99mTc标记丝状噬菌体肽库及其在正常小鼠体内的分布

利用噬菌体展示技术已选出了多条与靶结合的肽.然而,即使是体内直接筛选得到的,肽与肿瘤或靶器官的体内结合并不理想.为了更好地理解噬菌体在体内的循环,通过MAG399mTc标记噬菌体肽库,研究了肽库在体内分布.体内分布实验结果显示,99mTc-噬菌体主要分布在肝和脾中,而心脏、肌肉、脑和胰腺这些器官或组织中的分布非常低.99mTc-噬菌体在胃、肠和骨中的累积,随着时间延长在不断升高,其他器官中的吸收则在不断降低.从5min到30min,99mTc-噬菌体在血中清除迅速.当噬菌体在体内循环足够长的时间后,一些噬菌体颗粒可以穿透血管进入并内化在器官或组织中.总之,为了筛选具有高特异性和亲和性的肽,应该根据靶器官和筛选部位的不同,在筛选前确定合适的噬菌体在体内的循环时间.     

Cd2+结合肽的筛选及与重金属离子的亲和作用*

利用噬菌体随机十二肽库和亲和层析技术对重金属Cd进行亲和筛选,共获得两条Cd结合肽序列。将展示有Cd²⁺结合肽的噬菌体单克隆扩增物对不同重金属离子(Cd²⁺、Cr²⁺、Cu²⁺、Co²⁺、Zn²⁺、Ni²⁺)螯合的树脂进行亲和测定,结果表明Cu²⁺、Co²⁺、Zn²⁺、Ni^2+<     

随机十肽库的构建及血管形成素结合肽的筛选

将合成的含有随机序列（NNK）₁₀的寡核苷酸片段,克隆入噬菌体呈现载体噬菌粒pCANTAB5E的SfiⅠ,NotⅠ位点,即cpⅢ蛋白信号肽与成熟肽之间,电转化E.coli TG1,构建了噬菌体表面呈现的十肽库,实际库容为3.53×10⁷,插入率为66.7%.经辅助噬菌体M13KO7超感染后,获得滴度为4.8 ×10¹¹ pfu/ml的噬菌体上清.经过两轮panning筛选和富集,从构建的随机十肽库中筛选到26个具有血管形成素结合活性的重组噬菌体克隆,对其中12个阳性噬菌体克隆的短肽序列进行了分析,ELISA检测结果显示12个阳性噬菌体克隆都能够与血管形成素特异性结合.     

短肽蝎毒素的结构分类与功能特征

大量的资料已证实蝎毒中主要致死成分是一类由60～70个残基组成, 选择性地作用于电压门控Na⁺通道的长肽毒素.另一类由30～40个残基组成的短肽蝎毒素,由于其具有结构致密,易于合成改造的优点,特别是具有选择性地阻遏K⁺或Cl^－通道的特异药理功效,近年来倍受学术界的关注,并在结构与功能方面取得了很大的研究进展.     

噬菌体展示技术体内筛选的研究进展

噬菌体展示技术体内筛选是直接将噬菌体肽库注射到动物体内,筛选与活体内某些器官或组织有特异结合活性的小肽。噬菌体展示技术的体内筛选在血管靶向肽的筛选、肿瘤组织靶向肽的筛选、免疫反应的研究和相关疾病治疗、监测方面都有广泛的应用前景。     

HLA-A*2402-BSP在大肠杆菌中的优化表达及其四聚体的制备和鉴定

HLA-A^*2402是中国人群中最常见的等位基因之一,为研究该基因型人群的人巨细胞病毒（HCMV）特异性细胞毒T细胞（CTL）免疫应答,需要制备负载相应抗原肽的HLA-A^*2402四聚体。以RT-PCR方法克隆HLA-A^*2402重链基因的cDNA,并构建了羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A^*2402重链胞外域融合蛋白（HLA-A^*2402-BSP）的表达载体,但该载体不能在大肠杆菌(E. coli)中有效表达HLA-A^*2402-BSP融合蛋白;通过对氨基端（N端）区域编码区的密码子进行优化,构建了同义突变的HLA-A^*2402-BSP表达载体,融合蛋白在E. coli中获得了高效表达。进而制备了负载HLA-A^*2402限制性HCMV pp65_341-349抗原肽(QYDPVAALF, QYD)的可溶性HLA-A^*2402-QYD单体分子和四聚体,获得的四聚体具有与HLA-A24⁺供者抗原特异性CTL的结合活性,特异性CTL的频率为总CD8⁺T细胞的0.09％～0.37％。这些结果为进一步研究HLA-A^*2402限制性的特异性CTL免疫应答规律奠定基础。     

