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muc1/y特异表位肽在大肠杆菌中的可溶性表达及其抗体的制备  [PDF]
张立新* 李春海 孙丽亚 王淼 路浩军
生物工程学报 , 2003,
Abstract: 为获得muc1/y的特异表位肽,制备抗体,用pcr扩增其编码序列,克隆到pgex-2t中,转化dh5α感受态,0.2mmol/liptg诱导表达,超声破碎或bper-tmⅱ试剂处理诱导菌,亲和层析和阴离子交换纯化目的蛋白,sdspage及westernblotting鉴定;免疫家兔制备多抗,初步用于免疫组化分析。结果表明,转化菌经诱导后表达融合蛋白gsty30,约占菌体总蛋白的20%,大多以可溶形式存在,与诱导温度无关。免疫家兔获得多抗,效价为1∶320000,纯化的抗体具有特异性,可识别肿瘤细胞表面的muc1/y蛋白。所得蛋白和抗体可用于muc1/y的表达特征及其生物学功能研究。
Chemoresistance Is Associated with MUC1 and Lewis y Antigen Expression in Ovarian Epithelial Cancers  [PDF]
Danye Zhang,Jian Gao,Liancheng Zhu,Zhenhua Hu,Rui Hou,Shuice Liu,Mingzi Tan,Juanjuan Liu,Bei Lin
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms140611024
Abstract: Objective: The aim of this study was to analyze the correlation and clinical significance between the expression of Mucin-1 (MUC1) and the Lewis y antigen with chemoresistance in ovarian epithelial cancers. Methods: Ovarian cancer patients ( n = 92) treated at our hospital from May 2005 to July 2009 were divided, according to their treatment and follow-up outcomes, into a resistant group ( n = 37) or sensitive group ( n = 55). The expression of MUC1 and Lewis y antigen in ovarian cancer tissues was detected using immunohistochemistry and correlated with chemoresistance. Results: The positive rates of MUC1 and Lewis y antigen in the resistant group were both 91.89%, significantly higher than their positive rates in the sensitive group (65.45% and 69.09%, respectively, and both p < 0.05). MUC1 or Lewis y expression and the pathological stage of the tissue were independent risk factors for chemoresistance (all p < 0.05). Conclusion: The increased expression of MUC1 and the Lewis y antigen is a significant risk factor for chemoresistance in patients with ovarian epithelial cancer.
Breast cancer humoral immune response: involvement of Lewis y through the detection of circulating immune complexes and association with Mucin 1 (MUC1)
Marina Larrain, Sandra Demichelis, Marina Crespo, Ezequiel Lacunza, Alberto Barbera, Aldo Cretón, Francisco Terrier, Amada Segal-Eiras, María Croce
Journal of Experimental & Clinical Cancer Research , 2009, DOI: 10.1186/1756-9966-28-121
Abstract: Pretreatment serum and tissue breast samples from 76 adenocarcinoma, 34 benign and 36 normal specimens were analyzed. Anti-MUC1 and anti-Lewis y MAbs were employed. To detect Lewis y/CIC and MUC1/CIC, ELISA tests were developed; serum samples containing MUC1 were previously selected by Cancer Associated Serum Antigen (CASA). Immunoprecipitation (IP) was performed in 9 malignant, benign and normal samples and analyzed by SDS-PAGE and Western blot. Lewis y and MUC1 expression was studied by immunohistochemistry (IHC). Statistical analysis was performed employing principal component analysis (PCA), ANOVA, Tukey HSD, Chi square test and classical correlation (p < 0.05).By ELISA, Lewis y/IgM/CIC levels showed statistically significant differences between breast cancer versus benign and normal samples; mean ± SD values expressed in OD units were: 0.525 ± 0.304; 0.968 ± 0.482 and 0.928 ± 0.447, for breast cancer, benign disease and normal samples, respectively, p < 0.05. Lewis y/IgG/CIC did not show any statistically significant difference. MUC1/IgM/CIC correlated with Lewis y/IgM/CIC. By CASA, 9 samples with MUC1 values above the cut off were selected and IP was performed, followed by SDS-PAGE and Western blot; bands at 200 kDa were obtained with each MAb in all the samples. By IHC, with C14 MAb, 47.5%, 31% and 35% of malignant, benign and normal samples, respectively, showed positive reaction while all the samples were positive with anti-MUC1 MAb; in both cases, with a different pattern of expression between malignant and non malignant samples.Our findings support that in breast cancer there was a limited humoral immune response through Lewis y/IgM/CIC levels detection which correlated with MUC1/IgM/CIC. We also found that Lewis y might be part of circulating MUC1 glycoform structure and also that Lewis y/CIC did not correlate with Lewis y expression.Worldwide, breast cancer is the most common cause of mortality by cancer in female population (GLOBOCAN, 2002, IARC). In o
Progress, applications and prospects of epitope vaccine
表位疫苗及相关技术研究进展

