Aptamers directly radiolabeled with technetium-99m as a potential agent capable of identifying carcinoembryonic antigen (CEA) in tumor cells T84

Aptamers are small oligonucleotides that are selected to bind with high affinity and specificity to a target molecule. Aptamers are emerging as a new class of molecules for radiopharmaceutical development. In this study a new method to radiolabel aptamers with technetium-99m (^99mTc) was developed. Two aptamers (Apt3 and Apt3-amine) selected against the carcinoembryonic antigen (CEA) were used. Labeling was done by the direct method and the developed complex was subjected to quality control tests. Radiochemical purity and stability were monitored by Thin Layer Chromatography. Binding and specificity assays were carried out in the T84 cell line (CEA+) to evaluate tumor affinity and specificity after radiolabeling. Aptamers were successfully labeled with ^99mTc in high radiochemical yields, showing in vitro stability in presence of plasma and cystein. In binding assays the radiolabeled aptamer Apt3-amine showed the highest affinity to T84 cells. When evaluated with HeLa cells (CEA−), lower uptake was observed, suggesting high specificity for this aptamer. These results suggest that the Apt3-amine aptamer directly labeled with ^99mTc could be considered a promising agent capable of identifying the carcinoembryonic antigen (CEA) present in tumor cells.     

A cell-based single-stranded DNA aptamer specifically targets gastric cancer

Gastric cancer is one of the most prevailing cancers with high morbidity and mortality. Limitations in the current diagnosis and therapy, specially lacking of specific molecular therapeutic targets, ask for the development of new strategies. Aptamer, a newly developed adaptive molecule, could be used in clinical detection and therapy because of its high affinity and specificity. As no aptamer has ever been developed in preventing gastric cancer so far, we were the first who cloned such an aptamer specifically targeting gastric cancer. The aptamer was selected by systematic evolution of ligands by exponential enrichment with gastric cancer cell-line HGC-27 as target cell line and immortalized gastric epithelial cell-line GES-1 as control cell line. The affinity and specificity of candidate aptamers were examined by flow cytometry, confocal imagining and aptamer-based histochemistry staining. After 19 cycles of systematic evolution of ligands by exponential enrichment and subsequent cloning and sequencing, an aptamer with the highest affinity and specificity (nominated as AGC03) among candidates was screened out from a random single-stranded DNA pool. Moreover, AGC03 could not only specifically bind to gastric cancer cells (the equilibrium dissociation constant value was 16.49 ± 0.40 nM) in vitro, but also recognize cancer cells in human cancer tissue. Our most important finding is that AGC03 could even be internalized into cells automatically. In conclusion, we obtained a novel aptamer specifically targeting gastric cancer, which is an effective tool for both gastric cancer diagnosis and drug delivery.     

Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX

The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K _d value) of 292.8 ± 53.1 nM with 47.27 ± 5.58% cells fluorescent (bound) in a 1.48-μM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25–36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1–5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets.     

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K_d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.     

研究报告: 基于Ser-SELEX技术的结肠癌血清适配体的筛选及其应用研究

目的 为了解决癌症早期诊断困难的问题，实现结肠癌的早期检测。方法 采用Ser-SELEX技术筛选了结肠癌血清的特异性适配体。通过前期结肠癌血清正筛-正常人血清反筛步骤和后期结肠癌血清正筛-其他癌症血清反筛步骤循环的方式，经过16轮筛选流程，共选取4条候选适配体，并对其进行序列分析、二级结构和三级结构模拟、特异性分析等。结果 候选适配体主要形成的结构是茎环和假结两种，qPCR法测试亲和力，K_d约为10 nmol/L，适配体与结肠癌患者血清特异性结合情况分析表明，候选适配体APT-2具有良好特异性，且此种方法检出率约为 82.5%。结论 适配体（APT-2）应用于结肠癌早期诊断具有良好的发展前景。     

Selection of a high-affinity and in vivo bioactive ssDNA aptamer against angiotensin II peptide

Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52 ± 2.44E-10 and 5.87 ± 1.3E–9 M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33 ± 1.15E-9 and 4.11 ± 1.09E–9 M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (P < 0.05). They also significantly reduced the serum sodium level and increased the urine volume (P < 0.05). The core regions of aptamers did not show high inhibitory potential against Ang II. It can be a spotlight that ssDNA aptamers have high potential for blocking Ang II. In conclusion, it appears that the researches focusing on high affinity and bioactive aptamers may lead to excellent results in blocking Ang II activity.     

