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国家自然科学基金(30572199)

作品数:14 被引量:30H指数:4
相关作者:何贤辉徐丽慧欧阳东云查庆兵高琦更多>>
相关机构:暨南大学暨南大学附属第一医院中国医学科学院北京协和医学院更多>>
发文基金:国家自然科学基金广东省自然科学基金中国科学院知识创新工程更多>>
相关领域:医药卫生生物学更多>>

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14 条 记 录,以下是 1-10
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Preparation and Identification of HLA-A*1101 Tetramer Loading with Human Cytomegalovirus pp65 Antigen Peptide被引量:1
2007年
MHC/peptide tetramer technology has been widely used to study antigen-specific T cells, especially for identifying virus-specific CD8^+ T cells in humans. The tetramer molecule is composed of HLA heavy chain, β2-microglobulin (β2m), an antigenic peptide, and fluorescent-labeled streptavidin. To further investigate the HLA-A*1101-restricted CD8^+ T cell responses against human cytomegalovirus (HCMV), we established an approach to prepare HLA-A*1101 tetramer complexed with a peptide from HCMV. The cDNA encoding HLA-A*1101 heavy chain was cloned and the prokaryotic expression vector for the ectodomain of HLA-A*1101 fused with a BirA substrate peptide (HLA-A*1101-BSP) at its carboxyl terminus was constructed. The fusion protein was highly expressed as inclusion bodies under optimized conditions in Escherichla coli. Moreover, HLA-A*1101-BSP protein was refolded in the presence of β2m and an HCMV peptide pp6516.24 (GPISGHVLK, GPI). Soluble HLA-A*1101-GPI monomer was biotinylated and purified to a purity of 95%, which was subsequently combined with streptavidin to form tetramers at a yield of 〉 80%. The HLA-A*1101-GPI tetramers could bind to virus-specific CD8^+ T cells, suggesting soluble HLA-A*1101-GPI tetramers were biologically functional. This study provides the basis for further evaluation of HLA-A*1101-restricted CD8^+ T cell responses against HCMV infection.
Fengyao LiLihui XuQingbing ZhaXiaoyun ChiQiantao JiaKianhui He
关键词:TETRAMERCYTOMEGALOVIRUSEPITOPE
Phenotypic and Functional Analysis of LCMV gp33-41-Specific CD8 T Cells Elicited by Multiple Peptide Immunization in Mice Revealed the Up-regulation of PD-1 Expression on Antigen-Specific CD8 T Cells被引量:1
2007年
The phenotype and function of antigen-specific CD8 T cells are closely associated with the efficacy of a therapeutic vaccination. Here we showed that multiple immunizations with LCMV gp33-41 peptide (KAV) in Freund's adjuvant could induce KAV-specific CD8 T cells with low expression of CD127 and CD62L molecules. The inhibitory receptor PD-1 was also expressed on a substantial part of KAV-specific CD8 T cells, and its expression level on KAV-specific CD8 T cells in spleen and lymph nodes was much higher when compared to those in peripheral blood. Furthermore, KAV-specific CD8 T cells could specifically kill KAV-pulsed target cells in vivo but the efficiency was low. These data suggest that prime-boost vaccination schedule with peptide in Freund's adjuvant can elicit antigen-specific CD8 T cells of effector-like phenotype with partial functional exhaustion, which may only provide short-term protection against the pathogen. Cellular & Molecular Immunology.
Yi LiuLihui XuYiqun JiangJianfang SunXianhui He
关键词:LCMVPD-1
抗人PD-1高亲和力抗血清的制备及其在PD-1免疫印迹分析中的应用
2008年
利用纯化的可溶性人PD-1胞外域(sPD-1)蛋白为免疫原免疫小鼠,获得抗血清.免疫印迹显示该抗血清与原核表达的sPD-1有特异性反应,但与某些菌体蛋白存在交叉反应.经菌体蛋白吸附法处理后,交叉反应明显减弱.以商业化抗人PD-1抗体和小鼠sPD-1抗血清分析经亲和层析富集的活化的Jurkat T细胞表达的PD-1,只有自制的抗血清鉴定到特异反应条带,其表观分子质量为49 ku.
