ORP4L 多克隆抗体的制备及其在蛋白质组学研究中的应用

目的:原核表达并纯化人氧化固醇结合蛋白相关蛋白4(ORP4L)肽段,制备兔抗人ORP4L多克隆抗体,并利用其进行蛋白质组学研究。方法:应用PCR技术扩增人ORP4L 382-485氨基酸(ORP4Lm)的基因序列并插入到PGEX-4T-1载体中,在大肠杆菌RosettaTM(DE3)中表达融合蛋白GST-ORP4Lm。利用所表达的融合蛋白中含有的GST标签进行亲和纯化。用所获得的纯化蛋白免疫新西兰大白兔,获得兔抗人ORP4L多克隆抗体。用Western blotting检测抗体免疫特异性。将亲和纯化后的抗体偶联到CNBr-actived sepharose beads上,利用免疫沉淀的方法,通过质谱仪分析鉴定可能与ORP4L存在相互作用的蛋白质。通过West-ern blotting进一步确证特异性的相互作用蛋白。结果:在大肠杆菌中表达并纯化了GST-ORP4Lm重组蛋白,用其免疫新西兰大兔,成功制备了相应兔源多克隆抗体,Western blotting证实该抗体可以特异识别内源性及外源性的ORP4L蛋白。质谱分析和Western blotting的结果表明所制备的多克隆抗体可以用于蛋白质组学研究。结论:利用重组的GST-ORP4Lm融合蛋白成功制备了有良好特异性的ORP4L多克隆抗体,并可将其用于ORP4L的蛋白组学研究。     

人生长分化因子15的原核表达、纯化与多克隆抗体制备

目的:原核表达并纯化、鉴定人生长分化因子15(GDF-15),制备其多克隆抗体。方法:从人结肠癌细胞系HT29的cDNA扩增出GDF-15基因片段并插入pET-32a(+)原核表达载体,转化大肠杆菌BL21,IPTG诱导表达重组GDF-15,用镍亲和柱纯化,SDS-PAGE、Western印迹鉴定重组蛋白。用纯化的重组GDF-15免疫BALB/c小鼠制备多克隆抗体,鉴定并检测其效价。结果:制备了pET-32a(+)-GDF-15表达载体;经IPTG诱导重组蛋白表达后,采用Ni亲和柱纯化蛋白,并经SDS-PAGE和免疫印迹鉴定;免疫BALB/c小鼠后获得了GDF-15多克隆抗体,ELISA检测抗体效价为1∶100000,并应用于肿瘤细胞的GDF-15检测中。结论:用基因工程和免疫学方法制备了重组人GDF-15及其多克隆抗体,为后续的分子机制和靶向治疗研究奠定了基础。     

抗人DR5多克隆抗体的制备与纯化

目的:分离纯化融合蛋白GST-eDR5,免疫小鼠制备抗人DR5多克隆抗体.方法:用GST纯化试剂盒和电泳两次纯化的融合蛋白GST-eDR5免疫小鼠,制备抗CST-eDR5抗血清,然后用GST纯化得到抗人DR5抗血清.通过Westem blot、ELISA方法鉴定抗血清特异性和效价.结果:通过亲和纯化得到了高纯度的融合蛋白GST-eDR5,其浓度为0.65μg/μl.免疫产生的DR5抗血清特异性高,且效价高达1:25600,纯化后的抗人DR5抗血清不再识别GST,只识别人DR5.结论:成功制备了高特异性、高效价的抗人DR5多克隆抗体,为深入研究其对肿瘤细胞的生长抑制和凋亡作用提供了实验工具.     

