人扁桃体B淋巴细胞mRNA的差异显示分析及活化B细 …

目的 分离新的Ｂ细胞活化基因。方法 采用差异显示反转录ＰＣＲ技术（ＤＤＲＴ－ＰＣＲ）对人扁桃体活化和静止Ｂ细胞 ｍＲＮＡ的差异显示情况进行分析，对显示片段进行克隆并行Ｎｏｒｔｈｅｒｎ分析，对Ｎｏｒｔｈｅｒｎ杂交阳性的ｃＤＮＡ片段进行测序并比较同源性。     

编码人分化抗原5C5全长cDNA的克隆及功能初探

目的 编码５Ｃ５分化抗原全长ｃＤＮＡ的克隆及功能探索。方法 构建人活化Ｂ细胞株３Ｄ５细胞的λｇｔ１１ ｃＤＮＡ文库，以核苷酸杂交法从ｃＤＮＡ文库中筛选阳性克隆、作核苷酸序列分析，编码蛋白质氨基酸序列的亲疏水分析，Ｎｏｒｔｈｅｒｎ ｂｌｏｔ测５Ｃ５ ｍＲＮＡ转录本长度，用ＲＴ－ＰＣＲ检测５Ｃ５ ｍＲＮＡ在不同细胞株的表达，观察单抗５Ｃ５－Ｇ１对３Ｄ５细胞增殖的影响。结果 从人活化Ｂ细胞株３Ｄ５的λｇ     

MPTP诱导的褐鼠黑质基因差异表达的研究

本研究目的在于获取１－甲基－４－苯基－１，２，３，６－四氢吡啶（ＭＰＴＰ）诱导的褐鼠黑质差异表达的表达序列标签（ＥＳＴ）。通过ＭＰＴＰ腹腔注射建立褐鼠帕金森病模型，利用差别显示反转录ＰＣＲ（ＤＤＲＴ－ ＰＣＲ）技术分析ＭＰＴＰ 处理褐鼠与正常褐鼠黑质基因差异表达的状况，并筛选差异表达基因片段。结果经ＤＤＲＴ－ＰＣＲ 筛选并通过反Ｎｏｒｔｈｅｒｎ 杂交去除假阳性，最终获得２ 个差异表达基因片段。对其中一个片段进行克隆、测序和同源性比较发现，该片段为一个新的ＥＳＴ 序列，并且与胰岛素原样α肽具有同源性。本研究的结果提示，差异表达序列的分析有可能为研究ＭＰＴＰ诱导的褐鼠黑质变性的分子机理提供线索。     

B7—1（CD80）基因的克隆及其在小鼠黑色素瘤细胞中的表达

应用ＲＴ－ＰＣＲ技术从人Ｂ淋巴瘤细胞系Ｒａｊｉ中克隆到Ｂ７－１（ＣＤ８０）ｃＤＮＡ，并经测序证实，将其插入至真核表达载体ｐＬＸＳＮ中，用脂质体转染小鼠黑色素瘤细胞Ｂ１６，Ｇ４１８筛选，并经流式细胞仪检测，得到了细胞表面表达Ｂ７－１的重组克隆。ＰＣＲ从其基因组ＤＮＡ中扩增到Ｂ７－１基因，提示Ｂ７－１ ｃＤＮＡ已整合至宿主细胞的基因组ＤＮＡ中，本文为研究Ｂ７－１在肿瘤免疫中所起的作用奠定了基础。     

THANK基因的克隆和序列分析

目的 克隆ＴＨＡＮＫ基因全长及胞外区片段并测序。方法 采用ＰＴ－ＰＣＲ技术,唑人白血病细胞系ＨＬ－６０细胞总ＰＡＮ中扩增人ＴＨＡＮＫ ｃＤＮＡ,并定向克隆于ｐＭＤ－１８Ｔ载体,转染大肠杆菌、抽提质粒后,用ＡＢＩ ＰＲＩＳＭ＾ＴＭ３７７ＸＬＤＮＡ 自动测义测序。结果ＲＴ－ＰＣＲ扩增出一个８５８ｂｐ的ＤＮＡ片段,限制性内切酶图谱分析和测序结果显示：该８５８ｂｐ片段为编码入ＴＨＡＮＫ的ｃＤＮＡ,与公布     

U937细胞表达成纤维细胞生长因子受体

以逆转录－ＤＮＡ聚合酶链反应（ＲＴ－ＰＣＲ）自Ｕ９３７细胞钓出了两条ｃＤＮＡ片段。将其克隆入ＴＡ克隆载体后进行ＤＮＡ测序。结果证明ｃＤＮＡ片段分别是人成纤维细胞生长因子受体１（ＦＧＦＲ１）的细胞外段和细胞内段ｃＤＮＡ。这一结果表明，Ｕ９３７细胞可以表达ＦＧＦＲ１ｍＲＮＡ。     

