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目的分离新的B细胞活化基因。方法采用差异显示反转录PCR技术(DDRT-PCR)对人扁桃体活化和静止B细胞mRNA的差异显示情况进行分析,对显示片段进行克隆并行Northern分析,对Northern杂交阳性的cDNA片段进行测序并比较同源性。结果差异显示分析共获得明显差异表达的cDNA片段62条,选择主要表达于活化B细胞上的20条cDNA片段克隆到pGEM-T载体上,并逐一对静止和活化B细胞RNA进行Northern分析,其中6条cDNA片段在活化B细胞中呈Northern杂交阳性且与差异显示情况相符,对它们进行测序,然后通过国际互联网与GenBank,EMBL和DDBJ的DNA数据库比较序列同源性,发现其中4个克隆与已发现的基因具明显同源性,一个克隆是新的,另一个克隆虽然与人T细胞分泌的趋化因子I-309同源性达95%,但二者转录本不同。结论通过DDRT-PCR分离到两个可能为新的B细胞活化基因的cDNA片段,这为进一步克隆新的B细胞活化基因奠定了基础。
Objective To isolate new B cell activation genes. Methods Differential display reverse transcriptase-PCR (DDRT-PCR) was used to analyze the differences of human tonsil activation and resting B cell mRNA. The displayed fragments were cloned and analyzed by Northern analysis. Northern hybridization positive cDNA fragments were sequenced and compared Homology. Totally, 62 differentially expressed cDNA fragments were obtained. Twenty cDNA fragments, mainly expressed on activated B cells, were selected and cloned into pGEM-T vector. Northern analysis of stationary and activated B-cell RNA was performed one by one, among which Six of the cDNA fragments were positive for Northern hybridization in activated B cells and were consistent with the differences, and they were sequenced. Sequence homology was then compared with the DNA databases of GenBank, EMBL and DDBJ over the Internet and four of the clones were found The discovered genes have obvious homology, one clone is new, and the other clone has different transcripts though it has 95% homology with chemotactic I-309 secreted by human T cells. Conclusion The cDNA fragments of two new B cell activation genes were isolated by DDRT-PCR, which laid the foundation for further cloning of new B cell activation genes.