摘要
背景:通过筛选小鼠反转录病毒cDNA文库和质谱分析的方法鉴定出一种跨膜的胶原XVII是白细胞相关免疫球蛋白样受体1和白细胞相关免疫球蛋白样受体2的配体之一,然而,二者的其他配体尚未得到鉴定。目的:采用生物信息学法鉴定白细胞相关免疫球蛋白样受体1和白细胞相关免疫球蛋白样受体2(CD305/306)的候选配体分子。设计、时间及地点:生物信息学预测及生物学方法验证,于2006-03/2007-09在第四军医大学免疫教研室完成。材料:人WISH和Hela细胞系由第四军医大学免疫教研室冻存。方法:应用生物信息学方法搜索白细胞相关免疫球蛋白样受体1和白细胞相关免疫球蛋白样受体2序列同源分子,并通过文献检索初步推测白细胞相关免疫球蛋白样受体1和白细胞相关免疫球蛋白样受体2的配体候选分子。最后应用候选分子基因转染K562细胞,并应用白细胞相关免疫球蛋白样受体1-Fc/白细胞相关免疫球蛋白样受体2-Fc融合蛋白进行活细胞荧光染色,流式细胞术分析鉴定候选分子是否与融合蛋白结合。同时,合成两对候选分子的引物,提取高表达白细胞相关免疫球蛋白样受体1和白细胞相关免疫球蛋白样受体2配体分子的细胞系WISH的cDNA,进行巢式PCR,检测WISH中候选分子的表达情况。主要观察指标:应用人类基因组数据库对白细胞相关免疫球蛋白样受体1和白细胞相关免疫球蛋白样受体2分子氨基酸序列同源性比对结果,流式细胞术分析白细胞相关免疫球蛋白样受体分子是否与可能配体基因转染细胞结合,巢式PCR分析白细胞相关免疫球蛋白样受体配体高表达细胞是否表达可能配体基因。结果:应用生物信息学方法和文献检索初步推测白细胞相关免疫球蛋白样受体1和白细胞相关免疫球蛋白样受体2的配体候选分子为人类白细胞抗原G分子。经流式细胞术鉴定,白细胞相关免疫球蛋白样受体1-Fc/白细胞相关免疫球蛋白样受体2-Fc融合蛋白对人类白细胞抗原G基因转染的K562细胞的荧光染色结果为阴性;经巢式PCR鉴定,高表达白细胞相关免疫球蛋白样受体1和白细胞相关免疫球蛋白样受体2配体分子的WISH细胞系中未发现人类白细胞抗原G基因。结论:人类白细胞抗原G不是白细胞相关免疫球蛋白样受体1和白细胞相关免疫球蛋白样受体2的配体。
BACKGROUND： By screening a mouse retroviral cDNA library and subsequent protein sequencing by mass spectrum, transmembrane collagen XVII was identified as one of ligands for leukocyte-associated immunoglobulin-like receptor-1 （LAIR-1） and LAIR-2. However, the other ligands for LAIR- 1 or LA1R-2 have not been identified. OBJECTIVE： To screen the ligand candidate of human LAIR- 1 and LAIR-2 （CD305/306） by bioinformatics. DESIGN, TIME AND SETTING： Prediction by bioinformatics and verification through biological method were performed at the Department of Immunology of Fourth Military Medical University of Chinese PLA from March 2006 to September 2007. MATERIALS： Human WISH and Hela cell lines were from Department of Immunology of Fourth Military Medical University. METHODS： The homologous sequences of human LAIR-1 and LAIR-2 were screened by similarity BLAST sequence, and the ligand candidate of human LAIR- 1 and LAIR-2 was selected by document retrieval. The ligand candidate gene was transfected into K562 cells, then the cells were incubated with LAIR-1 and/or LAIR-2 fusion proteins, and the influorescent staining and flow cytometric analysis were performed, In addition, total RNA of ligand-expressing cell line WISH was isolated, mRNA was purified, and cDNA was synthesized. Two couples of primers of the ligand candidate were also synthesized and the nested PCR were performed. MAIN OUTCOME MEASURES： To observe LAIR-I and LAIR-2 protein sequences Blasting results by similarity BLAST sequence similarity search tool; whether the LAIR- 1 and LAIR-2 fusion proteins bind to the ligand candidate gene transfected cells and if the ligand candidate gene is found in the ligand-expressing cell line. RESULTS： The ligand candidate of LAIR- 1 and LAIR-2 is HLA-G molecule through similarity BLAST sequence similarity search tool and document retrieval. However, the LAIR-1 and/or LAIR-2 fusion proteins could not bind to the K562 cells transfected with the HLA-G gene by the flow cytometric analysis. Moreover, the HLA-G gene was not found in the ligand-expressing cell llne WISH. CONCLUSION： HLA-G is not the ligand of human LAIR-1 and LAIR-2.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第40期7865-7869,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
中国博士后科学基金一等面上资助金(20070420078)资助项目~~