HLA-A*2402-BSP在大肠杆菌中的优化表达及其四聚体的制备和鉴定

HLA-A^*2402是中国人群中最常见的等位基因之一,为研究该基因型人群的人巨细胞病毒（HCMV）特异性细胞毒T细胞（CTL）免疫应答,需要制备负载相应抗原肽的HLA-A^*2402四聚体。以RT-PCR方法克隆HLA-A^*2402重链基因的cDNA,并构建了羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A^*2402重链胞外域融合蛋白（HLA-A^*2402-BSP）的表达载体,但该载体不能在大肠杆菌(E. coli)中有效表达HLA-A^*2402-BSP融合蛋白;通过对氨基端（N端）区域编码区的密码子进行优化,构建了同义突变的HLA-A^*2402-BSP表达载体,融合蛋白在E. coli中获得了高效表达。进而制备了负载HLA-A^*2402限制性HCMV pp65_341-349抗原肽(QYDPVAALF, QYD)的可溶性HLA-A^*2402-QYD单体分子和四聚体,获得的四聚体具有与HLA-A24⁺供者抗原特异性CTL的结合活性,特异性CTL的频率为总CD8⁺T细胞的0.09％～0.37％。这些结果为进一步研究HLA-A^*2402限制性的特异性CTL免疫应答规律奠定基础。     

夏季台湾海峡南部海域上层水体的生物固氮作用

2011年6-7月,利用¹⁵N₂示踪法实测了台湾海峡南部海域的生物固氮速率,结合温度、盐度、天然颗粒物氮同位素组成的分布,分析并讨论了影响研究海域生物固氮速率的环境因素。结果表明,夏季台湾海峡南部海域的生物固氮速率介于 168-1080 nmol m^-3 d^-1之间,平均为537 nmol m^-3 d^-1,较高的生物固氮速率大多出现在次表层水体中。研究站位的积分固氮速率变化范围为11-40 μmol m^-2 d^-1,平均为23 μmol m^-2 d^-1。积分固氮速率的空间变化与不同水团的影响有关,在受黑潮水影响的海域,生物固氮速率较高,而在上升流和受河流冲淡水影响的海域,生物固氮速率较低,说明较低的水温及较高的无机氮营养盐可能会抑制研究海域的生物固氮作用。研究海域天然颗粒物δ¹⁵N与生物固氮速率之间呈现良好的负相关关系,表明天然颗粒物氮同位素组成可定性指征研究海域生物固氮作用的强弱。     

m6A修饰在植物生长发育和抗逆境胁迫中的功能

真核生物mRNA存在多种甲基化修饰,其中N⁶-腺苷酸甲基化(N⁶-methyladenosine, m⁶A)修饰是最为常见的一种动态内部修饰。m⁶A是指RNA腺嘌呤的第6位氮原子上发生甲基化修饰,它能够动态的被甲基转移酶添加,被去甲基化酶去除,以及被甲基化阅读蛋白识别。近年来,植物m⁶A修饰相关的酶被陆续鉴定,研究发现m⁶A修饰调控植物胚胎发育、茎尖分生组织分化、开花等生长发育过程,在植物抗逆境胁迫响应中也具有重要调控作用。本文就m⁶A修饰相关酶的组成及其在植物生长发育和植物抗逆境胁迫过程中的功能相关研究进展进行综述,并对甘蓝型油菜中m⁶A修饰相关的酶进行了生物信息学分析。     

