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目的:构建人Bcl-2基因的转基因表达载体用于转基因动物模型的建立,以便深入探讨脑缺血再灌注损伤中的基因机制。方法:利用RT-PCR技术,用包含bcl-2cDNA的引物从慢性淋巴细胞白血病患者的血液扩增得到一约为708bp的DNA片段,克隆后测序和同源性分析。结果:经RT-PCR扩增获得的产物大小与预计的目的片段大小一致,目的基因测序与国外报道的参考序列相比,序列同源性为99%。结论:以pEGFP-C1载体作为真核表达载体,形成以绿色荧光蛋白作为报告基因的融合基因,可以用于转基因的研究。
OBJECTIVE: To construct a transgenic expression vector for human Bcl-2 gene and establish a transgenic animal model in order to further investigate the gene mechanism of cerebral ischemia-reperfusion injury. Methods: A DNA fragment of about 708bp was amplified from the blood of patients with chronic lymphocytic leukemia using primers containing bcl-2 cDNA by RT-PCR. Sequencing and homology analysis were performed after cloning. Results: The size of the product obtained by RT-PCR amplification was consistent with the expected size of the target fragment. Compared with the reported foreign reference sequence, the sequence of the target gene was 99% homologous. Conclusion: The pEGFP-C1 vector as a eukaryotic expression vector, the formation of green fluorescent protein as a reporter gene fusion gene can be used for transgene research.