表达超抗原SEA基因的溶瘤腺病毒载体的构建与鉴定

目的:构建表达超抗原SEA基因的溶瘤腺病毒载体并鉴定.方法:采用PCR技术,从产SEA的葡萄球菌标准菌株ATCC13565基因组DNA中获得SEA全长基因序列,酶切后克隆入pCA13质粒,构建重组病毒质粒pCA13-SEA.将鉴定正确的pCA13-SEA与含有腺病毒右臂的质粒pBHGE3通过Lipofectamine2000共转染HEK293细胞,经同源重组产生重组腺病毒Ad-SEA.Ad-SEA在293细胞中大量扩增并通过氯化铯密度梯度离心法纯化、测定其滴度.结果:经PCR扩增、酶切鉴定、序列测定证实,SEA基因成功克隆到溶瘤腺病毒载体中,可实现SEA基因的表达.结论:成功构建了表达超抗原SEA基因的溶瘤腺病毒载体,为进一步研究该病毒对膀胱肿瘤靶向治疗的作用奠定了基础.     

超抗原SEA增强小鼠对HBV DNA 疫苗的免疫反应

观察超抗原SEA(D227A)的真核表达载体(pmSEA),对HBVDNA疫苗诱导Balbc小鼠(H2d)免疫应答的调节作用。肌内注射空载体pcDNA3、HBVDNA疫苗加pmSEA佐剂(pHBVS2S+pmSEA)或不加佐剂(pHBVS2S);ELISA法测定血清抗HBs;ELISPOT检测分泌IFNγ的脾淋巴细胞;4h51Cr释放法检测小鼠脾细胞CTL活性。HBVDNA佐剂组免疫小鼠抗HBsAg抗体滴度明显高于不加佐剂组,其IgG1IgG2a的比例不同于多肽免疫组,二者分别为0.282与10。HBVDNA佐剂组均能增强IgG1和IgG2a的产生,是不加佐剂组的1.36、1.73倍。佐剂组小鼠脾淋巴细胞IFNγ的分泌量是不加佐剂组2～3倍。CTL细胞杀伤活性(E:T=100)佐剂组与不加佐剂组分别为:69.77%±7.5%、42.81%±7.7%,差异显著(P<0.05)。HBVDNA疫苗具有较强的免疫原性,能够诱导机体产生特异性的抗体及CTL反应;pmSEA佐剂能够提高小鼠对DNA疫苗的免疫应答,有望成为DNA疫苗的免疫佐剂。     

超抗原SEA增强小鼠对HBV DNA 疫苗的免疫反应

观察超抗原SEA(D227A)的真核表达载体（pmSEA）, 对HBV DNA 疫苗诱导Balb/c 小鼠(H2d）免疫应答的调节作用。 肌内注射空载体pcDNA3、HBV DNA 疫苗加pmSEA佐剂（pHBVS2S+pmSEA）或不加佐剂（pHBVS2S）; ELISA 法测定血清抗HBs; ELISPOT检测分泌IFN-γ的脾淋巴细胞;4 h51Cr 释放法检测小鼠脾细胞CTL 活性。HBV DNA佐剂组免疫小鼠抗HBsAg抗体滴度明显高于不加佐剂组,其IgG1/IgG2a的比例不同于多肽免疫组,二者分别为0.282与10。HBV DNA佐剂组均能增强IgG1和IgG2a的产生,是不加佐剂组的1.36、1.73倍。佐剂组小鼠脾淋巴细胞IFN-γ的分泌量是不加佐剂组2～3倍。CTL 细胞杀伤活性（E:T=100）佐剂组与不加佐剂组分别为：69.77%±7.5%、 42.81%±7.7%,差异显著(P<0.05)。HBV DNA 疫苗具有较强的免疫原性, 能够诱导机体产生特异性的抗体及CTL反应;pmSEA佐剂能够提高小鼠对DNA 疫苗的免疫应答,有望成为DNA 疫苗的免疫佐剂。     

