水牛CD14 cDNA的克隆、表达及鉴定

目的:克隆水牛白细胞分化抗原14(buffalo cluster of differentiation antigen14,bCD14)基因,表达bCD14蛋白,并进行Western Blot鉴定.方法:采用RT-PCR方法从水牛外周血白细胞中扩增bCD14基因,构建重组质粒pET28a-bCD14,转化入E coli BL21,IPTG诱导表达,对表达蛋白进行可溶性分析及Western blot鉴定.结果:bCD14基因含有一个1 122bp的开放阅读框,编码373个氨基酸;与印度水牛、挪威大鼠和人CD14的cDNA序列同源性分别为97.95%、68.78%、78.60%,氨基酸同源性分别为96.78%、61.27%、72.34%;主要以包涵体形式表达,表达蛋白经Western Blot鉴定,得到了一条约46 kD的特异性条带.结论:该文成功克隆了bCD14基因,表达了bCD14蛋白,为进一步揭示水牛抵抗革兰氏阴性菌感染的免疫机制奠定了基础.     

布鲁菌抗原的快速克隆与高效表达

目的:通过Gateway技术构建布鲁菌抗原表达载体,并筛选出高效可溶性表达载体。方法:以山羊布鲁菌16M株染色体DNA为模板,扩增4个布鲁菌抗原基因BMEI2002、BMEI1069、BMEI1483和BMEI0748,利用GatewayBP反应将基因克隆到入门载体pDONR201中,构建重组质粒,然后用Gateway LR反应将基因重组到3种表达载体(pDEST17、pHXGWA、pHGGWA)中,构建相应的重组表达质粒,将重组质粒转化大肠杆菌ER2566(DE3)并诱导表达,分析利用3个不同载体所表达蛋白的表达量及表达形式。结果:利用BP反应构建了4个基因的重组质粒,用LR反应将这些基因分别克隆到表达载体,构建得到了相应的表达载体;诱导表达后的可溶性分析显示,含6×His和TRX标签的pHXGWA所表达的蛋白在表达量和可溶性方面均优于pDEST17和pHGGWA。结论:通过Gateway技术实现了布鲁菌抗原的快速克隆,筛选到的pHXGWA可作为后续大规模克隆表达载体,为布鲁菌抗原的大规模克隆表达和保护性抗原的筛选奠定了基础。     

Expression Cloning of Camelid Nanobodies Specific for Xenopus Embryonic Antigens

Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.     

Establishment of HLA-DR4 Transgenic Mice for the Identification of CD4+ T Cell Epitopes of Tumor-Associated Antigens

Reports have shown that activation of tumor-specific CD4⁺ helper T (Th) cells is crucial for effective anti-tumor immunity and identification of Th-cell epitopes is critical for peptide vaccine-based cancer immunotherapy. Although computer algorithms are available to predict peptides with high binding affinity to a specific HLA class II molecule, the ability of those peptides to induce Th-cell responses must be evaluated. We have established HLA-DR4 (HLA-DRA*01:01/HLA-DRB1*04:05) transgenic mice (Tgm), since this HLA-DR allele is most frequent (13.6%) in Japanese population, to evaluate HLA-DR4-restricted Th-cell responses to tumor-associated antigen (TAA)-derived peptides predicted to bind to HLA-DR4. To avoid weak binding between mouse CD4 and HLA-DR4, Tgm were designed to express chimeric HLA-DR4/I-E^d, where I-E^d α1 and β1 domains were replaced with those from HLA-DR4. Th cells isolated from Tgm immunized with adjuvant and HLA-DR4-binding cytomegalovirus-derived peptide proliferated when stimulated with peptide-pulsed HLA-DR4-transduced mouse L cells, indicating chimeric HLA-DR4/I-E^d has equivalent antigen presenting capacity to HLA-DR4. Immunization with CDCA1_55-78 peptide, a computer algorithm-predicted HLA-DR4-binding peptide derived from TAA CDCA1, successfully induced Th-cell responses in Tgm, while immunization of HLA-DR4-binding Wilms'' tumor 1 antigen-derived peptide with identical amino acid sequence to mouse ortholog failed. This was overcome by using peptide-pulsed syngeneic bone marrow-derived dendritic cells (BM-DC) followed by immunization with peptide/CFA booster. BM-DC-based immunization of KIF20A_494-517 peptide from another TAA KIF20A, with an almost identical HLA-binding core amino acid sequence to mouse ortholog, successfully induced Th-cell responses in Tgm. Notably, both CDCA1_55-78 and KIF20A_494-517 peptides induced human Th-cell responses in PBMCs from HLA-DR4-positive donors. Finally, an HLA-DR4 binding DEPDC1_191-213 peptide from a new TAA DEPDC1 overexpressed in bladder cancer induced strong Th-cell responses both in Tgm and in PBMCs from an HLA-DR4-positive donor. Thus, the HLA-DR4 Tgm combined with computer algorithm was useful for preliminary screening of candidate peptides for vaccination.     