人抗酶抑制因子-1的原核表达纯化及多克隆抗体的制备

原核表达纯化人抗酶抑制因子-1((antizyme inhibition factor-1,AZIN1),制备并鉴定抗AZIN1多克隆抗体。p ET-28a/AZIN1表达质粒转化大肠杆菌BL21(DE3)后,IPTG诱导蛋白表达,利用Ni-NTA树脂于变性条件下亲和层析纯化人AZIN1蛋白。将重组AZIN1蛋白用作抗原免疫BALB/c小鼠以制备多克隆抗体,ELISA检测抗AZIN1抗体效价,Western bloting、细胞免疫荧光、细胞免疫化学方法检测抗体的应用。结果显示,重组p ET-28a/AZIN1表达质粒经酶切及测序鉴定构建正确。细菌内重组AZIN1蛋白可被IPTG诱导表达并以包涵体的形式存在。用亲和层析法能有效纯化原核表达的AZIN1蛋白,该蛋白在小鼠体内能够诱导抗AZIN1特异性抗体产生,血清效价达到1640 000。制备抗体能够特异性识别和结合人及小鼠瘤细胞中表达的AZIN1蛋白,并可有效用于AZIN1的Western blotting、细胞免疫荧光和细胞免疫化学分析。成功原核表达和纯化了人AZIN1蛋白并制备了抗AZIN1多克隆抗体,为深入研究AZIN1在调控细胞增殖及在疾病防治中的作用提供了研究基础。     

猪FcγRIII的原核表达及多克隆抗体的制备

目的：构建猪FcγRIII 基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪 FcγRIII 抗血清。方法：从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII 原核表达载体pET-FcγRIII ,转化大肠杆菌BL21 (DE3) ,IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His 为抗原免疫小鼠,获得抗血清。Western blotting、ELISA 法鉴定获得的抗血清,ELISA 结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果：成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA 法证实多克隆抗体制备成功。结论：成功获得了猪 FcγRIII 多克隆抗体,为进一步研究猪FcγRIII 蛋白的功能奠定了基础。     

DNA损伤检查点蛋白调节子1片段的表达纯化及抗体制备

目的：原核表达、纯化DNA损伤检查点蛋白调节子1（MDC1）片段,并制备其多克隆抗体。方法：设计特异引物,通过RT-PCR扩增编码MDC1 N端194个氨基酸残基的基因片段,测序正确后插入含GST基因的原核表达载体pGEX-KG中,以IPTG诱导表达,并经谷胱甘肽琼脂糖珠纯化融合蛋白;用纯化的蛋白免疫小鼠制备多克隆抗体,用ELISA测定抗体的效价,Western印迹鉴定抗体的特异性。结果：原核表达并纯化了MDC1 N端片段,并获得了抗MDC1的多克隆抗体,抗体效价达到1∶12800,Western印迹显示该抗血清能特异识别原核及真核细胞表达的MDC1。结论：MDC1 N端片段能够诱导小鼠产生具有较高效价和特异性的多克隆抗体,为进一步研究MDC1在Fhit特异信号通路中的作用奠定了基础。     

拟南芥转录因子NF-YC的原核表达及多克隆抗体制备

采用PCR方法扩增NF-YC基因得到其全长cDNA序列,并将其克隆至原核表达载体pET-48b中,在大肠杆菌BL21中用IPTG诱导出分子量约为45 kD的融合蛋白,SDS-PAGE和Western blotting检测鉴定表达产物。利用亲和层析技术对融合蛋白进行纯化,纯化后的目的蛋白免疫新西兰兔制备多克隆抗体。间接ELISA检测抗体效价大于1 62 500,Western blotting结果显示,该抗体可特异性识别NF-YC蛋白。     

Ataxin-3融合蛋白表达、纯化及抗血清的制备

目的:表达GST-ataxin-3-N融合蛋白并制备GST-ataxin-3特异性抗体,为深入研究其功能及其在SCA3发病机制中的作用提供重要的技术和材料保障.方法:将人ataxin-3氨基端基因克隆入原核表达载体pGEX-4T-2,在大肠杆菌(E.coli)BL21中表达,用Glutathione sepharose4B凝胶亲和柱纯化目的蛋白.利用纯化的GST-ataxin-3-N蛋白制备多克隆抗体.结果:成功构建了原核表达载体,得到高表达量的融合蛋白,经亲和层析柱纯化获得较高纯度的GST-ataxin-3-N融合蛋白.以融合蛋白免疫新西兰兔得到Ataxin-3-N多克隆抗体,Western Blotting及免疫荧光均证实该抗体能够识别Ataxin-3-myc蛋白,具有较高特异性.结论:利用原核表达人GST-ataxin-3-N融合蛋白制备的Ataxin-3多克隆抗体具有较好的特异性,可用于该蛋白的相关研究.     