Epstein—Barr病毒膜抗原在中国仓鼠卵巢（CHO）细胞中的表达

将切去３＇端穿膜序列的ＥＢ病毒膜抗原（ＭＡ）基因,插入ｐＳＶ２－ｄｈｆｒ质粒的ＳＶ４０早期启动子下游,构建了真核表达载体ｐＳＶ２－ｄｈｆｒＧＰＴＲ,使两个ＳＶ４０早期启动子分别调控ＭＡ和二氢叶酸还原酶（ｄｈｆｒ）基因。将该重组质粒转化ＣＨＯ－ｄｈｆｒ细胞。在选择培养基中筛选阳性克隆,用氨甲喋呤加压扩增,建立了表达ＥＢＶ－ＭＡ的克隆细胞系。Ｗｅｓｔｅｒｎ ｂｏｌｔ分析证明,所表达的蛋白的分子量大约为     

p53转染黑色素瘤细胞修饰的树突状细胞疫苗体外抗肿瘤作用的研究

利用野生型p53质粒转染黑色素瘤B16细胞,反复冻融法提取p53修饰的肿瘤抗原(p53-Ag),将抗原体外冲击同基因小鼠骨髓来源的树突状细胞(dendritic cells,DC)制备特异性DC肿瘤疫苗;观察DC诱导的淋巴细胞增殖反应和细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)对黑色素瘤细胞的细胞毒效应,分析其诱导肿瘤抗原特异性免疫应答的机制。结果显示,p53-肿瘤抗原冲击的DC可显著刺激淋巴细胞增殖,其诱导的CTL效应对肿瘤细胞也有很好的杀伤效果。     

超抗原葡萄球菌肠毒素A、B和C1的T细胞抗原HLA表位预测

为预测超抗原葡萄球菌肠毒素（SE）家族中的SEA、SEB和SEC1的HLAⅠ和HLAⅡ抗原结合表位,并对其活化T细胞作用的机理进行探讨,根据已发表的SEA、SEB和SEC1基因全序列,用T细胞抗原表位预测软件Guotif2.0对其进行T细胞抗原表位预测,统计与HLAⅠ和HLAⅡ各抗原位点结合的SEA、SEB和SEC1肽段的出现次数。结果显示,SEA、SEBT SEC1具有共同的特点,即都是主要与HLAⅠ类分子的A3位点和HLAⅡ类分子的DR1位点具有较强的结合。说明SEA、SEB和SEC1与HLAⅠ类分子和HLAⅡ类分子都有很强的结合性。三者在HLAⅠ和HLAⅡ结合位点上具有较强的同源性。本研究为SE活化T细胞作用机制的功能实验提供了依据。     

超抗原及靶向治疗肿瘤作用进展

超抗原作为一种强大的T细胞激活荆,单用极低浓度便可激活大量的T淋巴细胞克隆来杀伤肿瘤细胞,但这种杀伤作用缺乏特异性.靶向治疗是现阶段肿瘤治疗的新技术,可针对各种机制来抑制肿瘤的发生和发展或消除肿瘤.时此,通过单抗导向或将超抗原结合于肿瘤细胞表面以及基因工程等手段,国内外的学者已在超抗原的靶向抗肿瘤治疗方面开展了大量工作,为肿瘤的防治提供了参考依据.     

CCR7和B7-2在抗原负载树突状细胞诱导特异性CTL抗肿瘤效应中的表达和意义

目的:研究CCR7(趋化因子受体7)和B7-2(白细胞分化抗原86)与抗原负载树突状细胞(dentritic cell,DC)诱导特异性CTL(细胞毒性T淋巴细胞)抗肿瘤效应的关系.方法:分离和培养DC,制备B16黑色素瘤细胞抗原,进行共培养,即为抗原负载的DC,建立B16黑色素瘤小鼠模型,于肿瘤周围皮下注射抗原负载的DC.应用原位杂交和免疫组织化学方法检测CCR7和B7-2的表达情况.结果:原位杂交和免疫组织化学染色显示,CCR7和B7-2阳性细胞主要分布于肿瘤周围组织,随着注射抗原负载DC时间的进展,CCR7和B7-2呈强阳性表达.结论:CCR7和B7-2的表达与抗原负载树突状细胞诱导特异性CTL抗肿瘤效应有关.     

