禽类Ii基因的克隆、表达及其功能的初步研究

【作者】 仲大莲；

【导师】 刘兢； 余为一；

【作者基本信息】 中国科学技术大学 ， 分子与细胞生物学， 2005， 博士

【摘要】 Ii链是一种非多态性的Ⅱ型跨膜蛋白，在调节MHC Ⅱ类分子的表达和功能方面发挥重要作用。人类和小鼠的Ii基因都是单拷贝基因，其mRNA都能够以不同的拼接模式产生不同形式的Ii链异构体。但是禽类Ii链是否存在异构体还未见报道。我们利用RT-PCR和RACE的方法确定了两个鸡Ii链cDNA，并且用Southern blotting、Northern blotting和免疫荧光法分别检测了Ii基因的拷贝数、mRNA表达和蛋白表达情况。被命名为鸡Ii-1的cDNA长为1，151bp，含有一个由672bp组成的开放阅读框，它与一个已经被报道的鸡Ii链序列相一致；另一个Ii链cDNA被命名为鸡Ii-2，其大小为1，337bp，含有一个由861bp组成的开放阅读框。Ii-1与Ii-2在核苷酸和氨基酸水平上的同源性均为99％，两者的区别在于Ii-2比Ii-1多出一个Tg区。Southern blotting结果表明，这两个cDNA由一个单拷贝的基因产生。Northern blotting结果表明，Ii-1和Ii-2在6周龄鸡的脾脏和法氏囊中高表达，而在胸腺、心脏和肝脏中有Ii-1低水平表达，但未检测到Ii-2的表达；在鸡胚胎的晚期发育中，两种Ii链异构体在脾脏和法氏囊中均被检测到高水平表达。脾组织切片的免疫荧光染色结果表明，Ii链主要表达在细胞膜上。这些资料表明，鸡Ii链存在两种异构体，它们由mRNA的选择性拼接产生，并且主要在免疫器官中高表达。 利用一对自行设计的简并引物，通过RT-PCR和RACE方法，我们获得了鸭Ii链cDNA，并将其命名为鸭Ii-1。Ii-1长为1，190bp，含有一个41bp的5′不翻译区、一个669bp的开放阅读框和一个480bp的3′不翻译区。鸭Ii链也存在编码含有Tg区的异构体，我们将其命名为鸭Ii-2。鸭Ii-1与鸡Ii-1氨基酸序列的同源性为82％，与哺乳动物Ii P31／P33之间的同源性约为60％。鸭Ii-2 Tg区与鸡Ii-2Tg区在氨基酸水平上的相似性高达96％，与哺乳动物Ii链Tg区之间也达到70％。利用一对能同时扩增鸭Ii链两种异构体的引物，通过半定量RT-PCR检测不同组织Ii mRNA的分布情况，结果表明，Ii链两种异构体在不同组织中广泛表达，尤其在脾脏和法氏囊中高水平表达。应用comparative protein modeling分析了鸭Ii链三聚体区和Tg区的三维结构，它们与人Ii链相应domain的结构完全相似。这些结果表明鸭Ii链也存在两种异构体，它们主要在免疫器官中高表达，并且更多还原

【Abstract】 The invariant chain (Ii) is a non-polymorphic type II transmembrane protein and plays a central role in regulating the expression and function of class II major histocompatibility complex (MHC) molecules. The biosynthesis of distinct forms of the invariant chain (Ii) protein from a unique gene as the result of differential splicing patterns has been observed in humans and mice. However, there have been no reports on the existence of Ii isoforms in avian species. In the present study, we identified two chicken Ii cDNAs by RT-PCR and RACE, and examined the Ii gene copy number, mRNA expression and protein expression by Southern blotting, Northern blotting and immunofluorescence confocal microscopy, respectively. One of the Ii cDNAs, named Ii-1, was 1,151 bp in length, and had an open reading frame (ORF) of 672 nucleotides, in agreement with a previously identified chicken Ii sequence; the other, named Ii-2, was 1,337 bp long and had an ORF of 861 nucleotides. The Ii-1 and Ii-2 isoforms are similar to each other where they overlap, and the sequence similarity between them is 99% at either the nucleotide or amino acid level. However, they differ in the presence or absence of a Tg domain. Southern blotting confirmed that these cDNAs were derived from a single copy gene. Northern blotting performed with total RNA from various tissues of 6-week-old chickens revealed high levels of Ii-1 and Ii-2 mRNA expression in the spleen and bursa of Fabricius, and low levels of Ii-1 expression in the thymus, heart and liver, while Ii-2 was not expressed in these tissues. High levels of expression of both Ii isoforms were detected in the spleen and bursa of Fabricius during late embryogenesis. Immunofluorescence staining showed that Ii proteins were expressed in the cell membranes of the splenocytes. These data suggest that chicken Ii exists in two isoforms resulting from alternative splicing, and is strongly expressed in the major immune organs.With a pair of degenerate primers, we further identified a duck invariant chain (Ii)) cDNA, named duck Ii-1, by RT-PCR and RACE. It was 1,190 bp in length and contained a 41 bp 5’ untranslated region, a 669 bp open reading frame and a 480 bp 3’ untranslated region. An alternative transcript encoding a thyroglobulin更多还原