13.2,P ＜ 0.05).The apoptosis of combined

借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体(LacI HPM)高亲和力结合DNA的特性,构建新型基因转导载体。方法:PCR扩增LacI、LacI基因前头肽序列、前头肽序列突变体、TAT序列的编码基因,构建前头肽序列突变体和TAT的原核表达载体,可溶性表达TAT-LacI HPM融合蛋白并纯化,在缓冲液中氧化获得TATLacI HPM二聚体并浓缩,PCR检测二聚体融合蛋白与质粒的体外结合能力。结果:获得了pET-28a(+)-LacI HPM及pET-28a(+)-TAT-LacI HPM表达质粒,表达纯化并获得二聚化融合蛋白,体外结合实验确定TAT-LacI HPM二聚体融合蛋白与检测质粒DNA具有特异的高亲和力结合活性。结论:构建了穿膜肽TAT-LacI HPM,为进一步研究其作为新型DNA转运载体的可行性奠定了基础。

Objective: To construct the novel DNA delivery vectors by using cell-penetrating peptides with the ability of high efficient cellular penetration and LacI to specifically bind to its recognizing DNA sequence with high affinity. Methods: The DNA sequence encoding the LacI, LacI HP(headpiece), LacI HPM(headpiece mu-tant) and TAT were firstly amplified by PCR respectively, then the expression plasmids of TAT-LacI HPM were cloned into pET-28a(+) vectors. Fusion proteins expressed in E.coli were purified by Ni-NTA beads, and were di-alyzed against buffer to form the TAT-LacI HPM dimers, then the dimers were concentrated by PEG-8000. DNA-binding activities were examined. Results: The expression plasmids of pET-28a(+)-LacI HPM and pET-28a (+)-TAT-LacI HPM were obtained, after expression and purification, the TAT-LacI HPM dimers were concentrat-ed, and the results showed that TAT-LacI HPM dimers have high affinity with its recognizing DNA sequences. Conclusion: The TAT-LacI HPM fusion proteins