Gene Cloning of Nuclear Antigen(s) with Anti-nuclear Antigen(s) SSE Antibodirs: Molecular and Biological Analysis and its Clinical Application

利用系统性红斑狼疮患者的血清，我们从牛视网膜cDNA文库中分离到一个克隆，该克隆能产生具有免疫活性的血清抗体之一的融合蛋白。我们还获得了一个覆盖整个编码区的同源人胎盘基因克隆。测定了该基因的核苷酸序列及其预测的一个Poen阅读框的氨基酸序列。在GenBank和NBRF数据库中没有发现与这些cDNA有显著同源性的序列。这些人和牛的cDNA克隆中的氨基酸序列除了一个氨基酸交换外完全相同。在进化上的这种严格的保守性证明了这个新基因在正常细胞生理中的重要性。我们从重组牛的E.Coli环表达的提取液中纯化了融合蛋白在兔…中制备了该融合蛋白的多克隆抗血清。用这些抗体对培养细胞的细胞核进行特异性染色，在预测的开放阅读框中识别出一个核定位信号氨基酸序列，这些发现提示该基因产物可能是核抗原之一。用固相微滴度ELISA对2 5 0份自身免疫血清进行筛选，发现该融合蛋白的滴度分布与已知的核抗原之一的KI抗原非常相似。用免疫印迹法检测融合蛋白和KI抗原(高崎博士亲手提供的)的结果表明，两者的主要条带在SDSPAGE上具有非常相似的迁移率，并具有共同的抗原表位(S)。综上所述，所克隆的cDNA克隆可能是KI抗原的基因或类似基因(S)核抗原共享交叉反应免疫表位(S)。KI抗原的克隆尚未见报道。较少

Using sera of patients with systemic lupus erythematosus, we have isolated from bovine retina cDNA library a clone that produces a fusion protein immunologically reactive one of the antibodies in the sera.Using this cDNA as a probe,we have also obtained a homologous human placenta cDNA colne covering a full portion of coding region.Nucleotide sequences of the cDNAs and its predicted amino acid sequences of an poen reading frame were determined.No significant homologous sequences to these cDNAs were not detected in the sequences enlisted in the Data-Base of GenBank and NBRF.Comparable amino acid sequences in these human and bovine cDNA clones were exactly the same except one amino acid exchange.Such a strict conservation in evolution argues for the importance of this new gene in normal cell physiology.We have purified the fusion protein from the extract of E.coli carring expression recombinant of the bovine cDNA clone.Polyclonal antisera to this fusion protein were generated in rabbits … More and monoclonal antibody with mouse hybridoma cells.Nuclei of the cultured cells were specifically stained with these antibodies and a nucoear localization signal amino acid sequence was recognized in the predicted open-reading frame.These findings suggest that this gene product may be one of the nuclear antigens. Autoimmune sera (250 samples) were screened against the purified fusion protein by using a solid-phase microtiter ELISA.It was found that the titer distribution for this fusion protein was quite similar to that of the Ki antigen already known as one of the nuclear antigens.already known as one of the nuclear antigens.The fusion protein and Ki antigen (kindly given by Dr Y.Takasaki) were examined by western immunoblots to show that both main bands have a quite similar mobility on SDS-PAGE and share common antigenic epitope(s).In summary isolate cDNA clones seem to be the gene of Ki antigen or analogous gene(s) of nuclear antigen sharing cross-reactive immunological epitope(s). Cloning of the Ki antigen has not been reported. Less