斜带石斑鱼CD4基因克隆和组织表达分析

CD4 作为TCR的共受体可以提高TCR/抗原-MHC复合体的稳定性,辅助TCR识别抗原,并且参与T细胞活化.本研究从斜带石斑鱼Epinephelus coioides头肾中克隆得到全长2240 bp的CDM cDNA序列,该序列包含长1410 bp的ORF,编码469个氨基酸,预测蛋白分子包含一段信号肽,4个Ig样区...     

Wistar大鼠CD9的cDNA克隆、原核表达及生殖腺中的检测

为了用Wistar大鼠研究CD9对精卵融合的影响和与其它精卵融合相关蛋白的作用,克隆了Wistar大鼠的CD9cDNA.测序结果显示,Wistar大鼠的CD9 cDNA编码区与GenBank中发布的SD( Sprague-Dawley)大鼠相同,但在3′非翻译区多一个T.用Western blotting方法检测Wistar大鼠睾丸和卵巢总蛋白发现睾丸和卵巢里均表达内源性CD9蛋白,分子量相同.此外,在大肠杆菌中表达了GST-CD9融合蛋白,并用GST标签纯化CD9蛋白,为体外研究CD9与其它精卵融合相关蛋白的作用提供参考.     

结核分枝杆菌4种抗原的基因克隆、表达、纯化和抗原性初步检测

目的:克隆38kD、ESAT-6、CFP10和MPT64等4种结核分枝杆菌抗原基因,利用大肠杆菌表达系统分别表达重组蛋白,纯化并初步评价其抗原性。方法:通过PCR方法从结核分枝杆菌H37Rv株基因组中扩增38kD、ESAT-6、CFP10和MPT64抗原的基因,连接入pBVIL1表达载体,在大肠杆菌HB101株中进行表达,以间接ELISA方法初步评价其抗原性。结果:获得了结核分枝杆菌抗原38kD、ESAT-6、CFP10和MPT64的基因,并在大肠杆菌中进行了高效表达,初步验证所纯化获得的抗原具有良好的抗原性。结论:pBVIL1表达载体可以高效表达多种结核分枝杆菌抗原,38kD、ESAT-6和CFP10抗原均可作为结核病血清学诊断的候选抗原。     

Molecular Cloning and Expression Analysis of CD82 in Pig

CD82, which was originally referred to as KAI1 (kangai 1), is a member of the tetraspanin protein family, which contains four transmembrane domains. CD82 is implicated in a variety of biological processes, including apoptosis, cell adhesion, and cell migration. In this study, the full-length cDNA of pig CD82 was cloned and sequenced. Pig Cd82 cDNA contains an open reading frame (801 bp) encoding 266 amino acids. Sequence alignment results indicated that pig CD82 cDNA evidenced 85.45%, 85.63%, 77.03%, and 77.78% identity with human, cattle, rat, and mouse, respectively. In the expression study, the constitutive expression of swine Cd82 mRNA was detected in a variety of tissues, including lymphoid tissues as well as nonlymphoid tissues. Future studies will be focused on the functional role of CD82 during the course of pig infectious diseases or tumor development.     