树突状细胞和抗原负载树突状细胞在肿瘤模型鼠体内的分布和形态学观察

目的:研究体外培养的小鼠树突状细胞(dentritic cells,DCs)和抗原负载树突状细胞在肿瘤模型鼠体内的分布和形态,为肿瘤的生物学治疗提供形态学基础.方法:分离和培养DC,制备B16黑色素瘤细胞抗原,进行共培养,即为抗原负载的DC,Brdu标记DC和抗原负载的DC,建立B16黑色素瘤小鼠模型,于瘤周围皮下注射Brdu标记的DC和抗原负载的DC.应用光镜、免疫组化方法和透射电镜观察DC和抗原负载DC在肿瘤模型鼠体内的分布和形态.结果:免疫组化染色显示Brdu标记的抗原负载DC与DC比较,体积较大.实验组Brdu标记的DC和抗原负载DC分布的数密度和面密度,分别与对照组比较,有显著差异(P＜0.01).电镜下抗原负载DC细胞与DC比较体积较大,核有切迹,细胞表面的突起较粗大弯曲,形态较成熟.结论:抗原负载DC比DC更易集聚于肿瘤组织周围,推测抗原负载DC比Dc可能诱导抗肿瘤效应更强.     

超抗原SEA与抗黑色素瘤ScFv融合基因的构建及表达

采用酶切连接和重叠PCR连接两种方法将抗黑色素瘤单链抗体基因和去除N端信号肽的金黄色葡萄球菌肠毒素A基因进行融合,并将融合基因克隆于pET28a表达载体上,转化大肠杆菌BL21(DE3)。用NiNTA系统对表达产物进行分离、纯化。MTT法检测融合蛋白对黑色素瘤细胞的体外抑制率。结果表明6HisScFvSEA融合蛋白可在E.coli BL21(DE3)中稳定表达,表达量占菌体蛋白的30%,主要以包涵体的形式存在。融合蛋白可通过激活效应细胞对表达相关抗原的黑色素瘤细胞发挥抑制作用。     

Intercellular transfer regulation of the paracrine activity of GPI-anchored Cripto-1 as a Nodal co-receptor

Cripto-1 (CR-1) is a glycosylphosphatidylinositol-anchored glycoprotein which acts as an obligate co-receptor of a TGFβ family ligand, Nodal. Previous studies have demonstrated that CR-1 functions in a paracrine fashion by a cellular mechanism which has not been fully described. This paracrine activity was observed only when CR-1 was expressed as a membrane-bound form and was abolished when CR-1 was expressed in a soluble form. In the current study, we found that there were few biochemical differences in post-translational modifications between membrane-anchored and soluble forms of CR-1. Flow cytometric analysis revealed an intercellular transfer of the membrane-bound form of CR-1 between cells. CR-1-expressing cells formed unique membrane extensions, generated more membrane fragments than control cells, and exhibited enhanced cellular adhesion. Thus, expression of CR-1 may alter the physiochemical properties of the plasma membrane resulting in an enhancement of intercellular transfer of cellular signaling components which may account for the paracrine activity of CR-1.     

Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts within the membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factors controlling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins in supported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The transmembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains. Replacement of the transmembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted the association of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with the rafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchored proteins in supported lipid bilayers was investigated further using membrane dipeptidase (MDP) whose GPI anchor contains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used but associated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on the distribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supported lipid bilayers provide the first direct evidence that the nature of the membrane-anchoring domain influences the association of a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchored proteins in rafts.     