FANG Zhong,LUO Wen-xin,XIA Ning-shao,
方钟
,罗文新,夏宁邵

中国生物工程杂志 , 2007,
Abstract: Epitope vaccine is one of the emerging vaccine techniques developing in past decade years.Particularly the advantages of this vaccine on preventing and therapy illness,as cancer and virus,are espacially outstanding.The most importent elements about the vaccine,namely T/B-epitopes obtainment and identification,vehicles for epitope and vaccine construction,are reviewed.In addition,applications of the vaccine technique in some refractory diseases,such as cancer,virus and pathogen infection,are depicted.
Novel MUC1 Aptamer Selectively Delivers Cytotoxic Agent to Cancer Cells In Vitro  [PDF]
Yan Hu, Jinhong Duan, Qimin Zhan, Fengdan Wang, Xin Lu, Xian-Da Yang
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031970
Abstract: Chemotherapy is a primary treatment for cancer, but its efficacy is often limited by the adverse effects of cytotoxic agents. Targeted drug delivery may reduce the non-specific toxicity of chemotherapy by selectively directing anticancer drugs to tumor cells. MUC1 protein is an attractive target for tumor-specific drug delivery owning to its overexpression in most adenocarcinomas. In this study, a novel MUC1 aptamer is exploited as the targeting ligand for carrying doxorubicin (Dox) to cancer cells. We developed an 86-base DNA aptamer (MA3) that bound to a peptide epitope of MUC1 with a Kd of 38.3 nM and minimal cross reactivity to albumin. Using A549 lung cancer and MCF-7 breast cancer cells as MUC1-expressing models, MA3 was found to preferentially bind to MUC1-positive but not MUC1-negative cells. An aptamer-doxorubicin complex (Apt-Dox) was formulated by intercalating doxorubicin into the DNA structure of MA3. Apt-Dox was found capable of carrying doxorubicin into MUC1-positive tumor cells, while significantly reducing the drug intake by MUC1-negative cells. Moreover, Apt-Dox retained the efficacy of doxorubicin against MUC1-positive tumor cells, but lowered the toxicity to MUC1-negative cells (P<0.01). The results suggest that the MUC1 aptamer may have potential utility as a targeting ligand for selective delivery of cytotoxic agent to MUC1-expressing tumors.
Study on the Methods for Virus Epidemiology in Epitope Level
表位水平研究病毒流行病学的方法探讨

Lai Dazhi Du Yong Du Guixin Chen Wei Wang Haitao,
来大志
,杜勇,杜桂鑫,陈薇,王海涛

微生物学报 , 2002,
Abstract: A method for studying virus epidemiology in epitope level was established via phage random peptide library and thioredoxin surface display technique and the method was proved by test with core protein of HCV.
加权贝叶斯线性B细胞表位特征提取方法
An weighted linear B-cell epitope Bayes feature extraction
 [PDF]

,,,
福州大学学报(自然科学版) , 2015, DOI: 10.7631/issn.1000-2243.2015.01.0040
Abstract: 特征提取方法对线性B细胞表位预测起到非常重要的作用,但贝叶斯特征提取方法忽略了氨基酸之间的相互关系. 为了更准确地描述表位序列的关系,提出一种基于氨基酸对量表加权的贝叶斯特征提取方法,该方法对单个氨基酸在序列分布的基础上充分考虑了氨基酸之间的关系,并使用支持向量机作为分类器进行分类. 在El-Manzalawy,Saha数据集上的测试表明改进的贝叶斯特征提取方法. 相比传统的贝叶斯特征提取方法,提取精度有一定的提升.
Feature extraction method play a very important role in linear B-cell epitope prediction. The bayes feature extraction methods ignore the relationship between the amino acids. In order to describe the epitope sequence more accurately,proposed an weighted bayes feature extraction(WBFE) based on amino acid antigen scale,apart to the distribution of the individual amino acids in the sequence the method is fully taking into account the relationship between the amino acid,and using support vector machine as classifier for classification. The test on the the El-Manzalawy,Saha data set shows that,compared to the traditional the Bayes feature extraction method,using the proposed method predicted effects enhance
Molecular Mimics of the Tumour Antigen MUC1  [PDF]
Tharappel C. James, Ursula Bond
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0049728
Abstract: A key requirement for the development of cancer immunotherapy is the identification of tumour-associated antigens that are differentially or exclusively expressed on the tumour and recognized by the host immune system. However, immune responses to such antigens are often muted or lacking due to the antigens being recognized as “self”, and further complicated by the tumour environment and regulation of immune cells within. In an effort to circumvent the lack of immune responses to tumour antigens, we have devised a strategy to develop potential synthetic immunogens. The strategy, termed mirror image phage display, is based on the concept of molecular mimicry as demonstrated by the idiotype/anti-idiotype paradigm in the immune system. Here as ‘proof of principle’ we have selected molecular mimics of the well-characterised tumour associated antigen, the human mucin1 protein (MUC1) from two different peptide phage display libraries. The putative mimics were compared in structure and function to that of the native antigen. Our results demonstrate that several of the mimic peptides display T-cell stimulation activity in vitro when presented by matured dendritic cells. The mimic peptides and the native MUC1 antigenic epitopes can cross-stimulate T-cells. The data also indicate that sequence homology and/or chemical properties to the original epitope are not the sole determining factors for the observed immunostimulatory activity of the mimic peptides.
草莓镶脉病毒外壳蛋白抗原表位预测、克隆、表达及鉴定
Prediction, cloning, expression and identification of an epitope of the coat protein of the Strawberry vein banding virus
 [PDF]

