Cloning ofagene encoding 70 kDa heat shock protein expressed on the cell surface of mammalian cells

(1)建立并表征了检测细胞表面上的HSP 70样分子的单克隆抗体#067和NT 22。利用重组缺失突变体HSP 70将NT 22的表位定位于HSP 70上。使用来自NT 22表位的氨基酸序列的简并引物进行编码NT 22抗原的基因的PCR克隆。从Daudi细胞cDNA文库中扩增出一个新基因。该基因的序列和表达正在阐明中。(2)编码#067抗原或NT 22抗原的基因的表达克隆通过信号序列捕获法进行。目前尚未获得阳性克隆。逆转录病毒表达克隆仍在进行中。(3)#067抗原的表征表明其可将肽抗原呈递给双阴性T细胞。建立了对#067抗原有应答的G/d T细胞杂交瘤克隆。对克隆的TCR基因使用情况的分析表明，在克隆中使用了一组独特的g/d TCR基因， ...更多信息 内斯表明#067抗原可能将肽呈递给特定的T细胞亚群。(4)TAP和HSP 70之间的分子相互作用进行了研究。发现HSP 70与TAP有关。此外，能够与HSP 70结合的合成多胺化合物抑制了这种结合，从而减少了TAP介导的胞质肽的移位。对各种抗原肽的HSP 70亲和力的分析表明，具有高HSP 70亲和力的肽可以被TAP优先转运。我们的研究表明，第一次，MHC I类呈递肽可能被选择的HSP 70在胞质溶胶中。(5)克隆了编码内质网HSP 40家族蛋白HEDJ-1的基因。通过免疫重组蛋白制备了识别HEDJ-1的特异性抗体。分析了HEDJ-1的表达和功能。提示HEDJ-1可能参与上皮细胞和浆细胞分泌蛋白的质量控制。少

(1) Monoclonal antibodies #067 and NT22, which detected HSP70-like molecules on the cell surface, were established and characterized. The epitope of NT22 was mapped on HSP70 by using recombinant deletion mutant HSP70. PCR cloning of a gene encoding NT22 antigen was performed using degenerated primers from the amino acid sequence of the NT22 epitope. A novel gene was amplified from cDNA library of Daudi cells. The sequence and expression of the gene are on the way to elucidation.(2) Expression cloning of a gene encoding #067 antigen or NT22 antigen was performed by a signal sequence trap method. No positive clone has been obtained so far. Retroviral expression cloning is still on the way.(3) Characterization of #067 antigen suggested that it could present peptide antigens to double negative T-cells. G/d T-cell hybridoma clones that were responsive to #067 antigen were established. Analysis of TCR gene usage of the clones revealed that a unique set of g/d TCR gene was utilized in the clo … More nes. It was indicated that #067 antigen might present peptides to a specific subset of T-cells.(4) Molecular interaction between TAP and HSP70 was examined. It was found that HSP70 was associated with TAP. In addition, synthetic polyamine compound that was capable of binding to HSP70 inhibited the ssociation, thereby decreasing TAP-mediated translocation of cytosolic peptides. Analysis of HSP70 affinity of various antigenic peptides revealed that peptides with high HSP70 affinity could be preferentially transported by TAP. Our study showed for the first time that MHC class I-presented peptides might be selected by HSP70 in the cytosol.(5) A gene encoding HEDJ-1, HSP40 family protein in the endoplasmic reticulum, was cloned. Specific antibody recognizing HEDJ-1 was developed by immunizing a recombinant protein. Expression and fuction of HEDJ-1 have been analyzed. It was indicated that HEDJ-1 might be involved in the quality control of secretary proteins in epitheial cells and plasma cells. Less