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树突状细胞(dendritic cells,DC)是体内功能最强的抗原提呈细胞,从DC中克隆并发现新的免疫分子基因并研究其免疫学功能是目前免疫学领域的前沿热门课题.本课题通过大规模随机测序和同源比较,从人树突状细胞.DNA文库中克隆到全长1101 bp的新基因,根据 GCG软件预测N端有一77个氨基酸组成的信号肽,提示其为分泌性蛋白,成熟分子由209个氨基酸组成,理论上计算其分子量约为23kD,其蛋白水平与人和小鼠TNF有15%左右的同源性,因此推测可能是一新型细胞因子,命名为树突状细胞衍生的TNF-α样细胞因子(DC derived TNF-αlike cytokine,DDTLC).DDTLC在多种组织和细胞中都有不同程度的表达.为开展其生物学功能的研究,进行了DDTLC的原核重组表达.利将DDTLC定向插入pBV220表达载体,构建成重组表达载体pBV-DDTLC;转化DH5_α细菌,进行热诱导表达并纯化出纯度达95%以上的DDTLC重组蛋白.以上结果为进一步开展DDTLC生物学功能研究奠定了基础.
Dendritic cells (DCs) are the most potent antigen-presenting cells in the body. It is a hot frontier issue in the field of immunology to clone and discover new immune molecule genes from DCs and to discover their immunological functions. A novel full-length 1101 bp gene was cloned from human dendritic cell DNA library by large-scale random sequencing and homology comparison. According to GCG software, a signal peptide of 77 amino acids at the N-terminus was predicted, indicating that it is secreted Protein, mature molecule consists of 209 amino acids, theoretically calculated molecular weight of about 23kD, the protein level and human and mouse TNF about 15% homology, it is speculated that may be a new type of cytokine, named dendrites DC derived TNF-αlike cytokine (DDTLC) .DDTLC is expressed in many tissues and cells to varying degrees.For the study of its biological function, the prokaryotic expression of DDTLC DDTLC was inserted into pBV220 expression vector and constructed into recombinant expression vector pBV-DDTLC.DH5_α was transformed into DH5_α bacteria, and the DDTLC recombinant protein with more than 95% purity was purified by heat-induced expression.The above results were The biological function of DDTLC step to carry out the foundation.