杨菊梅,马建忠,潘博,李印武,Yang Jumei,Ma Jianzhong,Pan Bo,Li Yinwu
- , 2018, DOI: 10.13802/j.cnki.zwbhxb.2018.2016189
Abstract: 为实现草莓镶脉病毒(Strawberry vein banding virus,SVBV)的简单和快速检测,应用生物信息学方法分析了SVBV外壳蛋白的线性抗原表位及其理化性质,以大肠杆菌的优势密码子对所预测的优势抗表原位进行设计并合成,同时进行了最佳异源表达条件优化。结果表明,第218~238位的氨基酸残基序列为SVBV外壳蛋白的优势抗原表位,其编码片段为5-AE。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)检测结果表明,重组融合蛋白的分子量约为24.8 kD,与其理论分子量20.5 kD基本一致。异源表达优化的结果表明,当IPTG浓度为0.5 mmol/L、温度为40℃、诱导时间为4 h时,重组融合蛋白的表达量最高,为23.6 mg/L。Western blot结果表明重组融合蛋白能与His多克隆抗体起特异性反应。串联质谱结果表明,5-AE肽段氨基酸残基序列正确。
For realize the simple and rapid detection of Strawberry vein banding virus (SVBV), the linear antigen epitope and physicochemical properties of SVBV coat protein were analyzed with bioinformatics method. The dominant codon of Escherichia coli was used to design and synthesize the predicted superior antigen epitope, and the heterologous expression condition was optimized. The results showed that the sequence of amino acid residues at position 218-238 was the dominant epitope of SVBV coat protein, and its coding fragment was 5-AE. Detection results of sodium dodecyl sulfonatepolyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weight of the recombinant fusion protein was about 24.8 kD, which was basically consistent with its theoretical molecular weight of 20.5 kD. The expression of heterologous expression showed that, when the concentration of IPTG was 0.5 mmol/L, the temperature was 40℃, and the induction time was four hours, the expression of the recombinant fusion protein was the highest (23.6 mg/L). Western blot results showed that the recombinant fusion protein could react specifically with His polyclonal antibody. Tandem mass spectrometry showed that the amino acid sequence of 5-AE peptide fragment was correct.
Epitope Tagging Chromosomal Genes of Y. pestis By recombineering technique
利用重组工程技术标记鼠疫菌rpoS基因

ZHANG Jian-shan,CHEN Ze-liang,SONG Ya-jun,GUO Zhao-biao,WANG Jin,WANG Hong-xia,ZHAI Jun-hui,YANG Rui-fu,
张建山
,陈泽良,宋亚军,郭兆彪,王津,王红霞,翟俊辉,杨瑞馥

中国生物工程杂志 , 2006,
Abstract: Objective: To facilitate the functional analysis of chromosomal genes and their products, the recombineering technique to epitope tagging of chromosomal genes of Y. pestis was adapted. Methods: The epitope tag was generated by primer annealing and then fused with resistance gene by fusion PCR. The epitope-resistance cassette was inserted into pBluecript, resulted in the template plasmid, pBS-MH. The tagging cassette for rpoS was obtained by PCR amplification from pBS-MH with primers containing homology specific to the target gene. PCR products were transformed into recombination competent cells and recombinants were selected. PCR and DNA sequencing were used to confirm the correct tagging event. The expression of the tagged protein was detected with Western blot by using monoclonal antibody to the epitope. Results: The template plasmid containing fusion of epitope and resistance gene was successfully constructed. The sigma factor gene, rpoS, was tagged with a myc-his tag at the COOH terminus. Expression of the tagged rpoS was successfully detected indirectly by the antibody against His tag. Conclusion: The chromosomal gene tagging by recombineering technique represents a powerful tool in the functional study of bacterial genes and their products.
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