MHC GENES INVOLVED IN ANTIGEN PROCESSING & PRESENTATION

T淋巴细胞活化，从而引发免疫反应， 需要T细胞受体识别由肽组成的复合物 与主要组织相容性复合体结合的外源抗原片段 (MHC)抗原呈递细胞（APC）表面的分子。 尽管 免疫识别第一步的关键性，却鲜为人知 了解抗原加工的机械方面，例如 抗原片段的产生，以及它们如何以及在APC内的何处产生， 遇到MHC分子。 越来越多的证据表明，MHC含有的基因， 在将加工的抗原转运到膜囊泡中的功能 用于与MHC类分子结合的分泌系统和随后的 囊泡运输到细胞表面。 最近克隆的两个MHC 基因，称为HAM1和HAM2，这是同源的一个大的超家族， 原核和真核转运蛋白都代表了 了解抗原加工的本质。 这项建议 涉及这两个的结构和功能特征， 基因. 将进行两种类型的实验，以提供正式的证据， HAM1和HAM2基因参与抗原加工。 第一、 将全长野生型HAM1和HAM2 cDNA导入细胞 携带MHC连锁突变的细胞系， 通路 第二，HAM1和HAM2表达缺陷的小鼠将被 通过将这些基因的缺陷拷贝引入胚胎干细胞而产生 细胞通过同源重组，并随后注射这些 将修饰的细胞转化为小鼠胚泡。 为了进一步表征 HAM1和HAM2基因产物的功能，抗体将产生 衍生自推导的氨基酸序列的合成肽。 这 抗体将被化学地用于定位相应的 细胞内的蛋白质（从而识别亚细胞区室 其中肽与MHC分子相遇），并有助于它们的纯化。 将尝试在体外重建肽转运 系统，并分析运输过程的特殊性。 除上述外，两种新的MHC的功能意义 II类基因将通过检查它们的多态性来解决， 通过产生抗体， 基因产物

T lymphocyte activation, and hence the initiation of an immune response, requires recognition by T cell receptors of complexes consisting of peptide fragments of foreign antigens bound to major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APC). Despite the critical nature of this first step in immune recognition, little is known about the mechanistic aspects of antigen processing, such as how the antigen fragments are produced, and how and where within the APC they encounter MHC molecules. A growing body of evidence suggests that the MHC contains genes which function in the transport of processed antigen into vesicles of the secretory system for binding by MHC class molecules and subsequent vesicular transport to the cell surface. The recent cloning of two MHC genes, called HAM1 and HAM2, which are homologous to a large superfamily of both prokaryotic and eukaryotic transporters represents a major step towards understanding the nature of antigen processing. This proposal deals with the structural and functional characterization of these two genes. Two types of experiments will be done to provide formal proof of the involvement of the HAM1 and HAM2 gene in antigen processing. First, full length, wild type HAM1 and HAM2 cDNAs will be introduced into cell lines carrying MHC-linked mutations that inactivate the antigen processing pathway. Second, mice deficient in HAM1 and HAM2 expression will be produced by introducing defective copies of these genes into embryonic stem cells by homologous recombination, and subsequently injecting these modified cells into mouse blastocysts. In order to further characterize the function of the HAM1 and HAM2 gene products, antibody will be produced to synthetic peptides derived from the deduced amino acid sequences. This antibody will be used immunohistochemically to localize the corresponding proteins within the cell (thus identifying the subcellular compartment where peptide meets MHC molecule), and to aid in their purification. Attempts will be made to reconstitute peptide transport in in vitro systems, and to analyze the specificity of the transport process. In addition to the above, the functional significance of two novel MHC class II-like genes will be addressed by examining their polymorphism and by producing antibody which would allow identification of the corresponding gene products.