Hiraki D D, See-Tho K, Filvaroff E, Krishnaswamy S, de Bello W, Taidi-Laskowski B, Grumet F C
Department of Pathology, Stanford University School of Medicine, California.
Hum Immunol. 1994 Jul;40(3):235-46. doi: 10.1016/0198-8859(94)90074-4.
A soluble, secreted form of HLA-B7 was engineered by replacing the exons encoding the transmembrane and cytoplasmic domains of the B7 gene with a CI. The modified gene, gsB7, transfected into J27.2 or C1R cell lines, produced a secreted protein, sB7, serologically recognized as B7. Size fractionation showed one species of sB7 at the approximately 55 kD expected for an sB7 alpha-chain-beta 2m heteroduplex, and another at approximately 120 kD which had the same constituent chains and was a dimer of the 55-kD species. Dimer formation appeared to be related to protein concentration but not to disulfide bridging. The sB7 heavy chain on SDS-PAGE showed a doublet at approximately 39 and approximately 42 kD; enzyme analysis indicated that the two bands differed only by a carboxyl terminal polypeptide. Analysis of gsB7 transfectants' mRNA by Northern blots and PCR revealed message fully spliced or with retained CI, accounting for the 39- and 42-kD bands, respectively, and apparently untranslated message with I3 retained. sB7 was not detectable on the surface of gsB7 transfectants by CTLs, nor did it inhibit those CTLs. Production of the sB7 protein provides a ready, consistent source of soluble class I antigen for further study, including test materials for tolerogenicity studies in animal models.
通过用CI取代编码B7基因跨膜和胞质结构域的外显子，构建了一种可溶性分泌形式的HLA - B7。将修饰后的基因gsB7转染到J27.2或C1R细胞系中，产生了一种分泌蛋白sB7，血清学上被识别为B7。大小分级显示，一种sB7的分子量约为55 kD，这是sB7α链-β2m异二聚体预期的分子量，另一种约为120 kD，其组成链相同，是55 kD物种的二聚体。二聚体的形成似乎与蛋白质浓度有关，而与二硫键桥接无关。SDS - PAGE上的sB7重链显示在约39 kD和约42 kD处有一条双带；酶分析表明，这两条带仅在羧基末端多肽上有所不同。通过Northern印迹和PCR分析gsB7转染体的mRNA，发现有完全剪接或保留CI的信息，分别对应39 kD和42 kD的条带，并且明显保留了带有I3的未翻译信息。CTLs在gsB7转染体表面未检测到sB7，它也没有抑制那些CTLs。sB7蛋白的产生为进一步研究提供了一种现成、稳定的可溶性I类抗原来源，包括用于动物模型致耐受性研究的测试材料。
