摘要
目的 编码 5C5分化抗原全长cDNA的克隆及功能探索。方法 构建人活化B细胞株3D5细胞的λgt11cDNA文库 ,以核苷酸杂交法从cDNA文库中筛选阳性克隆 ,作核苷酸序列分析 ,编码蛋白质氨基酸序列的亲疏水分析 ,Northernblot测 5C5mRNA转录本长度 ,用RT PCR检测 5C5mRNA在不同细胞株的表达 ,观察单抗 5C5 G1对 3D5细胞增殖的影响。结果 从人活化B细胞株 3D5的λgt11cDNA文库筛选到长 90 5bp的 5C5 1cDNA及 75 4bp的 5C5 2cDNA。 5C5 1cDNA序列内有 1个开放阅读框架 (19～ 5 37bp) ,编码 179个aa的蛋白质。此蛋白质内有一穿膜螺旋结构 (94～ 114aa) ,其N端在胞内。 5C5 2cDNA从 16 3到 45 9为开放阅读框架 ,编码 99个aa的蛋白质。从Northernblot见0 9kb左右和 0 7kb左右 2条杂交带。由RT PCR见 5C5mRNA在 3D5细胞强表达 ,在Daudi细胞表达次之 ,在Jurkat细胞表达最弱。单抗 5C5 G1有增强cpBCGF诱导 3D5细胞增殖的作用。结论 长90 5bp的 5C5 1cDNA为 1个全长cDNA。编码 1个膜蛋白 ,可能参与 3D5细胞的增殖反应。
Objective Cloning and sequencing of the cDNA encoding a differentiation antigen 5C5. Methods Construction of a λgt11 cDNA library of 3D5 cells, a human activated B cell line; isolation of positive cDNA clones from the cDNA library with phage hybridization; sequencing of the cDNA; open reading frame analysis; hydrophobic analysis of the amino acid sequence of the deduced protein; determination of the length of 5C5 mRNA transcript with Northern blot hybridization test; determination of 5C5 mRNA transcription in various cell lines with RT PCR. Results 5C5 1 cDNA (0.9kb) and 5C5 2 cDNA (0.7kb) have been isolated from a λgt11 cDNA library of 3D5 cell, a human activated B cell line, by using a 467bp 5C5 cDNA as a probe. Nucleotide sequence analysis has shown that the 5C5 1 cDNA is 905bp long with an open reading frame (ORF) from 19 to 555bp in it. The TopPred 2 topology prediction of the membrane protein suggests that the 179aa protein deduced from the ORF has a helix membrane spanning segment from 94 to 114aa. The orientation is that the N terminal is inside the cell. 5C5 2 cDNA is 754bp long with an ORF from 163 to 459bp encoding a protein with a length of 99aa. Northern blotting shows two mRNA transcripts with lengths of 0.9kb and 0.7kb respectively, indicating that the 905bp cDNA is a full length cDNA. RT PCR reveals strong, medium and weak expressions of 5C5 mRNA in 3D5 cell, Daudi cell and Jurkat cell respectively. Conclusion The 905bp 5C5 1 cDNA is a full length cDNA encoding a human membrane protein.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2000年第3期248-252,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目!(39630300)