人MBL相关丝氨酸蛋白酶-1 cDNA的克隆与鉴定

目的 获得人甘露聚糖结合凝集素（ＭＢＬ）相关丝氨酸蛋白酶－１（ＭＡＳＰ－１）基因。方法 以人胎肝组织总ＲＮＡ为模板,采用ＲＴ－ＰＣＲ获取目的ｃＤＮＡ片段,克隆入ｐＧＥＭ－Ｔ载体,进行酶切图谱分析和测序鉴定。结果 以ＲＴ－ＰＣＲ方法获得了含信号顺序的全长ＭＡＳＰ－１ｃＤＮＡ,将其与ｐＧＥＭ－Ｔ载体连接,转化大肠杆菌ＴＧ１,建立了ＭＡＳＰ－１的ｃＤＮＡ克隆,酶切图谱与微机分析结果一致。序列分析表明。与     

差异显示逆转录PCR技术及其应用和进展

差异显示逆转录ＰＣＲ（ＤＤＲＴ－ＰＣＲ）技术是一种筛选和克隆差异表达基因的新方法。该方法利用ｃＤＮＡ逆转录技术、ＰＣＲ（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ）的高效扩增和凝胶电泳分离技术，能同时显示多种来自相关细胞的ｍＲＮＡ样品。可用于多种研究目的，如鉴别和观察基因表达的改变；差异表达基因的迅速测序并与基因库比较；单个的差异表达基因片段能迅速克隆并制成探针从ｃＤＮＡ文库或基因组文库中分离基因等。ＤＤＲＴ－ＰＣＲ技术具有迅速、简便、灵敏和高效等优点，已被广泛地应用于癌症、心脏疾病、细胞分化、衰老及其它领域的研究中，并且在应用中被不断地改进和发展而更趋合理和完善。     

B组轮状病毒ADRV与RDRV主要基因的克隆及其相关？…

对２株ＰＡＧＥ图型相似的Ｂ组轮状病毒－成人腹泻轮状病毒（ａｄｕｌｔｄａｒｒｈｅａｒｏｔａｖｉｒｕｓ,ＡＤＲＶ）和大白鼠服腹泻轮状病毒（ｒａｔｄｉａｒｒｈｅａｒｏｔａｖｉｒｕｓ,ＲＤＲＶ）主要基因的相关性进行研究。方法ＲＴ－ＰＣＲ扩增ＡＤＲＶ第４、５、９三个基因的ｃＤＮＡ全长拷贝,并获其ｃＤＮＡ克隆。用ＡＤＲＶ第９基因末端引物ＲＴ－ＰＣＲ成功扩增了ＲＤＲＶｄｓＲＮＡ,获得相对分子质量与ＡＤＲＶ第９基     

T细胞识别HLA—DRB1＊0901分子显示TCR BV基因取用的高 …

目的 分析ＤＲ９纯合细胞刺激下ＤＲ９阴性正常人ＴＣＲ ＢＶ基因片段的取用格局,确定和分离出ＤＲ９关联性疾病中的自身反应性Ｔ细胞史隆。方法 常规方法获取ＰＢＭＣ经ＰＣＲ－ＳＳＯ和ＰＣＲ－ＲＦＬＰ ＤＮＡ分型,确定ＤＲ、ＤＱ、ＤＰ等位基因后,进行单向混合淋巴细胞培养,抽提总ＲＮＡ并合成北一链ｃＤＮＡ,定量ＰＣＲ检测２２种ＴＣＲ ＢＶ 基因片段的取用格局。结果 Ｔ细胞对ＤＲ９分子的识别和扩增具有寡克隆笥     

人B细胞活化相关基因BC-1514全长cDNA的克隆与原核表达

目的 克隆与B细胞活化相关的新基因及其原核表达。方法 采用差异显示反转录PCR（DDRT-PCR）技术对人扁桃体活化和静止B细胞mRNA的差异表达进行分析。差异显示的片段经过Northern杂交验证后,作为探针进行入活化B细胞cDNA文库的筛选,将所获得的阳性克隆的编码区经PCR扩增后克隆到原核表达载体pGEX-5X-1中,重组质粒经酶切,测序鉴定后转化大肠杆菌BL-21,以IPTG诱导表达融合蛋白。结果 以在活化B细胞高表达的EST32为探针,经3轮筛选人活化B细胞文库获得一个新的全长为1514bp的cDNA克隆（命名为BC-1514）,重组的BC-1514蛋白可在E.coli中以融合蛋白的形式有效表达,其表达量约占细菌总蛋白量的14.3%左右,BC-1514cDNA的GenBank的登录号为AF304442。结论 获得了1条新的与B细胞活化相关的cDNA克隆并在E.coliBL-21中得到了有效表达。     