抗CD133胞外区骆驼多克隆抗体的制备及鉴定

目的:制备高效价、高特异性的抗CD133胞外区新疆双峰驼来源的多克隆抗体,为制备高亲和力的抗CD133纳米抗体做准备。方法:将CD133胞外区基因序列构建到原核表达载体pET28a中,诱导表达及纯化CD133蛋白,免疫新疆双峰驼及新西兰兔。通过酶联免疫吸附实验(ELISA)和Western blot检测多克隆抗体的效价及与CD133特异性结合活性。结果:ELISA测定抗-CD133骆驼源抗体的效价可达到百万以上,通过Western blot检测多克隆抗体可特异性结合CD133蛋白。结论:重组人CD133蛋白可以在骆驼体内激发高滴度抗体反应,为今后构建骆驼免疫单域抗体噬菌体展示文库奠定基础。     

ORP4L多克隆抗体的制备及其在蛋白质组学研究中的应用水

目的：原核表达并纯化人氧化固醇结合蛋白相关蛋白4（ORP4L）肽段,制备兔抗人ORP4L多克隆抗体,并利用其进行蛋白质组学研究。方法：应用PCR技术扩增人ORP4L382-485氨基酸（ORP4Lm）的基因序列并插入到PGEX-4T—1载体中,在大肠杆菌RosettaTM（DE3）中表达融合蛋白GST-ORP4Lm。利用所表达的融合蛋白中含有的GST标签进行亲和纯化。用所获得的纯化蛋白免疫新西兰大白兔,获得兔抗人ORP4L多克隆抗体。用Western blotting检测抗体免疫特异性。将亲和纯化后的抗体偶联到CNBr-actived sepharosC beads上,利用免疫沉淀的方法,通过质谱仪分析鉴定可能与ORP4L存在相互作用的蛋白质。通过West—ernblotting进一步确证特异性的相互作用蛋白。结果：在大肠杆菌中表达并纯化了GST-ORP4Lm重组蛋白,用其免疫新西兰大兔．成功制备了相应兔源多克隆抗体,Western blotting证实该抗体可以特异识别内源性及外源性的ORP4L蛋白。质谱分析和Western blotting的结果表明所制备的多克隆抗体可以用于蛋白质组学研究。结论：利用重组的GST-ORP4Lm融合蛋白成功制备了有良好特异性的ORP4L多克隆抗体,并可将其用于ORP4L的蛋白组学研究。     

Processing and localization of the african swine fever virus CD2v transmembrane protein

The African swine fever virus (ASFV)-encoded CD2v transmembrane protein is required for the hemadsorption of red blood cells around infected cells and is also required for the inhibition of bystander lymphocyte proliferation in response to mitogens. We studied the expression of CD2v by expressing the gene with a V5 tag downstream from the signal peptide near the N terminus and a hemagglutinin (HA) tag at the C terminus. In ASFV-infected cells, a full-length glycosylated form of the CD2v protein, which migrated mainly as a 89-kDa product, was detected, as well as an N-terminal glycosylated fragment of 63 kDa and a C-terminal nonglycosylated fragment of 26 kDa. All of these forms of the protein were localized in the membrane fraction of cells. The 26-kDa C-terminal fragment was also produced in infected cells treated with brefeldin A. These data indicate that the CD2v protein is cleaved within the luminal domain and that this occurs in the endoplasmic reticulum or Golgi compartments. Confocal microscopy showed that most of the expressed CD2v protein was localized within cells rather than at the cell surface. Comparison of the localization of full-length CD2v with that of a deletion mutant lacking all of the cytoplasmic tail apart from the 12 membrane-proximal amino acids indicated that signals within the cytoplasmic tail are responsible for the predominant localization of the full-length and C-terminal 26-kDa fragment within membranes around the virus factories, which contain markers for the Golgi compartment. Processing of the CD2v protein was not observed in uninfected cells, indicating that it is induced by ASFV infection.     