基于双层BiGRU网络的哺乳动物组织m6A甲基化位点预测

目的 N⁶-甲基化腺苷（N⁶-methyladenosine，m⁶A）是RNA中最常见、最丰富的化学修饰，在很多生物过程中发挥着重要作用。目前已经发展了一些预测m⁶A甲基化位点的计算方法。然而，这些方法在针对不同物种或不同组织时，缺乏稳健性。为了提升对不同组织中m⁶A甲基化位点预测的稳健性，本文提出一种能结合序列反向信息来提取数据更高级特征的双层双向门控循环单元（bidirectional gated recurrent unit，BiGRU）网络模型。方法 本文选取具有代表性的哺乳动物组织m⁶A甲基化位点数据集作为训练数据，通过对模型网络、网络结构、层数和优化器等进行搭配，构建双层BiGRU网络。结果 将模型应用于人类、小鼠和大鼠共11个组织的m⁶A甲基化位点预测上，并与其他方法在这11个组织上的预测能力进行了全面的比较。结果表明，本文构建的模型平均预测接受者操作特征曲线下面积（area under the receiver operating characteristic curve，AUC）达到93.72%，与目前最好的预测方法持平，而预测准确率（accuracy，ACC）、敏感性（sensitivity，SN）、特异性（specificity，SP）和马修斯相关系数（Matthews correlation coefficient，MCC）分别为90.07%、90.30%、89.84%和80.17%，均高于目前的m⁶A甲基化位点预测方法。结论 和已有研究方法相比，本文方法对11个哺乳动物组织的m⁶A甲基化位点的预测准确性均达到最高，说明本文方法具有较好的泛化能力。     

Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of <Superscript>99m</Superscript>Tc-labeled recombinant Affibody molecules

Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide ^99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of ^99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied ^99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of ^99mTc. The biodistribution of all ^99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of ^99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.     

99mTc-labeled monomeric and dimeric NGR peptides for SPECT imaging of CD13 receptor in tumor-bearing mice

CD13 receptor plays a critical role in tumor angiogenesis and metastasis. We therefore aimed to develop ^99mTc-labeled monomeric and dimeric NGR-containing peptides, namely, NGR1 and NGR2, for SPECT imaging of CD13 expression in HepG2 hepatoma xenografts. Both NGR-containing monomer and dimer were synthesized and labeled with ^99mTc. In vivo receptor specificity was demonstrated by successful blocking of tumor uptake of ^99mTc-NGR dimer in the presence of 20 mg/kg NGR2 peptide. Western blot and immunofluorescence staining confirmed the CD13 expression in HepG2 cells. The NGR dimer showed higher binding affinity and cell uptake in vitro than the NGR-containing monomer, presumably due to a multivalency effect. ^99mTc-Labeled monomeric and dimeric NGR-containing peptides were subjected to SPECT imaging and biodistribution studies. SPECT scans were performed in HepG2 tumor-bearing mice at 1, 4, 12, and 24 h post-injection of ~7.4 MBq tracers. The metabolism of tracers was determined in major organs at different time points after injection which demonstrated rapid, significant tumor uptake and slow tumor washout for both traces. Predominant clearance from renal and hepatic system was also observed in ^99mTc-NGR1 and ^99mTc-NGR2. In conclusion, monomeric and dimeric NGR peptide were developed and labeled with ^99mTc successfully, while the high integrin avidity and long retention in tumor make ^99mTc-NGR dimer a promising agent for tumor angiogenesis imaging.     

99mTc/Re complexes based on flavone and aurone as SPECT probes for imaging cerebral β-amyloid plaques

Two ^99mTc/Re complexes based on flavone and aurone were tested as potential probes for imaging β-amyloid plaques using single photon emission computed tomography. Both ^99mTc-labeled derivatives showed higher affinity for Aβ(1–42) aggregates than did ^99mTc-BAT. In sections of brain tissue from an animal model of AD, the Re-flavone derivative 9 and Re-aurone derivative 19 intensely stained β-amyloid plaques. In biodistribution experiments using normal mice, ^99mTc-labeled flavone and aurone displayed similar radioactivity pharmacokinetics. With additional modifications to improve their brain uptake, ^99mTc complexes based on the flavone or aurone scaffold may serve as probes for imaging cerebral β-amyloid plaques.     

In vivo phage display: identification of organ-specific peptides using deep sequencing and differential profiling across tissues

In vivo phage display is widely used for identification of organ- or disease-specific homing peptides. However, the current in vivo phage biopanning approaches fail to assess biodistribution of specific peptide phages across tissues during the screen, thus necessitating laborious and time-consuming post-screening validation studies on individual peptide phages. Here, we adopted bioinformatics tools used for RNA sequencing for analysis of high-throughput sequencing (HTS) data to estimate the representation of individual peptides during biopanning in vivo. The data from in vivo phage screen were analyzed using differential binding—relative representation of each peptide in the target organ versus in a panel of control organs. Application of this approach in a model study using low-diversity peptide T7 phage library with spiked-in brain homing phage demonstrated brain-specific differential binding of brain homing phage and resulted in identification of novel lung- and brain-specific homing peptides. Our study provides a broadly applicable approach to streamline in vivo peptide phage biopanning and to increase its reproducibility and success rate.     