糖基化磷脂酰肌醇锚定型EGFP真核表达质粒的构建及表达

构建与增强型绿色荧光蛋白基因相连的糖基化磷脂酰肌醇(glycosyl phosphatidylinositol,GPI)序列的真核表达质粒,并检测其在A549细胞中的表达.分离人外周血淋巴细胞,提取总RNA,以RT-PCR法扩增CD24基因的243 bp GPI锚定序列,双酶切后定向克隆入pEGFP-C1质粒中,构建并鉴定pEGFP-C1-GPI质粒.经脂质体介导转染A549细胞后,在荧光显微镜下观察目的蛋白在真核细胞内的表达情况.经酶切和测序鉴定证实,所克隆的CD24 GPI序列正确,荧光显微镜观察pEGFP-C1-GPI质粒转染A549细胞可见围绕细胞膜的强绿色荧光,而对照pEGFP-C1质粒转染A549细胞仅见胞内均匀荧光.成功构建与EGFP相连的GPI真核表达质粒,且能在A549细胞膜上锚定表达EGFP-GPI融合蛋白,为构建锚定表达型肿瘤疫苗奠定基础.     

Construction of an <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> cell-surface display system using a putative GPI-anchored protein

A novel cell-surface display system was constructed in Aspergillus oryzae. Each of the five genes encoding the putative cell-wall-localized protein from the A. oryzae genome was cloned and these cell-surface anchor functions were examined by fusion to the C-terminal of the green fluorescent protein (GFP). Using the MP1 and CWP proteins as anchor proteins, GFP signals were strongly observed on the cell surface of recombinant A. oryzae. When these proteins were used as anchor proteins for cell-surface display of β-glucosidase from A. oryzae, enzyme activity was detected on the cell surface. In particular, β-glucosidase activity of recombinant A. oryzae using MP1, a putative glycosylphosphatidylinositol (GPI) anchor protein was higher than CWP. Based on these results, it was concluded that the MP1 protein can act as a GPI-anchor protein in A. oryzae, and the proposed cell-surface display system using MP1 allows for the display of heterogeneous and endogenous proteins.     

GP2/THP gene family of self-binding, GPI-anchored proteins forms a cluster at chromosome 7F1 region in mouse genome

We investigated the genomic organization of pancreatic zymogen granule membrane-associated protein GP2, a GPI-anchored protein exhibiting self-aggregation at acidic pH, in order to construct a gene-knockout mouse. Cloning and analysis of lambda clones encoding GP2 from 129 Svj mouse genomic DNA libraries showed that the GP2 gene spans about 16.8 kb and includes 11 exons. Identifiable functional domains including a signal sequence, an EGF-like motif, a putative condensing ZP domain, a GPI-anchor attachment site, and a transmembrane sequence for GPI anchoring are encoded in separate exons. Using FISH, the GP2 gene was mapped to mouse chromosome 7F1 near the gene for THP, a GP2 homolog expressed in the cells of thick ascending loop of Henle (TALH) in the kidney. Further analysis of the mouse genome revealed that the THP and GP2 genes are adjacent to one another and are separated by only 3.5 kb in the 7F1 locus. Additionally, the overall structure of the THP gene, 16.2kb with 11 exons, was strikingly similar to that of GP2. This finding suggests that the GP2 and THP genes were generated by gene duplication and evolved separately to acquire regulatory elements leading to tissue-specific expression. Comparative analysis revealed that the 5' flanking region of the THP gene is similar to the first intron of NKCC2, a TALH cell-specific ion-transporter gene. The promoter region of the GP2 gene shares cis-elements found in other pancreas-specific genes. Using this genetic information, a GP2 null mutation was successfully introduced into an ES cell line, and an animal model was established without disruption of THP expression.     

Different glycoforms of the human GPI-anchored antigen CD52 associate differently with lipid microdomains in leukocytes and sperm membranes