Cloning and modeling of the first nonmammalian CD4

We have cloned and sequenced the first nonmammalian CD4 cDNA from the chicken using the COS cell expression method. Chicken CD4 contains four extracellular Ig domains that, in analogy to mammalian CD4, are in the order V, C2, V, and C2. The molecule is 24% identical with both human and mouse sequences. The extracellular domains were modeled using human and rat CD4 crystal structures as templates. In the first domain there are two extra Cys residues that are at suitable distance to form an intra-beta-sheet disulfide bridge in addition to the canonical one in the V domain. The region responsible for the interaction with MHC class II is relatively nonconserved in chicken. However, there are positively charged amino acids in the C" region of the N-terminal domain that may mediate the association to the negatively charged residues of the MHC class II beta-chain. Molecular modeling also implies that the membrane-proximal domain mediates dimerization of chicken CD4 in a similar way as it does for human CD4. Furthermore, the cytoplasmic tail is highly conserved, containing the protein tyrosine kinase p56lck recognition site that is preceded by an adjacent di-leucine motif for the internalization of the molecule. Interestingly, there are no Ser residues in the cytoplasmic part, which may explain the slow down-regulation of chicken CD4 after phorbol ester stimulation.     

Optimal Conditions for the Retrieval of CD4 and CD8 Antigens in Formalin-Fixed, Paraffin-Embedded Tissues

The effects of buffer, NaCl, EDTA, and urea on the retrieval of CD4 and CD8 antigens in formaldehyde-fixed, paraffin-embedded tissues with a microwave pressure-cooker were evaluated. The optimal retrieval conditions were found to be borate buffer at pH 8 containing 1 mM NaCl and 1 mM EDTA. Urea was found to be less effective.     

Local CD4 and CD8 T-Cell Reactivity to HSV-1 Antigens Documents Broad Viral Protein Expression and Immune Competence in Latently Infected Human Trigeminal Ganglia

Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1–infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.     

Identification of CD4 and transferrin receptor antibodies by CXCR4 antibody-guided Pathfinder selection.

To generate human antibodies against CXCR4, a seven-transmembrane chemokine receptor and a principal coreceptor for HIV-1, several rounds of Pathfinder and Step-back selection from a large phage display antibody library were performed on Jurkat cells. A mAb against CXCR4 or biotinyated phage antibodies were used as guide molecules. Over 100 pan-Jurkat-cell-positive antibodies were characterized, but none were CXCR4 specific. However, several antibodies against CD4 and the transferrin receptor were identified. Our results indicate that, although Pathfinder and Step-back selection can be used to select phage antibodies on whole cells, the successful selection of certain targets is still complex and limited. The reason is probably, in part, due to the inaccessibility of the targeted extracellular structures and the range of the horseradish peroxidase-labeled guide molecule. Refinements of these techniques are required to improve target specificity and selectivity.     

Cloning,expression, and characterization of fugu CD4, the first ectothermic animal CD4

We have cloned and sequenced the first ectothermic animal CD4 gene from fugu, Takifugu rubripes, using a public database of the third draft sequence of the fugu genome. The fugu CD4 gene encodes a predicted protein of 463 amino acids containing four extracellular immunoglobulin (Ig)-like domains, a transmembrane region, and a cytoplasmic tail. Fugu CD4 shares low identity of about 15–20% with avian and mammalian CD4 proteins. Unlike avian and mammalian CD4, fugu CD4 lacks the Cys pair of the first Ig-like domain, but has a unique possible disulfide bond in the third domain. These differences suggest that fugu CD4 may have a different structure that could affect binding of major histocompatibility complex class II molecules and subsequent T-cell activation. In the putative fugu cytoplasmic region, the protein tyrosine kinase p56^lck binding motif is conserved. The predicted fugu CD4 gene is composed of 12 exons, differing from other CD4 genes, but showing conserved synteny and many conserved sequence motifs in the promoter region. RT-PCR analysis demonstrated that the fugu CD4 gene is expressed predominantly in lymphoid tissues. We also show that fugu CD4 can be expressed on the surface of cells via transfection. Molecular characterization of CD4 in fish provides insights into the evolution of both the CD4 molecule and the immune system.     