抗人CD28单链抗体基因的构建及在BmN细胞和家蚕中的表达

采用TP-PCR法,将抗人CD28单链抗体的重、轻链可变区基因和人工设计的昆虫杆状病毒多角体蛋白基因(ph)偏好的连接肽序列,拼接成抗人CD28单链抗体(抗CD28-ScFv)基因,并将其插入昆虫杆状病毒转移载体pBacPAK8的ph启动子,构建成重组转移载体pBacPAK8/CD28-ScFv。为便于表达产物的纯化,在CD28-ScFv的C端增加了6×Histag序列。通过脂质体介导法将重组转移载体pBacPAK8/CD28-ScFv和线性化病毒Bm-BacPAK6共转染BmN细胞,经空斑分析和蓝白斑筛选,获得重组杆状病毒Bm-BacPAK6 CD28-ScFv。将重组病毒Bm-BacPAK6 CD28-ScFv感染BmN细胞和5龄家蚕,SDS-PAGE和Western Blotting分析表明,在分子量约为28kD处有一表达带。BmN细胞的表达始于24h,表达峰在72h,96h时表达量开始下降,而5龄家蚕的表达始于48h,表达峰在120h。结果证明,抗人CD28单链抗体基因在BmN细胞和家蚕中得到了特异性表达。上述研究成果为抗人CD28基因工程抗体的研制奠定了基础。     

Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.     

The CD24 protein inducible expression system is an ideal tool to explore the potential of CD24 as an oncogene and a target for immunotherapy in vitro and in vivo

CD24 is a cell surface, heavily glycosylated glycosylphosphatidylinositol-anchored mucin-like protein that is overexpressed in various human malignancies. To accurately analyze CD24 function and dissect its biological role in a defined genetic background, it is critical to tightly regulate its expression and be able to turn it on/off in a restricted environment and at a specific time. The tetracycline-induced expression system is most promising as it exhibits such regulation, lack of pleiotropic effects, and high and rapid induction levels. To evaluate the oncogenic and immunotherapeutic potential of CD24 by applying the Tet-On system, the human CD24 gene was cloned downstream to two tetracycline operator sequences, resulting in pCDNA4/TO-CD24, which was then transfected into tetracycline (Tet) repressor-expressing cells (293T-REx), allowing tight on/off regulation, thereby resulting in a very low background or leaky CD24 expression. Selected clones were chosen for further studies and characterized in vitro and in vivo, and several treatment modalities were examined. In addition, the role of CD24 in promoting cell proliferation and tumor growth was studied. The tetracycline-dependent system was successfully implemented. Tetracycline treatment induced CD24 expression in a dose- and time-dependent fashion, which was abrogated following treatment with anti-CD24 monoclonal antibodies (mAbs). CD24-induced expression led to an increased proliferation rate that was inhibited by mAb treatment. In vivo, significantly larger tumors were developed in tetracycline-fed mice. The CD24 Tet-On system is a good model to unravel the role and underlying CD24 pathogenesis in vivo. This valuable tool allows the successful study of novel treatment options, whose effectiveness depends on the CD24 expression level. This set of experiments supports CD24 oncogenic properties.     

CD59天然抗原的制备及纯化

目的 构建人matureCD59-mFC—pBOS真核表达载体,转染COSa细胞进行瞬时表达,对表达产物进行鉴定及纯化。方法在人Jurkat细胞中提取细胞总RNA,运用RT—PCR方法扩增matureCD59基因,定向克隆入带有mFC标签的真核表达载体pBOS中,大提质粒后通过电转仪转染COSa细胞,ELISA及特异抗体检测CD59的表达,并大量收集细胞上清用ProteinA进行纯化。结果构建r重组载体matureCD59．mFC—pBOS,瞬时转染COSa细胞,初步纯化得到天然表达的CD59抗原。结论带有mFC标签的CD59天然抗原的得到为制备抗人CD59单克隆抗体开辟了一条新的途径。     

Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery

Assembled modular complexes for targeted drug delivery can be based on strong non-covalent interactions between a cargo module containing an adapter protein and a docking tag fused to a targeting protein. We have recently constructed a completely humanized adapter/docking tag system based on interactions between 15 amino acid (Hu-tag) and 110 amino acid (HuS) fragments of human ribonuclease I (RNase I). Although recombinant HuS can be expressed and refolded into a functionally active form, the purification procedure is cumbersome and expensive, and more importantly, it yields a significant proportion of improperly folded proteins. Here we describe engineering, high-yield expression, and purification of a chimeric bovine/human RNase (BH-RNase) comprising 1-29 N-terminal amino acids of bovine ribonuclease A and 30-127 amino acids of human RNase I. Unlike RNase I, the chimeric BH-RNase can be cleaved by either subtilisin or proteinase K between A20 and S21, providing a functionally active HuS. The HuS obtained from chimeric BH-RNase differs from wild-type HuS by an N24T substitution; therefore, we have reverted this substitution by mutating N24 to T24 in BH-RNase. This BH-RNase mutant can also be cleaved by subtilisin or proteinase K yielding wild-type HuS. The affinity of HuS obtained from BH-RNase to Hu-tag is approximately five times higher than that for recombinant HuS, reflecting a higher percentage of properly folded proteins.     

CD24 Overexpression Is Associated with Poor Prognosis in Luminal A and Triple-Negative Breast Cancer

CD24 is associated with unfavourable prognoses in various cancers, but the prevalence of CD24 expression and its influence on clinical outcome in subtypes of breast cancers remain unclear. CD24 expression was analyzed by immunohistochemistry in 747 breast cancer tissues, and DNA methylation and histone modification status in the promoter region of CD24 were assessed using bisulfite sequencing and chromatin immunoprecipitation assay. 213 (28.5%) samples exhibited high CD24 expression in the membrane and/or cytoplasm of breast cancer cells, and CD24 overexpression was significantly correlated with the presence of lymph node metastasis and more advanced pathological stage. Patients with CD24-high tumours had significantly shorter patient survival than those with CD24-low tumours. Importantly, multivariate analysis that included tumour size, lymph node metastasis and chemotherapy demonstrated that high CD24 expression is independently associated with poorer survival in luminal A and triple-negative breast cancer (TNBC) subtypes. Furthermore, CD24 gene expression was associated with histone acetylation independent of DNA methylation, suggesting its epigenetic regulation in breast cancer. Our results suggest that CD24 overexpression is an independent unfavourable prognostic factor in breast cancer, especially for luminal A and TNBC subtypes, and CD24 may be a promising therapeutic target for specific subtypes of breast cancer.     

Phenotypic characterization of mammosphere-forming cells from the human MA-11 breast carcinoma cell line

The phenotypic diversity of breast carcinoma may be explained by the existence of a sub-population of breast cancer cells, endowed with stem cell-like properties and gene expression profiles, able to differentiate along different pathways. A stem cell-like population of CD44⁺CD24^−/low breast cancer cells was originally identified using cells from metastatic pleural effusions of breast carcinoma patients. We have previously reported that upon in vitro culture as mammospheres under stem cell-like conditions, human MA-11 breast carcinoma cells acquired increased tumorigenicity and lost CD24 expression compared with the parental cell line. We now report that upon passage of MA-11 mammospheres into serum-supplemented cultures, CD24 expression was restored; the rapid increase in CD24 expression was consistent with up-regulation of the antigen, and not with in vitro selection of CD24⁺ cells. In tumors derived from subcutaneous injection of MA-11 mammospheres in athymic nude mice, 76.1 ± 9.7% of cells expressed CD24, vs. 0.5 ± 1% in MA-11 cells dissociated from mammospheres before injection. The tumorigenicity of sorted CD44⁺CD24⁻ and CD44⁺CD24^high MA-11 cells was equal. Single cell-sorted CD24⁻ and CD24^high MA-11 gave rise in vitro to cell populations with heterogeneous CD24 expression. Also, subcutaneous tumors derived from sorted CD24⁻ sub-populations and single-cell clones had levels of CD24 expression similar to the unsorted cells. To investigate whether the high expression of CD24 contributed to the tumorigenic potential of MA-11 cells, we silenced CD24 by shRNA. CD24 silencing (95%) resulted in no difference in tumorigenicity upon s.c. injection in athymic nude mice compared with mock-transduced MA-11 cells. Since CD24 silencing was maintained in vivo, our data suggest that the level of expression of CD24 is associated with but does not contribute to tumorigenicity. We then compared the molecular profile of the mammospheres with the adherent cell fraction. Gene expression profiling revealed that the increased tumorigenicity of MA-11 mammospheres was associated with changes in 10 signal transduction pathways, including MAP kinase, Notch and Wnt, and increased expression of aldehyde dehydrogenase, a cancer-initiating cell-associated marker. Our data demonstrate that (i) the level of CD24 expression is neither a stable feature of mammosphere-forming cells nor confers tumorigenic potential to MA-11 cells; (ii) cancer-initiating cell-enriched MA-11 mammospheres have activated specific signal transduction pathways, potential targets for anti-breast cancer therapy.     