CD52 is a human GPI-anchored antigen, expressed exclusively in the immune system and part of the reproductive system (epididymal cells). Sperm cells acquire the antigen from the epididymal secretions when transiting in the epididymal corpus and cauda. The peptide backbone of CD52, consisting of only 12 aminoacids, is generally considered no more than a scaffold for post-translational modifications, such as GPI-anchor and especially N-glycosylation which occur at the third asparagine. The latter modification is highly heterogeneous, especially in the reproductive system, giving rise to many different glycoforms, some of which are tissue specific. A peculiar O-glycan-containing glycoform is also found in reproductive and immune systems. We determined to locate CD52 in microdomains of leukocytes and sperm membranes using two antibodies: (1) CAMPATH-1G, the epitope of which includes the last three aminoacids and part of the GPI-anchor of glycoforms present in leukocytes and sperm cells; (2) anti-gp20, the epitope of which belongs to the unique O-glycan-bearing glycoform also present in both cell types. Using a Brij 98 solubilization protocol and sucrose gradient partition we demonstrated that the CD52 glycoforms recognized by both antibodies are markers of typical raft microdomains in leukocytes, whereas in capacitated sperm the O-glycoform is included in GM3-rich microdomains different from the cholesterol and GM1-rich lipid rafts with which CAMPATH antigen is stably associated. The importance of the association between GM3 and O-glycans for formation of specialized microdomains was confirmed by heterologous CD52 insertion experiments. When prostasomes from human seminal fluid were incubated with rat sperm from different epididymal regions, the CD52 glycoform recognized by anti-gp20 decorated rat epididymal corpus and cauda sperm, associated with the same low-cholesterol GM3-rich sperm membrane fractions as in human sperm. The glycoforms recognized by CAMPATH-1G were not found in rat sperm. The relationship between this differential insertion and differences in glycosylation of rat and human CD52 is discussed.     

N-Glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin-Darby canine kidney cells.

Glycosyl-phosphatidylinositol (GPI)- anchored proteins are preferentially transported to the apical cell surface of polarized Madin-Darby canine kidney (MDCK) cells. It has been assumed that the GPI anchor itself acts as an apical determinant by its interaction with sphingolipid-cholesterol rafts. We modified the rat growth hormone (rGH), an unglycosylated, unpolarized secreted protein, into a GPI-anchored protein and analyzed its surface delivery in polarized MDCK cells. The addition of a GPI anchor to rGH did not lead to an increase in apical delivery of the protein. However, addition of N-glycans to GPI-anchored rGH resulted in predominant apical delivery, suggesting that N-glycans act as apical sorting signals on GPI-anchored proteins as they do on transmembrane and secretory proteins. In contrast to the GPI-anchored rGH, a transmembrane form of rGH which was not raft-associated accumulated intracellularly. Addition of N-glycans to this chimeric protein prevented intracellular accumulation and led to apical delivery.     

Plasmodium falciparum Pf34, a novel GPI-anchored rhoptry protein found in detergent-resistant microdomains

Apicomplexan parasites are characterised by the presence of specialised organelles, such as rhoptries, located at the apical end of invasive forms that play an important role in invasion of the host cell and formation of the parasitophorous vacuole. In this study, we have characterised a novel Plasmodium falciparum rhoptry protein, Pf34, encoded by a single exon gene located on chromosome 4 and expressed as a 34kDa protein in mature asexual stage parasites. Pf34 is expressed later in the life cycle than the previously described rhoptry protein, Rhoptry Associated Membrane Antigen (RAMA). Orthologues of Pf34 are present in other Plasmodium species and a potential orthologue has also been identified in Toxoplasma gondii. Indirect immunofluorescence assays show that Pf34 is located at the merozoite apex and localises to the rhoptry neck. Pf34, previously demonstrated to be glycosyl-phosphatidyl-inositol (GPI)-anchored [Gilson, P.R., Nebl, T., Vukcevic, D., Moritz, R.L., Sargeant, T., Speed, T.P., Schofield, L., Crabb, B.S. (2006) Identification and stoichiometry of GPI-anchored membrane proteins of the human malaria parasite Plasmodium falciparum. Mol. Cell. Proteomics 5, 1286-1299.], is associated with parasite-derived detergent-resistant microdomains (DRMs). Pf34 is carried into the newly invaded ring, consistent with a role for Pf34 in the formation of the parasitophorous vacuole. Pf34 is exposed to the human immune system during infection and is recognised by human immune sera collected from residents of malaria endemic areas of Vietnam and Papua New Guinea.