粘膜免疫相关基因的克隆与功能研究

【作者】 易绍琼；

【导师】 高杰英；

【作者基本信息】 中国人民解放军军事医学科学院 ， 免疫学， 2003， 博士

【摘要】 本课题包括对粘膜免疫相关新基因TIG-310和粘膜抗原提呈分子小鼠CD1d的研究。 本实验室在分析研究Ty21a免疫小鼠前后肠粘膜差异表达的基因时，发现了数条在Ty21a免疫后表达量发生改变的新表达序列标签(Expression Sequence Tag,ESTs)，为了深入研究相应新基因在粘膜免疫中的作用，我们采用了RACE-PCR技术以扩增这些ESTs所对应的基因全长。其中扩增得到了EST310的全长基因(TIG-310)。分析表明该基因序列与GenBank中的一条序列仅存在一处碱基突变，但未见该基因相关的功能报导。我们查询了该基因与其它基因的同源性，基因的ORF、电子表达谱和在染色体上的定位及其编码蛋白的信息。该基因位于小鼠的第11对染色体，含有一个ORF，编码的蛋白由267个氨基酸组成，可能表达于胃肠粘膜部位。 为了研究TIG-310是否与粘膜免疫相关，我们利用pQE30原核表达系统，获得TIG-310基因在M15受体菌株中的高效表达。利用RT-PCR技术分析了该基因在正常Balb／c小鼠脑、心脏、胸腺、肺、胃、肾、肠、肝、脾、PP结、MLN、子宫等12种组织的表达情况，发现该基因高表达于胃肠粘膜部位，在PP结上有较弱表达。由于肠粘膜含有多种细胞，其功能又各不相同，我们用原位杂交方法分析肠粘膜中表达TIG-310的细胞，发现该基因主要表达在肠粘膜的上皮细胞。将TIG-310的编码区与绿色荧光蛋白进行融合，分别转染小鼠成纤维细胞系L细胞和人COS-7细胞，筛选G418抗性克隆，在共聚焦显微镜和荧光显微镜下观察荧光蛋白的细胞定位，发现融合蛋白主要定位在细胞浆，但有的细胞也存在全细胞荧光分布。由以上结果我们可以推测，TIG-310主要表达在正常Balb／c小鼠小肠上皮细胞的胞浆。 由于Ty21a灌胃免疫后TIG-310在小肠细胞的表达发生改变，因此，我们利用RT-PCR系统检测了Ty21a每次免疫后第二、第五天该分子在胃、肠和PP结的表达情况。发现1．首次免疫后，胃肠粘膜与PP结中TIG-310表达均有显著性增高，且胃肠增高比PP更为明显；2．随免疫次数增加TIG-310的表达逐次逐天下降，均低于正常组。将纯化的pEGFP-310质粒注入小鼠的股四头肌，发现该重组质粒可促进大量炎性细胞向注入部位聚集，用RT-PCR检测注入局部14种炎性相关细胞浮目卜斑气丈因子的表达，发现重组质粒可使局部IFN一丫和IL一12p40的表达明显上调。说明该基因可能通过影响IFN一:和IL一12p40的表达，从而促进免疫局部的炎性反应。由于TIG一310主要表达在粘膜上皮细胞，并在初次免疫后显著上调，含有该基因的重组质粒可刺激肌注局部炎性细胞聚集及IFN一丫和IL一12p40细胞因子的表达上调，推测TIG一310可能与维持粘膜上皮细胞的正常功能或自然防御机制相关。 tetramer标记是一种新建立的可对抗原特异的T细胞进行鉴定的技术。NKT细胞是一群新近发现的具有免疫调节功能的细胞。在本实验中，我们尝试制备mCD 1 dtetramer，以标记CDI分子限制的NKT细胞，研究这群细胞在粘膜免疫中的作用。我们选用的表达系统是昆虫杆状表达系统。将mCDld和pZm先后连入穿梭质粒pFastBac Dual，获得重组质粒pBD一CDI一pZm;在DHlobae菌株中通过同源重组，将eD一和pZm整合到Bacmid基因组;重组Bacmid通过Cellfeetin转染Sf-9细胞，并在Sf-9细胞中包装成病毒颗粒。利用SDS一PAGE检测目的蛋白的表达。载体构建成功，但未获得蛋白表达。 为观察CDI分子是否对不同来源的淋巴细胞具有刺激活性，将表达mCDld的CHO细胞分别与从脾、肝、胸腺、PP和MLN等不同部位新分离的淋巴细胞进行共育，7天后利用MTT法检测淋巴细胞增殖，并用流式细胞术检测培养体系中NKI.1和CD3双阳性淋巴细胞的比例，结果发现表达mCDld的CHO细胞可刺激肝内和中心淋巴结的淋巴细胞发生增殖，并使CD3十细胞中NKI.1阳性细胞的比例增加。更多还原