Synthesis,radiolabeling, and preclinical evaluation of a new octreotide analog for somatostatin receptor‐positive tumor scintigraphy

Radiolabeled somatostatin analogs have become powerful tools in the diagnosis and staging of neuroendocrine tumors, which express somatostatin receptors. The aim of this study was to evaluate a new somatostatin analog, 6‐hydrazinopyridine‐3‐carboxylic acid‐Ser³‐octreotate (HYNIC‐SATE) radiolabeled with ^99mTc, using ethylenediamine‐N,N′‐diacetic acid and tricine as coligands, to be used as a radiopharmaceutical for the in vivo imaging of somatostatin receptor subtype 2 (SSTR2)‐positive tumor. Synthesis of the peptide was carried out on a solid phase using a standard Fmoc strategy. Peptide conjugate affinities for SSTR2 were determined by receptor binding affinity on rat brain cortex and C6 cell membranes. Internalization rate of ^99mTc‐HYNIC‐SATE was studied in SSTR2‐expressing C6 cells that were used for intracranial tumor studies in rat brain. A reproducible in vivo C6 glioma model was developed in Sprague–Dawley rat and confirmed by histopathology and immunohistochemical analysis. Biodistribution and imaging properties of this new radiopeptide were also studied in C6 tumor‐bearing rats. Radiolabeling was performed at high specific activities, with a radiochemical purity of >96%. Peptide conjugate showed high affinity binding for SSTR2 (HYNIC‐SATE IC₅₀ = 1.60 ± 0.05 n m ) and specific internalization into rat C6 cells. After administration of ^99mTc‐HYNIC‐SATE in C6 glioma‐bearing rats, a receptor specific uptake of radioactivity was observed in SSTR‐positive organs and in the implanted intracranial tumor and rapid excretion from nontarget tissues via kidneys. ^99mTc‐HYNIC‐SATE is a new receptor‐specific radiopeptide for targeting SSTR2‐positive brain tumor and might be of great promise in the scintigraphy of SSTR2‐positive tumors. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Radiochemical and radiobiological assessment of a pyridyl-S-cysteine functionalized bombesin derivative labeled with the 99mTc(CO)3+ core

Bombesin is a neuropeptide widely studied due to its ability to target various types of cancers. Technetium-99m on the other hand is ideal for diagnostic tumor targeting. The aim of the present study is the investigation of the coupling of the ligand (S)-(2-(2′-pyridyl)ethyl)-d,l-cysteine with the BN-peptide Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met(CONH₂) through the spacer aminohexanoic acidand the labeling of the resulting derivative MBN with the synthon [M(CO)₃(H₂O)₃]⁺ (M = ^99mTc, Re). The peptide was synthesized according to the SPPS method, purified and characterized by ESI-MS. The new ^99mTc-labeled biomolecule was stable in vitro, showed high affinity for the human GRP receptor expressed in PC3 cells and the rate of internalization was found to be time-dependent tissue distribution of the radiopeptide was evaluated in normal mice and in prostate cancer experimental models and significant radioactivity uptake was observed in the pancreas of normal mice as well as in PC3 tumors. Dynamic studies of the radiopeptide showed satisfactory tumor images.     

Identification and characterization of an anti-isoaspartic acid monoclonal antibody