Activation of tumor-specific T lymphocytes after laser-induced thermotherapy in patients with colorectal liver metastases

Purpose  To asses if laser-induced thermotherapy (LITT) induces a specific cytotoxic T cell response in patients treated with LITT for colorectal cancer liver metastases. Methods  Eleven patients with liver metastases of colorectal cancer underwent LITT. Blood was sampled before and after LITT. Peripheral T cell activation was assessed by an interferon gamma (IFNg) secretion assay and flow cytometry. Test antigens were autologous liver and tumor lysate obtained from each patient by biopsy. T cells were stained for CD3/CD4/CD8 and IFNg to detect activated T cells. The ratio of IFNg positive to IFNg negative T cells was determined as the stimulation index (SI). To assess cytolytic activity, T cells were co-incubated with human colorectal cancer cells (CaCo) and cytosolic adenylate kinase release was measured by a luciferase assay. Results  IFNg secretion assay: before LITT SI was 12.73 (±4.83) for CD3+, 4.36 (±3.32) for CD4+ and 3.64 (±1.77) for CD8+ T cells against autologous tumor tissue. Four weeks after LITT SI had increased to 92.09 (±12.04) for CD3+ (P < 0.001), 42.92 (±16.68) for CD4+ (P < 0.001) and 47.54 (±15.68) for CD8+ T cells (P < 0.001) against autologous tumor tissue. No increased SI was observed with normal liver tissue at any time point. Cytotoxicity assay: before LITT activity against the respective cancer cells was low, with RLU = 1,493 (±1,954.68), whereas after LITT cytolytic activity had increased to RLU = 7,260 [±3,929.76 (P < 0.001)]. Conclusion  Patients with liver metastases of colorectal cancer show a tumor-specific cytotoxic T cell stimulation and a significantly increased cytolytic activity of CD3+, CD4+ and CD8+ T cells after LITT against an allogenic tumor (CaCo cell line).     

In vitro selection of aptamer S1 against MCF-7 human breast cancer cells

Breast cancer is the most common female cancer. However, the known effective specific biomarkers for breast cancer are still scarce. Abnormal membrane proteins serve as ideal biomarkers for disease diagnoses, therapeutics and prognosis. Thus aptamers (single-stranded oligonucleotide molecules) with molecular recognition properties can be used as efficient tools to sort cells based on differences in cell surface architecture between normal and tumor cells. In this study, we aimed to screen specific aptamer against MCF-7 human breast cancer cells. Cell-SELEX process was performed to isolate aptamers from a combinatorial single-stranded nucleic acid library that selectively targeting surface proteins of MCF-7 cells in contrast with MCF-10A human mammary epithelial cells. The process was repeated until the pool was enriched for sequences that specifically recognizing MCF-7 cells in monitoring by flow cytometry. Subsequently, the enriched pool was cloned into bacteria, and positive clones were sequenced to obtain individual sequences. Representative sequences were chemically synthesized and evaluated their binding affinities to MCF-7 cells. As a result, an aptamer S1 was finally identified to have high binding affinity with equilibrium dissociation constant (K_d) value of 29.9 ± 6.0 nM. FAM-labeled aptamer S1 induced fluorescence shift in MCF-7 cells but not in MCF-10A human mammary epithelial cells, or MDA-MB-453 and MDA-MB-231 human breast cancer cells. Furthermore, result of cell imaging observed from laser confocal fluorescence microscope showed that MCF-7 cells exhibited stronger fluorescence signal resulted from Cy5-labeled aptamer S1 than MCF-10A cells. The above findings suggested that S1 may be a specificity and selectivity aptamer for MCF-7 cells and useful for the breast cancer detection and diagnosis.     

