cDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody.
cDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody.
复制标题
通过单克隆抗体对多核糖体进行免疫纯化后获得 HLA-DR 抗原重链的 cDNA 克隆。
DOI：
10.1073/pnas.79.6.1844
复制
发表时间：
1982
影响因子：
11.1
通讯作者：
Strominger,JL
中科院分区：
文献类型：
--
作者：
Korman,AJ;Knudsen,PJ;Kaufman,JF;Strominger,JL
制备了针对分离的人类白细胞抗原-DR重链的单抗(HC-2.1)。通过将HC 2.1与人B淋巴母细胞多聚体结合，然后使用Protein A-Sepharose柱作为免疫吸附剂，我们已经纯化了编码人类白细胞抗原-DR重链的mRNA，几乎是均一的。免疫提纯后的mRNA已用于制备标记的cDNAs，并用其检测cDNA库。从免疫纯化的mRNA中扩增出双链cDNA，直接克隆到pBR322中。两个克隆，每一个来自上述程序，肯定选择DR重链信息，通过无细胞翻译和免疫沉淀检测。一个克隆pDRH-2[500个碱基对+75个碱基对的聚(A)]包含整个3‘非翻译区以及羧基末端亲水性胞内区和部分疏水性跨膜区的编码信息。洗涤剂增溶的(P34)和木瓜酶处理的(P33)的人类白细胞抗原-DR重链的羧肽酶消化结果与预测的蛋白质序列一致。用特定的单抗对多聚体进行特异性免疫纯化，然后将其直接克隆到高纯度的mRNA上，是获得鉴定克隆的一种有效方法。
A monoclonal antibody (HC 2.1) directed against the separated heavy chain of HLA-DR has been prepared. By binding HC 2.1 to polysomes from human B lymphoblastoid cells followed by the use of a protein A-Sepharose column as an immunoadsorbent, we have purified the mRNA coding for the HLA-DR heavy chain nearly to homogeneity. The immunopurified mRNA has been used to prepare labeled cDNA with which to probe cDNA libraries. Double-stranded cDNA was also made from the immunopurified mRNA and cloned directly into pBR322. Two clones, one from each of the above procedures, positively selected DR heavy chain message as assayed by cell-free translation and immunoprecipitation. One clone, pDRH-2 [500 base pairs plus 75 base pairs of poly(A)] contains the entire 3' untranslated region as well as coding information for the carboxy-terminal hydrophilic intracellular domain and part of the hydrophobic transmembrane region. Results of carboxypeptidase digestion of the heavy chains from detergent-solubilized (p34) and papain-treated (p33) HLA-DR antigen were consistent with the predicted protein sequence. Specific immunopurification of polysomes by defined monoclonal antibodies followed by direct cloning of cDNA to the highly purified mRNA is a powerful method for obtaining identified cDNA clones.