施焕敬徐丽慧韩捷卢宏松何贤辉
关键词:JURKAT抗血清免疫印迹
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被引量:2
2007年
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HCMVpp65341-349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。
贾仟涛徐丽慧李丰耀查庆兵何贤辉
关键词:四聚体巨细胞病毒密码子优化原核表达包涵体
负载野生型p53抗原肽HLA-A^*2402四聚体的制备和鉴定
2010年
目的:制备负载野生型p53抗原肽的HLA-A*2402四聚体,用于检测卵巢肿瘤患者p53特异性细胞毒T淋巴细胞(CTL)。方法:在p53125-134抗原肽TYS和轻链β2微球蛋白存在时,利用稀释法复性获得负载p53125-134抗原肽(TYSPALNKMF,TYS)的可溶性单体,经生物素酰化并纯化后与荧光素标记的链亲和素按4∶1的比例混合形成HLA-A*2402/TYS四聚体,以流式细胞仪检测特异性CTL的频率。结果:流式细胞术分析显示,卵巢癌患者外周血中可检测减低频率的p53125-134TYS抗原肽特异性CTL。结论:成功制备HLA-A*2402/TYS四聚体。
王笑迎查庆兵何贤辉欧阳东云徐丽慧郭贺高琦
关键词:四聚体细胞毒T淋巴细胞
H-2D^b四聚体的制备及其在检测LCMV特异性CD8^+T细胞中的应用被引量:4
2008年
MHC四聚体技术是研究抗原特异性淋巴细胞应答的关键技术之一。为研究H-2D^b基因型(如C57BL/6)小鼠的特异性CD8^+T细胞免疫应答,需要建立H-2D^b四聚体制备技术平台。首先以RT-PCR方法克隆H-2D^b重链基因的cDNA,进而构建H-2D^b胞外域与生物素化酶BirA底物肽(BSP)融合蛋白的表达载体,并在大肠杆菌中获得表达。在LCMV GP_(33-41)抗原肽(KAVYNFATC,KAV)和人β_2-微球蛋白存在时,通过稀释法复性获得H-2D^b/KAV单体。该单体经生物素化并纯化后与PE-链亲和素按4:1的比例混合,即形成四聚体。通过流式细胞术检测经KAV肽免疫的C57BL/6小鼠体内的LCMV特异性CD8^+T细胞的频率.结果表明在外周血、引流淋巴结和脾脏中均可检测到一定频率的LCMV特异性CD8^+T细胞,其中以对外周血标本染色的效果最佳。成功建立了小鼠H-2D^b四聚体制备技术平台,为监测及分析基于H-2D^b基因型小鼠的实验性免疫治疗创造了条件。
刘毅徐丽慧曾学思孙建方何贤辉
关键词:淋巴细胞性脉络丛脑膜炎病毒原核表达
重组人ASCT2胞外结构域ECL2在大肠杆菌中的表达及其鉴定被引量:1
2010年
中性氨基酸转运蛋白(ASCT2)是人类内源性病毒的包膜蛋白合胞素在细胞膜上的主要受体,ECL2是该受体的其中一个较大的胞外结构域.通过RT-PCR方法从人乳腺癌MCF-7细胞中克隆ASCT2基因编码区全长序列,再从中扩增ASCT2的胞外区ECL2序列,与pET-41b连接构建原核表达载体,重组质粒在大肠杆菌中获得高效表达,重组蛋白在N-和C-端分别融合谷胱甘肽转移酶(GST)和His6标签,融合蛋白在上清液和包涵体中均有表达,可溶性部分经亲和层析纯化获得高纯度的重组蛋白,该蛋白可结合在表达合胞素的MCF-7细胞表面,具有结合合胞素的潜在活性,这些结果为进一步研究ASCT2与合胞素的相互作用奠定了基础.