Humanisation and characterisation of PR1A3, a monoclonal antibody specific for cell-bound carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is highly expressed by most tumours of gastrointestinal origin, but its use as a target for tumour therapy is complicated by the high levels of soluble CEA that are found circulating in the blood of cancer patients. A monoclonal antibody PR1A3 has been prepared, which binds preferentially to cell-surface rather than soluble CEA, this cell selectivity should make PR1A3 an ideal candidate for antibody-targeted tumour therapy. PR1A3 has been humanised and shown to retain its cell-surface specificity and affinity. Stable expression of the humanised antibody from chinese hamster ovary (CHO) cells has been achieved after transfection and amplification. Since PR1A3 binds preferentially to cell-associated CEA, a cell-free enzyme-linked immunosorbent assay (ELISA) has been developed to allow characterisation and routine assay of the antibody. This assay was developed using a recombinant chimeric protein constructed by cloning the domain of CEA that is bound by PR1A3 (the B3 domain) into a hybrid gene containing the Fc portion of IgG and three domains of biliary glycoprotein. Stable expression of this hybrid protein has been achieved from CHO cells. In ELISA both humanised and murine PR1A3 bound strongly to this antigen but only at a minimal level to soluble CEA. Two binding sites for the antibody were found on the gastric carcinoma cell line MKN45, one of higher affinity (1 nM) and the other at lower affinity (60 nM). Similar affinities were found for both murine and humanised antibodies. The data presented make it unlikely that the differential binding to cell-surface as distinct from soluble CEA can be accounted for by low affinity of PR1A3 for CEA, and provides further support for the hypothesis that some conformational change takes place on CEA release from cells and that it is this change that blocks PR1A3 binding to its epitope. Received: 5 October 1998 / Accepted: 19 November 1998     

Immunological characteristics of monoclonal antibodies against human carcinoembryonic antigen (CEA)

Four hybridomas secreting monoclonal antibodies (MAbs) of the IgG1 subclass against human carcinoembryonic antigen (CEA) were obtained from fusion of P3-NS1/1-Ag4 myeloma cells with splenic cells from mice immunized with purified CEA. None of the MAbs showed cross-reactivity to perchloric acid extractable antigens from the normal human colon by an inhibition radioimmunoassay. However, MAb C27 showed the highest affinity to CEA. The intensity of immunofluorescence staining of human colorectal cancer cells with MAb C27 correlates well to the cellular CEA content of cancer cells. LS174T showed the highest intensity of fluorescence (95%) while COLO320DM and COLO320HRS were the lowest (0.5%). None of the normal human organs - colon, lungs, liver, spleen or kidneys-showed positive staining by immunoperoxidase anti-peroxidase (PA) techniques, while tissues from colorectal carcinoma (CRC), gastric carcinoma, hepatoma and lung cancer gave a positive rate of 100% (30/30), 96.6% (28/29), 32.1% (9/28) and 82.1% (69/84) respectively. Results suggest that MAb C27 can be used in immunodetection and radiolocalization of colorectal carcinoma.     

IgY‐binding peptide screened from a random peptide library as a ligand for IgY purification

Chicken egg yolk immunoglobulin (IgY) is a functional substitute for mammalian IgG for antigen detection. Traditional IgY purification methods involve multi‐step procedures resulting in low purity and recovery of IgY. In this study, we developed a simple IgY purification system using IgY‐specific peptides identified by T7 phage display technology. From disulfide‐constrained random peptide libraries constructed on a T7 phage, we identified three specific binding clones (Y4‐4, Y5‐14, and Y5‐55) through repeated biopanning. The synthetic peptides showed high binding specificity to IgY‐Fc and moderate affinity for IgY‐Fc (K_d: Y4‐4 = 7.3 ± 0.2 μM and Y5‐55 = 4.4 ± 0.1 μM) by surface plasmon resonance analysis. To evaluate the ability to purify IgY, we performed immunoprecipitation and affinity high‐performance liquid chromatography using IgY‐binding peptides; the result indicated that these peptides can be used as affinity ligands for IgY purification. We then used a peptide‐conjugated column to purify IgY from egg yolks pre‐treated using an optimized delipidation technique. Here, we report the construction of a cost‐effective, one‐step IgY purification system, with high purity and recovery. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.     