Heterogeneity of Expression of CD68 and Other Macrophage-associated Antigens in Human Long-term Bone Marrow Culture

The authors have performed an immunohistochemical study of intact human long-term bone marrow cultures (hLTBMC) grown directly onto glass slides. Between 4 and 12 weeks of growth, such cultures consist of a complex stromal layer supporting foci of haemopoietic cells which undergo granulocytic and monocytic differentiation. As part of a large panel of antibodies employed to characterize monocytes and macrophages within hLTBMC, we included six different anti-CD68 reagents and three antibodies representing a putative new CD group recognizing a macrophage-associated antigen of 130 kDa molecular weight. These gave heterogeneous immunostaining patterns with macrophages and stromal myofibroblastic cells.     

人ING4的基因克隆及真核表达

目的克隆人生长抑制因子家族（inhibitor of growth famility member4,ING4）基因,构建其真核表达载体pEGFP—ING4。方法提取人胎盘总RNA,经RT—PCR扩增出ING4 cDNA,克隆至pEGFP—C2载体,构建的真核表达载体pEGFP—ING4用双酶切、基因测序进行序列鉴定;转染MCF-7细胞用荧光显微镜和免疫组化检测重组质粒的表达。结果RT—PCR产物为750bp的条带,双酶切和基因测序正确,转染可见目的蛋白融合表达。结论从人胎盘组织中成功克隆了ING4基因并构建其真核表达质粒在人MCF-7细胞中表达,为进一步研究1NG4基因的作用及抗肿瘤机制奠定了基础。     

酵母PHO4基因的克隆与表达

用原位杂交方法从酵母菌染色体ＤＮＡ中分段克隆,拼接,获得完整的ＰＨＯ４基因,全长约３．４ｋｂ,利用体内同源重组的方法,构建了ＰＨＯ４基因缺失突变株。ＰＨＯ４基因的缺失导致酸性磷酸酯酶活性明显下降,将完整的ＰＨＯ４基因转入这种缺陷细胞,能使酶活性回复到野生型水平,ＰＨＯ４起正调控作用,构建ＰＨＯ４－ＬａｃＺ融合基因,以β－半乳糖苷酶的活力表示ＰＨＯ４的表达水平。融合基因不同名的表达表明,ＰＨＯ４基因     

Cloning, Expression, and Affinity Purification of RecombinantShigella flexneriInvasion Plasmid Antigens IpaB and IpaC

Shigella flexneriand related enteropathogenic bacteria are important agents of bacillary dysentery, a potentially life-threatening illness for children in underdeveloped regions of the world. Onset of shigellosis stems fromS. flexneriinvasion of colonic epithelial cells, leading to localized cell death and inflammation. Invasion plasmid antigens (Ipa) B, C, and D are three secreted proteins encoded by the large virulence plasmid ofS. flexnerithat have been implicated as essential effectors of this cell invasion process. These proteins are expressed as part of theipaoperon and are among the major targets of the host immune response to shigellosis. Biochemical characterization of the Ipa invasins has been complicated by the fact they have not been purified in the quantities needed for detailedin vitroanalysis. Here we describe the first cloning, expression, and extensive purification of IpaB and IpaC fusion proteins fromEscherichia colifor use in dissecting of the protein biochemistry ofS. flexneripathogenesis. A variety of approaches were used to prepare significant quantities of these proteins in their soluble forms, including the use of different host cell lines, modification of bacterial growth conditions, and the use of alternative plasmid expression vectors. Now that these Ipa proteins are available in a highly pure form, it will be possible to initiate studies on their important biological and immunological properties as well as their recruitment into high-molecular-weight protein complexes. Together with IpaD (purified as part of a previous study), these purified proteins will be useful for: (a) exploring properties of the host immune response toS. flexneriinvasion, (b) elucidating the specific biochemical properties that lead to pathogen internalization, (c) analyzing the importance of specific Ipa protein complexes in host cell invasion, and (d) monitoring, or perhaps even augmenting, the efficacy of live oral vaccines in human trials.