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

(1)建立了检测细胞表面hsp70样分子的单克隆抗体#067和NT22，并对其进行了鉴定。利用重组缺失突变体HSP70将NT22抗原表位定位到HSP70上。利用NT22抗原表位氨基酸序列的退化引物进行了NT22抗原编码基因的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研究中。(2)采用信号序列陷阱法克隆编码#067抗原或NT22抗原的基因。到目前为止还没有获得阳性克隆。逆转录病毒表达克隆仍在进行中。(3) 067抗原对双阴性t细胞可呈递肽抗原。建立了对067抗原有应答的G/d t细胞杂交瘤克隆。对这些克隆的TCR基因使用分析表明，克隆中使用了一组独特的g/d TCR基因。结果表明，067抗原可能向特定的t细胞亚群呈递肽。(4)检测TAP与HSP70的分子相互作用。发现HSP70与TAP相关。此外，合成的能够与HSP70结合的多胺化合物抑制了这种结合，从而减少了tap介导的胞质肽的易位。对不同抗原肽的HSP70亲和力分析表明，具有高HSP70亲和力的肽可被TAP优先转运。我们的研究首次表明MHC i类呈递肽可能被细胞质溶胶中的HSP70选择。(5)克隆了内质网HSP40家族蛋白hej -1的编码基因。通过免疫重组蛋白制备了HEDJ-1特异性抗体。分析了HEDJ-1的表达和功能。提示HEDJ-1可能参与了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