实验性脑脓肿组织中G蛋白信号系统相关基因的克隆

目的 筛选和克隆实验性脑脓肿的早期相关基因，研究脑组织对病原菌的免疫反应过程中所涉及的分子机制。方法通过脑组织内直接注射金黄色葡萄球菌获得大鼠脑脓肿，利用mRNA荧光差异显示PCR技术比较脑脓肿组脑组织中mRNA的表达与正常对照组、手术对照组之间的差异，获得的差异表达cDNA片段经克隆、测序及BLAST软件进行同源性比较分析，并采用Noahem杂交和RT-PCR进行鉴定。结果共获得25条差异表达的cDNA条带，经Noahem杂交和RT-PCR鉴定其中17条为阳性（阳性率为68％）。克隆和测序结果显示其中3个差异表达片段与G蛋白相关的细胞信号传导系统有关：片段G18-1代表的鸟苷二磷酸解离抑制因子3（GD13）、片段G20-1代表的交集素（ITSN）以及片段C2-1代表的Ras相关蛋白（Rap1）。结论在实验性脑脓肿早期GD13、ITSN和Rap1差异表达，推导G蛋白信号系统的激活可能与脑脓肿的发病相关。     

cDNA代表性差异分析法分离鼻咽癌上皮细胞株HNE1表达差异cDNA序列的初步研究

目的寻找鼻咽癌中差异性表达基因，包括与鼻咽癌发病相关的候选抑瘤基因。方法应用ｃＤＮＡ代表性差异分析法（ＲＤＡ），分离原代培养的正常人鼻咽上皮细胞与鼻咽癌细胞株ＨＮＥ１中差异表达的ｃＤＮＡ序列，Ｓｏｕｔｈｅｒｎ杂交和Ｎｏｒｔｈｅｒｎ杂交被用来分析差异性表达产物的来源，最后，将这些序列克隆到ｐＧＥＭ－Ｔｅａｓｙ载体中，并用链终止法测序。结果在第４轮杂交及扩增反应后，获得４条差异性条带。Ｓｏｕｔｈｅｒｎ杂交及Ｎｏｒｔｈｅｒｎ杂交证明，这些差异性片段来自作为“检测”扩增子的正常人鼻咽上皮，在鼻咽癌细胞株ＨＮＥ１中不表达或表达降低。序列分析这些差异性片段的克隆，发现一些序列是与已知基因高度同源的基因，包括一些看家基因；另有一些基因则为新基因序列。结论鼻咽癌的发生是一个多基因参与的过程，所获得的差异性片段中，与之同源的一些已知基因具有抑瘤功能。     

Identification of an alpha2,6-sialyltransferase induced early after lymphocyte activation.

We have used mRNA differential display PCR to search for genes induced in activated T cells and we identified a gene encoding an alpha2,6-sialyltransferase (ST6GalNAc IV) that is rapidly induced in lymphocytes after antigen or mitogen stimulation. The 3.6 kb full-length cDNA clone (MK45) obtained contained a single open reading frame encoding a 302 amino acid protein and a 2.5 kb 3' untranslated region. MK45 expression in in vivo-activated CD8 T cells reached the highest level 4 h after antigen triggering and then declined rapidly to nearly base levels within 45 h. Northern blot analysis further revealed that MK45 expression was also induced in LPS-activated B cells and antigen-triggered CD4 T cells in vitro. MK45 expression was low or undetectable in most other mouse tissues examined, when compared to activated lymphocytes. Importantly, the mRNA expression level of other sialyltransferases remained largely unchanged during the early stage of lymphocyte activation. Finally, increased ecto-sialyltransferase activity and an altered sialylation pattern were demonstrated on the cell surface of early activated CD8 T cells. Our report identifies a candidate sialyltransferase gene that is involved in the early alteration of the sialylation pattern of cell surface molecules in activated lymphocytes.     

锌对大鼠脾脏淋巴细胞功能的影响

通过大鼠锌（ＺＤ），过量锌（ＺＥ）模型，研究３锌对脾脏Ｔ、Ｂ淋巴细胞功能的影响，结果：①ＺＤ、ＺＥ组的脾重及脾脏指数，与对喂（ＰＦ）组、自由喂养（ＡＬ）组，缺锌后再补锌（ＺＤ＋ＡＬ）组比较，差别非常显著。②ＺＤ、ＺＥ组外周血白细胞减低，以淋巴细胞为主。③ＺＤ、ＺＥ组Ｔ、Ｂ淋巴细胞增殖率非常显著低于ＰＦ、ＡＬ和ＺＤ＋ＡＬ组。④ＺＤ、ＺＥ组的总脾淋巴细胞，Ｔ、Ｂ淋巴细胞细胞周期Ｇ０／Ｇ１％增高，而Ｓ％     