Multiple amino acid substitutions in hemagglutinin are necessary for wild-type measles virus to acquire the ability to use receptor CD46 efficiently

Measles virus (MV) possesses two envelope glycoproteins, namely, the receptor-binding hemagglutinin (H) and fusion proteins. Wild-type MV strains isolated in B-lymphoid cell lines use signaling lymphocyte activation molecule (SLAM), but not CD46, as a cellular receptor, whereas MV vaccine strains of the Edmonston lineage use both SLAM and CD46 as receptors. Studies have shown that the residue at position 481 of the H protein is critical in determining the use of CD46 as a receptor. However, the wild-type IC-B strain with a single N481Y substitution in the H protein utilizes CD46 rather inefficiently. In this study, a number of chimeric and mutant H proteins, and recombinant viruses harboring them, were generated to determine which residues of the Edmonston H protein are responsible for its efficient use of CD46. Our results show that three substitutions (N390I and E492G plus N416D or T446S), in addition to N481Y, are necessary for the IC-B H protein to use CD46 efficiently as a receptor. The N390I, N416D, and T446S substitutions are present in the H proteins of all strains of the Edmonston lineage, whereas the E492G substitution is found only in the H protein of the Edmonston tag strain generated from cDNAs. The T484N substitution, found in some of the Edmonston-lineage strains, resulted in a similar effect on the use of CD46 to that caused by the E492G substitution. Thus, multiple residues in the H protein that have not previously been implicated have important roles in the interaction with CD46.     

CD24 on the resident cells of the central nervous system enhances experimental autoimmune encephalomyelitis

CD24 is a cell surface glycoprotein that is expressed on both immune cells and cells of the CNS. We have previously shown that CD24 is required for the induction of experimental autoimmune encephalomyelitis (EAE), an experimental model for the human disease multiple sclerosis (MS). The development of EAE requires CD24 expression on both T cells and non-T host cells in the CNS. To understand the role of CD24 on the resident cells in the CNS during EAE development, we created CD24 bone marrow chimeras and transgenic mice in which CD24 expression was under the control of a glial fibrillary acidic protein promotor (AstroCD24TG mice). We showed that mice lacking CD24 expression on the CNS resident cells developed a mild form of EAE; in contrast, mice with overexpression of CD24 in the CNS developed severe EAE. Compared with nontransgenic mice, the CNS of AstroCD24TG mice had higher expression of cytokine genes such as IL-17 and demyelination-associated marker P8; the CNS of AstroCD24TG mice accumulated higher numbers of Th17 and total CD4+ T cells, whereas CD4+ T cells underwent more proliferation during EAE development. Expression of CD24 in CD24-deficient astrocytes also enhanced their costimulatory activity to myelin oligodendrocyte glycoprotein-specific, TCR-transgenic 2D2 T cells. Thus, CD24 on the resident cells in the CNS enhances EAE development via costimulation of encephalitogenic T cells. Because CD24 is increased drastically on resident cells in the CNS during EAE, our data have important implications for CD24-targeted therapy of MS.