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目的 研究 Fas配体 (Fas ligand,Fas L )在移植免疫方面的作用 ,克隆其编码的全部 c DNA序列 ,并添加 Kozak序列 ,以便在真核细胞高效表达大鼠 Fas L。 方法 从大鼠的睾丸组织中提取总 RNA,设计上、下游引物以 RT- PCR的方法扩增出一 931bp的 DNA片段 ,将这一片段重组于 p BK - RSV载体中 ,酶切鉴定插入片段正确后进行全序列分析。 结果 证实该研究所克隆的 c DNA是编码正确的大鼠 Fas L基因 ,与发表于 Gene Bank上的序列相比 ,完全正确。已构建成用于真核表达的重组质粒 p BK- RSV/r Fas L。 结论 克隆到了编码正确的大鼠Fas L的 c DNA全序列 ,对移植免疫和帕金森病基因治疗的基础研究及临床应用具有重要意义。
OBJECTIVE: To study the role of Fas ligand (Fas L) in graft immunity, clone the entire cDNA sequence encoding it, and add Kozak sequence to efficiently express rat FasL in eukaryotic cells. Methods The total RNA was extracted from the testis of rats. The upstream and downstream primers were designed to amplify a 931 bp DNA fragment by RT - PCR. The fragment was recombined into pBK - RSV vector and the inserted fragment was identified by restriction enzyme digestion Correct after the full sequence analysis. The results confirm that the c DNA cloned from this study is the correct rat Fas L gene, which is completely correct compared with the sequence published on Gene Bank. The recombinant plasmid pBK-RSV / r FasL has been constructed for eukaryotic expression. Conclusion Cloning of the complete c DNA sequence encoding the rat Fas L gene is of great importance to the basic research and clinical application of gene therapy of transplantation and Parkinson ’s disease.