【Abstract】 During the research on variations of gene expression before or after Ty21a immunization, we found several novel ESTs, which were expressed differently after Ty21a immunization. The sequence of one gene, which is corresponding to EST3 10, was acquired by RACE-PCR and was named Ty21a immunization-associated gene, TIG-310. We blasted it in the GenBank and found one gene that is similar to it. But we did not get any published information about its function. We analyzed its homogenicity to other genes, its ORF, expression profile, locus on the chromosome and so on. We also get some information about the protein coded by the gene. The gene is localized on the 11th chromosome and may be expressed in the stomach and intestine. The gene has one ORF, coding a protein composed of 267 amino acids.To identify the function of TIG-310 in mucosal immune responses, we expressed the gene by pQE30 vector. After induction by IPTG, high level of target protein was expressed in M15 host strain. It was proved that the gene was expressed in stomach and intestine of normal mouse. There was low level of expression that can be detected in the Peyer’s patch. Then cells expressing the TIG-310 gene in the intestinal mucosa were analyzed by in situ hybridization. It seems that the gene is mainly expressed in the intestine epithelial cells. In addition, the TIG-310 was fused to GFP and expressed in L cells and COS-7 cells. It seems that the TIG-310 gene may be a cytoplastic protein, for the fluorescence is mainly found in the cytoplasm of the transfected cells. So it was presumed that the gene might be expressed in the cytoplasm of intestine epithelial cells of normal Ba1b/c mouse.The expression of TIG-310 varied in the intestine epithelial cells after Ty21a immunization. We detailed the expression of the gene in stomach, intestine and Peyer’s patch by RT-PCR. The results show that the expression of the gene in stomach and intestine was much higher than that in Peyer’s patch. The expression in stomach and intestine after first Ty21a immunization was much higher than that after second and third immunization. And the expression in stomach and intestine after second and third immunization was lower than that in normal mice. From these results, we can draw some conclusion that immunization with Ty21a can induce the expression of TIG-310 instomach and intesine. The Ty21a may interact with the intestine epithelial cells and regulate the expression of TIG-310.After the injection of pEGFP-310 plasmid in muscle, there were more inflammatary cells at the injection site. Then the expression of many cytokines associated with inflammation was analyzed by RT-PCR. These results show that the expression of IFN-y and IL-12P40 was upregulated. It implied that TIG-310 might promote the inflammation response by upregulating the expression of IFN-y and IL-12P40.From the above results, we can presume that the TIG-310 may be associated with innate defence of mucosal epithelial cells.To study the function of CD1d in mucosal antigen presentation and the role of NK T cells in mucosal immunity, we need to prepare CD1d tetramer to stain the NK T cells. CD1d tetramer is a complex consisting of four CD1d and four P2m. The baculovirus expression system was selected to express mCD1d and 2m. First, the two genes were cloned into shuffle vector pFastBac Dual one after another. The recombinant plasmid was named as pBD-CDl- 2m. After homologous recombination, the GD1 and 2m were inserted into the Bacmid genome. Then the recombinant Bacmid was transfected into Sf-9 cells by Cellfectin. The expression of target protein was analyzed by SDS-PAGE. But protein expression was not detected.To study the stimulating function of GDI on lymphocytes isolated from different tissues, we detected the proliferation of lymphocytes co-cultured with CHO cells expressing CD1 molecule by MTT method and analyzed the ratio of NK1.1 and CD3 double positive cells. We found out that mCDld expressed on CHO cells could stimulate lymphocytes proliferation isolated from liver and mes更多还原