Chip-based detection of hepatitis C virus using RNA aptamers that specifically bind to HCV core antigen

The development of reagents with high affinity and specificity to the antigens of hepatitis C virus (HCV) is important for the early stage diagnosis of its infection. Aptamers are short, single-stranded oligonucleotides with the ability to specifically recognize target molecules with high affinity. Herein, we report the selection of RNA aptamers that bind to the core antigen of HCV. High affinity aptamers were isolated from a 10(15) random library of 60 mer RNAs using the SELEX procedure. Importantly, the selected aptamers specifically bound to the core antigen, but not to another HCV antigen, NS5, in a protein chip-based assay. Using these aptamers, we developed an aptamer-based biosensor for HCV diagnosis and detected the core antigen from HCV infected patients' sera with good specificity. This novel aptamer-based antigen detection sensor could be applied to the early diagnosis of HCV infection.     

A cross-linked anti-TNF-α aptamer for neutralization of TNF-α-induced cutaneous Shwartzman phenomenon: A simple and novel approach for improving aptamers' affinity and efficiency

To increase the efficiency of aptamers to their targets, a simple and novel method has been developed based on aptamer oligomerization. To this purpose, previously anti-human TNF-α aptamer named T1–T4 was trimerized through a trimethyl aconitate core for neutralization of in vitro and in vivo of TNF-α. At first, 54 mer T1–T4 aptamers with 5′-NH₂ groups were covalently coupled to three ester residues in the trimethyl aconitate. In vitro activity of novel anti-TNF-α aptamer and its dissociation constant (K_d) was done using the L929 cell cytotoxicity assay. In vivo anti-TNF-α activity of new oligomerized aptamer was assessed in a mouse model of cutaneous Shwartzman. Anchoring of three T1–T4 aptamers to trimethyl aconitate substituent results in formation of the 162 mer fragment, which was well revealed by gel electrophoresis. In vitro study indicated that the trimerization of T1–T4 aptamer significantly improved its anti-TNF-α activity compared to non-modified aptamers (P < 0.0001) from 40% to 60%. The determination of K_d showed that trimerization could effectively enhance K_d of aptamer from 67 nM to 36 nM. In vivo study showed that trimer aptamer markedly reduced mean scar size from 15.2 ± 1.2 mm to 1.6 ± 0.1 mm (P < 0.0001), which prevent the formation of skin lesions. In vitro and in vivo studies indicate that trimerization of anti-TNF-α aptamer with a novel approach could improve the anti-TNF-α activity and therapeutic efficacy. According to our findings, a new anti-TNF-α aptamer described here could be considered an appropriate therapeutic agent in treating several inflammatory diseases.     

大肠癌相关抗原LEA的血清学诊断

为了探讨抗人大肠癌相关抗原LEA在大肠癌患者的血清学中诊断价值。本文采用双抗夹心ELISA方法,并应用抗人大肠癌单克隆ND-1对大肠癌患者和正常人的血清进行了LEA抗原水平的检测,以及与CEA抗原水平检测的对比研究。结果表明:LEA在大肠癌患者血清学诊断的阳性率为68.7％,正常人为3.1％,CEA分别为56.6％和6.25％,在大肠癌的早期诊断中Dukes(A B)期LEA的阳性率为66.7％,明显高于CEA的36.1％,两者相比有显著统计学意义(P<0.05)。本研究还发现,大肠癌患者血清中LEA水平的表达与肿瘤的Dukes分期无关,而与肿瘤的分化程度密切相关,在高、中、低分化的大肠癌患者的血清中LEA的阳性表达率分别为86.8％、78.6％和11.8％,CEA则分别为71.1％、60.7％和17.6％。LEA在早期癌的阳性率为66.7％、晚期癌为70.2％,CEA分别为36.1％和72.3％。由此可见LEA在对人大肠癌患者血清学诊断的灵敏度和特异性均比CEA高,LEA对于高、中分化大肠癌患者的早期诊断,早期治疗和提高大肠癌患者的生存率方面将具有很重要的意义,是临床上具有应用价值的新型肿瘤标志物。     

Blocking interaction of viral gp120 and CD4-expressing T cells by single-stranded DNA aptamers