欧阳东云徐丽慧高琦郭贺陈清何贤辉
关键词:胞外结构域原核表达
细胞自噬与高迁移率族蛋白B-1(HMGB1)介导肿瘤耐药的研究进展被引量:6
2011年
恶性肿瘤可通过多种细胞机制,产生对抗癌药物和放疗的抗性,即所谓耐药现象。细胞自噬是肿瘤细胞耐药的一个重要原因。高迁移率族蛋白B-1(HMGB1)是高度保守的非组蛋白DNA结合蛋白,在DNA结构、基因转录、基因重组、DNA损伤修复以及细胞存活等方面发挥重要的调控作用;HMGB1在细胞内的功能,与其氧化还原状态和细胞定位息息相关;在应激条件下,可从核内转位至胞质,启动细胞自噬,介导肿瘤细胞对抗癌药物和放疗的抗性。因此,HMGB1是克服肿瘤细胞耐药的潜在靶标。
何贤辉欧阳东云
关键词:细胞自噬高迁移率族蛋白B-1抗癌药物肿瘤耐药
Construction of Soluble Mamu-B~*1703,a Class I Major Histocompatibility Complex of Chinese Rhesus Macaques,Monomer and Tetramer Loaded with a Simian Immunodeficiency Virus Peptide被引量:4
2009年
Chinese-descent rhesus macaques have become more prevalent for HIV infection and vaccine investigation than Indian-origin macaques. Most of the currently available data and reagents such as major histocompatibility complex (MHC) class I tetramers, however, were derived from Indian-origin macaques due to the dominant use of these animals in history. Although there are significant differences in the immunogenetic background between the two macaque populations, they share a few of common MHC class I alleles. We reported in this study the procedure for preparation of a soluble Mamu-B*1703 (a MHC class I molecule of Chinese macaques) monomer and tetramer loaded with a dominant simian immunodeficiency virus (SIV) epitope IW9 (IRYPKTFGW) that was identified to be Mamu-B*1701-restricted in Indian macaques. The DNA fragment encoding the Mamu-B*1703 extracellular domain fused with a BirA substrate peptide (BSP) was amplified from a previously cloned cDNA and inserted into a prokaratic expression vector. In the presence of the antigenic peptide IW9 and light chain β2-microglobulin, the expressed heavy chain was refolded into a soluble monomer. After biotinylation, four monomers were polymerized as a tetramer by phycoerythrin-conjugated streptavidin. The tetramer, having been confirmed to have the right conformation, was a potential tool for investigation of antigen-specific CD8^+ T-lymphocytes in SIV vaccine models of Chinese macaques. And our results also suggested that some antigenic peptides reported in Indian-origin macaques could be directly recruited as ligands for construction of Chinese macaque MHC tetramers.
Dongyun OuyangXiaoying WangXianhui HeLihui XuHuanjing ShiQi GaoHe Guo
关键词:TETRAMER
负载SIV抗原肽的中国恒河猴Mamu-B*1703可溶性单体及其四聚体的制备和鉴定被引量:2
2009年
目的:制备负载SIV抗原肽的中国恒河猴Mamu-B*1703可溶性单体及其四聚体。方法:以含Mamu-B*1703重链cDNA序列的pMD19-T克隆为模板,通过PCR的方法克隆Mamu-B*1703重链基因,进而构建羧基端融合生物素化酶BirA底物肽(BSP)的Mamu-B*1703重链胞外域融合蛋白的表达载体,并在大肠杆菌中获得表达。β微球蛋白、SIV抗原肽共存时,通过稀释法复性可溶性Mamu-B*1703单体,经生物素化并纯化后与荧光素标记的链亲和素按4∶1的比例混合形成四聚体。结果:ELISA检测显示获得具有正确构象的负载SIV抗原肽的Mamu-B*1703四聚体。结论:印度恒河猴Mamu-B*1701特异性抗原肽IW9,与中国恒河猴的Mamu-B*1703相结合形成可溶性Mamu-B*1703/IW9单体和四聚体。
王笑迎欧阳东云何贤辉徐丽慧施焕敬高琦郭贺
关键词:恒河猴猴免疫缺陷病毒四聚体
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