Distinct helper activities control growth or maturation of B lymphocytes

A clone (C-11) of C3H/HeJ Lyt-1+2-T cells with specificity for "minor" antigens of C3H/Tif has been isolated which, in contrast to other similarly derived clones, did not activate polyclonal plaque-forming cell (PFC) responses in T cell-depleted "target" spleen cells. This clone, however, showed unaltered proliferative responses to the naturally occurring antigen(s) on presenting cells, and strongly synergized with regular helper clones in the induction of PFC responses. Further analysis demonstrated that C-11 cells are competent to stimulate extensive "target" B cell proliferation, but lack the ability to produce (or participate in the production of) maturation factors for activated B cells. Thus, the defective PFC responses could be fully reconstituted with supernatants from regular clones stimulated with antigen, but not by supernatants prepared from the C-11 cells themselves. While it is not clear whether this clone represents a normal helper T cell subpopulation or a variant that has lost maturation-factor production, these results demonstrate that distinct factors control growth and maturation in T cell-dependent B lymphocyte responses.     

具有GPX活性的单克隆抗体可变区基因的克隆和序列分析

目的利用分泌具有GPX活性的单克隆抗体(mAb)的杂交瘤细胞3G5,克隆其mAb体的可变区基因。方法提取杂交瘤细胞的总RNA ,分离mRNA ,反转录合成cDNA。经PCR扩增VH 基因和VL 基因 ,将VH 基因和VL基因与载体 pGEM T连接后 ,进行酶切鉴定和序列分析。结果构建了2个分别含有VH 和VL 基因的重组质粒。序列分析表明 ,VH 和VL 分别属于mouscheavychainsubgroupIII和mouselightchainsubgroupV亚群 ,长度为372和324bp ,编码124和108个氨基酸 ,在高变区分别有4和2个丝氨酸。结论克隆的VH 和VL 基因符合功能性重排的鼠抗体可变区基因特征 ,为将来制备具有GPX活性的单链抗体提供了可靠的基因材料。     

Molecular characterization of the early activation antigen CD69: A type II membrane glycoprotein related to a family of natural killer cell activation antigens

CD69 is a disulfide-linked homo-dimer expressed on the surface of activated T cells, B cells, natural killer cells, neutrophils and platelets. Antibody cross-linking of CD69 in the presence of phorbol ester results in cellular activation events including proliferation and the induction of specific genes. Using an expression cloning strategy we have isolated cDNA encoding human CD69 from a CD4⁺ T cell clone. Transfection of the cDNA clone in CV-1/EBNA cells results in the expression of a covalently linked homodimer. The cDNA insert hybridizes to a 1.7-kb mRNA in phorbol 12-myristate 13-acetate- or phytohemoagglutinin-stimulated human T cells. Using the human clone we have isolated cDNA encoding mouse CD69, which, when expressed in human T cells allowed those cells to respond to anti-mouse CD69 antibodies by secreting interleukin-2 and interferon-γ. Sequence analysis showed that both mouse and human CD69 are type II membrane glycoproteins related to the NKR-P1 and Ly-49 families of natural killer cell activation molecules.     

Molecular cloning of a second phospholipase B gene, caPLB2 from Candida albicans.

Accumulating evidence suggests that phospholipase B, secreted by pathogenic fungi such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, functions as one of the virulence factors. In the present study, we have attempted to clone phospholipase B gene from C. albicans. By RT-PCR analysis with degenerate primers based on conserved regions of phospholipase B from Saccharomyces cerevisiae, Penicillium notatum and Torulaspora delbrueckii two similar but different cDNA fragments were obtained. One corresponded to the partial sequence of caPLB1, recently cloned phospholipase B gene from C. albicans by a different approach (Leidich et al.: J Biol Chem 1998; 273: 26078-86). The other fragments contained sequences similar to the corresponding sequences of phospholipase B from other fungi. The presence of two related genes was confirmed by Southern and Northern blot analyses. The full length of the second C. albicans phospholipase B gene (caPLB2) encoded a putative protein with 608 amino acids and contained a potential signal peptide sequence and a putative catalytic region, which are found in phospholipase B from other fungi. Consistent with the findings of caPLB1, caPLB2 also lacks a cluster of hydrophobic amino acids at the COOH-terminal, which may function as a signal of glycosylphosphatidylinositol anchor.     

A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2 fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed to plastic resulted in weak T cell activation, and these T cells did not induce B cell responses. Haptenated B cell populations, although recognized by E9.D4, were not activated. Separation of T and B cells by a 0.4-micron membrane prevented T-dependent B cell activation, although Th cell-derived B cell-activating lymphokines would be assayed across these membranes. These results suggest a polyclonal noncognate B cell activation that depends on physical contact between B cells and activated T cells. The requirement for a cognate interaction of Th with B cells for the production and delivery of B help can therefore be overcome by activating Th cells with high densities of T cell receptor ligands.