To investigate the potential clinical application of aptamers to prevention of HIV infection, single-stranded DNA (ssDNA) aptamers specific for CD4 were developed using the systematic evolution of ligands by exponential enrichment approach and next generation sequencing. In contrast to RNA-based aptamers, the developed ssDNA aptamers were stable in human serum up to 12 h. Cell binding assays revealed that the aptamers specifically targeted CD4-expressing cells with high binding affinity (K_d = 1.59 nM), a concentration within the range required for therapeutic application. Importantly, the aptamers selectively bound CD4 on human cells and disrupted the interaction of viral gp120 to CD4 receptors, which is a prerequisite step of HIV-1 infection. Functional studies showed that the aptamer polymers significantly blocked binding of viral gp120 to CD4-expressing cells by up to 70% inhibition. These findings provide a new approach to prevent HIV-1 transmission using oligonucleotide aptamers.     

Monoclonal antibodies for carcinoembryonic antigen (CEA) as a model system: identification of two novel CEA-related antigens in meconium and colorectal carcinoma tissue by Western blots and differential immunoaffinity chromatography

In a previous study, five monoclonal antibodies against the carcinoembryonic antigen (CEA) with different epitope specificities were delineated. One of these antibodies which exhibits a high affinity for CEA binds to different carcinoma tissues, to liver tissue, and to granulocytes. This antibody was selected for the immunoaffinity purification of CEA and related antigens from colorectal carcinoma tissue, from spleen tissues, from bile, and from meconium. After elution from the immunosorbent, the antigens were separated by SDS-PAGE, were transferred to nitrocellulose, and were incubated with the five different antibodies. Antibody T84.1 bound to the following antigens: 177 kD and 128 kD from colonic carcinoma, 81 kD from bile, 49 kD from spleen, as well as 165 kD and 100 kD from meconium. Two additional antibodies showed a similar binding pattern. The fourth antibody (CEA.11) bound to the 165 kD meconium antigen and to the two colorectal carcinoma antigens. The fifth antibody (T84.66) showed a strong reaction with the 177 kD colorectal carcinoma antigen and a faint reaction with a 183 kD antigen in meconium. As judged from m.w. and immunochemical properties, the 128 kD colorectal carcinoma antigen and the 100 kD meconium antigen are two novel CEA-related antigens. Because antibody CEA.11 did not bind to the 100 kD meconium antigen in Western blots, the 165 kD antigen could be eluted from a CEA.11 immunosorbent without contamination by the 100 kD antigen. Similarly, as predicted from the binding pattern in the Western blots, the two colorectal carcinoma antigens were separated from each other by a T84.66 immunosorbent.     

The Tumor Targeted Superantigen ABR-217620 Selectively Engages TRBV7-9 and Exploits TCR-pMHC Affinity Mimicry in Mediating T Cell Cytotoxicity

The T lymphocytes are the most important effector cells in immunotherapy of cancer. The conceptual objective for developing the tumor targeted superantigen (TTS) ABR-217620 (naptumomab estafenatox, 5T4Fab-SEA/E-120), now in phase 3 studies for advanced renal cell cancer, was to selectively coat tumor cells with cytotoxic T lymphocytes (CTL) target structures functionally similar to natural CTL pMHC target molecules. Here we present data showing that the molecular basis for the anti-tumor activity by ABR-217620 resides in the distinct interaction between the T cell receptor β variable (TRBV) 7-9 and the engineered superantigen (Sag) SEA/E-120 in the fusion protein bound to the 5T4 antigen on tumor cells. Multimeric but not monomeric ABR-217620 selectively stains TRBV7-9 expressing T lymphocytes from human peripheral blood similar to antigen specific staining of T cells with pMHC tetramers. SEA/E-120 selectively activates TRBV7-9 expressing T lymphocytes resulting in expansion of the subset. ABR-217620 selectively triggers TRBV7-9 expressing cytotoxic T lymphocytes to kill 5T4 positive tumor cells. Furthermore, ABR-217620 activates TRBV7-9 expressing T cell line cells in the presence of cell- and bead-bound 5T4 tumor antigen. Surface plasmon resonance analysis revealed that ABR-217620 binds to 5T4 with high affinity, to TRBV7-9 with low affinity and to MHC class II with very low affinity. The T lymphocyte engagement by ABR-217620 is constituted by displaying high affinity binding to the tumor cells (K_D approximately 1 nM) and with the mimicry of natural productive immune TCR-pMHC contact using affinities of around 1 µM. This difference in kinetics between the two components of the ABR-217620 fusion protein will bias the binding towards the 5T4 target antigen, efficiently activating T-cells via SEA/E-120 only when presented by the tumor cells.     

