Wang M, Stepkowski S M, Hebert J S, Tian L, Yu J, Kahan B D
Department of Surgery, University of Texas Medical School at Houston, USA.
Ann Transplant. 1996;1(3):26-31.
The polymerase chain reaction (PCR)-based method was used to obtain and sequence three H-2K and three H-2D mouse complementary DNAs (cDNA) of class I major histocompatibility complex (MHC) molecules.
Messenger RNA was isolated from Conconavalin A-activated splenocytes of C57BL/10 (H-2b), C3H (H-2k), and Balb/c (H-2d) mice. We designed H-2K- and H-2D-specific primers as well as a common downstream primer based on previously published mouse class I MHC sequences. Using the PCR method and selective primers we isolated and sequenced H-2Kb and H-2Db cDNAs of C57BL/10, H-2Kk and H-2k cDNAs of C3H, as well as H-2Kd and H-2Dd cDNAs of Balb/c strains.
Analysis of the nucleotide sequences documented similarity between our three H-2K cDNA sequences and all mouse MHC class I sequences available in the GenBank. Similarly, our three H-2D sequences were homologous with all mouse class I MHC sequences deposited in the GenBank. Our H-2K and H-2D sequences were also identical to numerous published sequences.
Using these mouse cDNAs, we plan to determine the localization of polymorphic in vivo immunogenic amino acids in class I MHC H-2K and H-2D alloantigens.
采用基于聚合酶链反应(PCR)的方法获取并测序3个I类主要组织相容性复合体(MHC)分子的H-2K和3个H-2D小鼠互补DNA(cDNA)。
从C57BL/10(H-2b)、C3H(H-2k)和Balb/c(H-2d)小鼠的伴刀豆球蛋白A激活的脾细胞中分离信使RNA。我们根据先前发表的小鼠I类MHC序列设计了H-2K和H-2D特异性引物以及一个通用的下游引物。使用PCR方法和选择性引物,我们分离并测序了C57BL/10的H-2Kb和H-2Db cDNA、C3H的H-2Kk和H-2k cDNA以及Balb/c品系的H-2Kd和H-2Dd cDNA。
核苷酸序列分析表明,我们的3个H-2K cDNA序列与GenBank中所有可用的小鼠MHC I类序列相似。同样,我们的3个H-2D序列与GenBank中所有储存的小鼠I类MHC序列同源。我们的H-2K和H-2D序列也与众多已发表的序列相同。
利用这些小鼠cDNA,我们计划确定I类MHC H-2K和H-2D同种异体抗原中多态性体内免疫原性氨基酸的定位。