摘要
近年来的研究表明 ,CD4分子于细胞膜表面不仅以单体形式存在还可以通过其D4和D1形成同源二聚化及寡聚化 ,并且二聚化及寡聚化的CD4分子才能稳定地与MHC Ⅱ类分子结合。通过分析CD4分子以及CD4缺失突变体分子融合Fas基因片段所诱导的转染靶细胞的凋亡 ,以及携带绿色荧光蛋白的CD4分子转染HEK2 93细胞所筛选出的稳定克隆的不同荧光强度和与MHC Ⅱ类分子阳性细胞Raji之间的不同黏附效应间接鉴定CD4同源二聚体或寡聚体的存在 ,并对二聚化或寡聚化CD4分子所介导的生物学功能进行初步分析。
To examine the self-association of CD4 molecules and preliminary stud ies on its biological function by several indirect methods. A series of CD4 chim eras were generated including truncated CD4 lacking the short cytoplasmic tail, deleted mutants-D1/D2 devoid of D3 and D4 and D3/D4 devoid of D1 and D2 by PCR techniques, as well as another three CD4 chimeric genes by fused human Fas cyto plasmic death domain to the downstream of the above chimeras respectively. All t hese molecules were subcloned into pEGFP-N1, forming the corresponding expressi o n vectors. After introducing into HEK293 cells, gene-modified cell morphologica l changes and target protein subcellular localization were observed and analyzed by a confocal microscopy. Moreover, stable 293/CD4 clones were obtained by trans fecting the truncated CD4 recombinant plasmid into the HEK293 cell line and sele cted by G418. The fluorescene intensity and rosette formation of different clon e s was each analyzed by a confocal microscopy and cell adhesive assays. It's seen that CD4-Fas fusion gene could induce approximately 80% cell apoptosis of tran s fected HEK293 cells, compared to FKBP12-Fas is about 30% and CD4 gene only is 7 % . Furthermore, both D1/D2-Fas and D3/D4-Fas chimeras could trigger nearly all tr ansfected HEK293 cells to death. Cell adhesion assays showed that neither the D1 /D2 nor D3/D4 chimeras when expression in HEK293 cells binds to MHC class Ⅱ + Ra ji B cells. Interestedly, there were two type stable clones among 293/CD4. Fluo r escence intensity analysis displayed that one' mean fluorescence intensity value is about twice of the other while cell-cell binding examination showed that th e former is capable of forming rosette with Raji cells but the latter. All these results suggest that CD4 molecules most likely could exist as a dimer or even an oligomer on transfected HEK293 cell surface, which constitute a functional form for stable binding to MHC class Ⅱ molecules.
出处
《生物工程学报》
CAS
CSCD
北大核心
2005年第2期239-243,共5页
Chinese Journal of Biotechnology
基金
国家 973重点基础研究发展资助项目 (No.2 0 0 3CB5 15 5 0 8)~~
关键词
同源二聚化
间接检测
激光共聚焦
细胞黏附
玫瑰花环
缺失突变
homodimerization, indirect detection, confocal, cell adhesion, rosette formation, deleted mutation