The deamidation and rearrangement of protein-bound asparagine residues occurs when peptides and proteins are exposed to acidic or alkaline aqueous media. Asn⁹⁹ of bovine growth hormone (bGH) is readily modified via these mechanisms. We have generated a monoclonal antibody (MAb) that interacts with a bGH fragment that contains an isoaspartyl residue. To obtain this antibody, CAF₁/J mice were immunized with [isoaspartyl⁹⁹]-bGH(96–112) conjugated to BSA. Using a competitive ELISA assay, the interaction of this MAb to [isoaspartyl⁹⁹]-bGH(96–112) has been observed to have an apparentK _m of 150 nM. The corresponding native peptide and other bGH fragments do not bind to this antibody with high affinity. For example, the binding affinities of [Asp⁹⁹]-bGH(96–112) and [Glu⁹⁹]-bGH(96–112) to this antibody are 54- and 78-fold lower than the corresponding isoaspartyl peptide. The antibody also binds to bGH that is enriched in isoaspartic acid at position 99, but not to the unmodified protein. The binding epitope of the peptide has been further characterized by comparing the binding of bGH(96–112) analogues to the MAb. Alanine substitution at residues 99, 100, 101, and 103 reduce binding affinity to the antibody by more than 10³-fold. Replacement of valine with alanine at position 102 has much less impact on antibody affinity. Further experiments suggest that the relative insensitivity to this substitution is due to the structural similarity of these sidechains. Other isoaspartic acid-containing peptides not derived from the bGH sequence do not bind to the antibody. We conclude that the epitope binding site of this MAb is highly specific for 99–103 of [isoaspartyl⁹⁹]-bGH(96–112). This strategy can be used to obtain monoclonal antibodies that selectively interact with other proteins that have been similarly modified, or that contain other chemical modifications. The potential for generating antibodies that universally recognize protein- and peptide-bound isoaspartic acid residues is discussed.     

Comparative Binding of 125I-and 99mTc-Labeled Native and Glycated Low-Density Lipoprotein to Human Microvascular Endothelial Cells-Potential for Atherosclerosis Imaging?

Native (n), glycated (g), and glycoxidated (go) low-density lipoproteins (LDL) were labeled with ¹²⁵I or ^99mTc, and the labeling efficiency and binding were assessed for potential use of these LDL compounds in imaging analysis of atherosclerotic lesions (PPAR-γ receptors) by determining the number of specific receptors for nLDL, gLDL or goLDL on human microvascular endothelial cells as well as the K_D s using either ¹²⁵I-or ^99mTc-labeled LDLs. The specific activity of labeled gLDL and goLDL was much higher (for goLDL 20 times higher) than that of nLDL. Gel filtration of labeled LDLs revealed, however, that ^99mTc–g/goLDL is significantly degraded by the labeling reaction. No fragmentation was observed for ^99mTc-nLDL and all the ¹²⁵I-labeled LDL forms. Binding studies using both ¹²⁵I-and ^99mTc-nLDL indicated a weak binding affinity (K_D 10^{? 7}mol/L) to human microvascular endothelial cells. The binding affinity of ¹²⁵I-g/goLDL to these cells was significantly higher (K_D 10^{? 9}mol/L) and could be increased further by preactivation of the endothelial cells using TNFα. Incubation with ^99mTc-goLDL, however, did not result in specific binding of the ligand, possibly as a consequence of the fragmentation of the lipoprotein during the labeling. Scatchard transformation of the binding data with ^99mTc-gLDL revealed the presence of only a few binding sites. This was in contrast to the results obtained with ¹²⁵I-labeled gLDL, which revealed a much higher membrane density of scavenger receptors for this ligand. We conclude that for in vitro binding studies as well as for potential in vivo imaging, only ¹²⁵I-labeled goLDL should be used, whereas nLDL may be applied as ¹²⁵I-or ^99mTc-labeled ligand.     

Biological distribution of 99mTc-labeled YIGSR and IKVAV laminin peptides in rodents: 99mTc-IKVAV peptide localizes to the lung

Two laminin-derived peptides containing either YIGSR or IKVAV (single amino acid code) sequences were radiolabeled with ^99mTc and their biological distribution evaluated in rodents. Both ^99mTC-peptides cleared rapidly from the circulation though the kidney, and to a lesser extent, through the liver. ^99mTC-YIGSR peptide did not accumulate in any organ examined in normal, tumored, and emphysemic mice. The ^99mTc-IKVAV peptide localized within 10 min to the lung of normal animals, resulting in lung-to-blood ratios of approximately 23:1. The ^99mTc-IKVAV peptide localized to lung after submicron filtration and after intraperitoneal injection, suggesting that particulates do not play a major role in localization. Pre-incubation of ^99mTc-IKVAV peptide in whole blood decreased lung localization, suggesting that margination of radiolabeled blood cells does not play a major role in the lung localization. When ^99mTc-IKVAV was injected into mice with tumored lungs (melanoma), the lung uptake was markedly increased (up to 20% injected dose higher than control lungs) at all time points examined (10, 30, and 120 min). When ^99mTc-IKVAV was injected into mice with genetic emphysema, the lung uptake was markedly decreased at all time points. The localization of the ^99mTc-IKVAV-containing peptide to the lung is consistent with a receptor-based mechanism.