Interaction of Synthetic Dipeptide Bestim with Mouse Thymocytes and Macrophages

Tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE) reaction. [³H]bestim was found to bind with high affinity to mouse peritoneal macrophages (K _d 2.1 ± 0.1 nM) and thymocytes (K _d 3.1 ± 0.2 nM) and also plasma membranes isolated from these cells (K _d 18.6 ± 0.2 and 16.7 ± 0.3 nM respectively). The specific bonding of [³H]bestim with macrophages and thymocytes was inhibited by unlabeled dipeptide thymogen (L-Glu-L-Trp) (K _i 0.9 ± 0.1 and 1.1 ± 0.1 nM respectively). Treatment of the macrophages and thymocytes with trypsin led to their loss of capacity to bind [³H]bestim. Bestim at concentrations range of 0.1–1000 nМ reduced the adenylate cyclase activity in macrophage and thymocyte membranes.     

哈维氏弧菌适配子的SELEX筛选及其亲和特异性研究

哈维氏弧菌是水产养殖中的重要条件致病菌,对其进行快速、准确地检测和鉴定是相关病害防治的基础和关键.适配子具有亲和力高、特异性强、稳定性好等优点,在微生物的检测和鉴定方面呈现出广泛的应用前景.本研究以哈维氏弧菌为靶目标,采用SELEX技术,即指数级富集配体的系统进化技术,筛选其特异性适配子.经15轮筛选后,随机ssDNA文库的亲和力从3.51上升到58.95,提高了15.8倍.筛选出的适配子富集库经克隆、测序后得到52条不同序列,根据同源性将这些序列分成8个家族,其中第1和第2家族的适配子数量最多,超过总数的50%.通过深入分析,筛选出6个对哈维氏弧菌有显著亲和特异性(P0.01)的高频适配子,其中5个高频适配子(S1、S25、S26、S27、S35)对哈维氏弧菌有较高的亲和力,相应的亲和常数Kd值分别为(32.6±7.1)、(45.3±10.1)、(24.7±5.8)、(34.8±5.6)、(12.9±4.0)nmol/L.本文还对高频适配子的产生机制及其应用价值进行了探讨.本文首次筛选出了对哈维氏弧菌具有较高亲和特异性的适配子,为后续利用适配子进行哈维氏弧菌的检测和鉴定奠定了基础.     

癌胚抗原(CEA)启动子在肿瘤细胞中的特异表达及介异bak基因诱发凋亡

构建由癌胚抗原 (CEA)启动子控制报道基因增强型绿色荧光蛋白 (EGFP)表达的重组表达质粒pCEA EGFP .用转染细胞后检测荧光的方法对CEA阳性细胞进行简便、直观的检测 ,并结合流式细胞计数对CEA启动子在人结直肠腺癌细胞LS 1 74T、结肠癌细胞SW 4 80、肺腺癌细胞A5 49、人宫颈癌细胞HeLa和人喉癌细胞HEp 2中的活性进行了分析 ,发现其在SW4 80、LS 1 74T、A5 49中活性较强 ,而在HeLa和HEp 2中无活性 .构建由CEA启动子控制凋亡基因bak表达的重组表达质粒pCEA bak ,转染HeLa及SW 4 80细胞 ,用Hoechst332 5 8染色及PI染色 流式细胞计数分析的方法证明 ,pCEA bak转染能够特异性引起SW 4 80细胞的凋亡 .结果表明 ,CEA启动子具有很好的特异性 ,CEA介导bak基因的方法可望用于CEA阳性癌细胞的靶向性基因治疗 .