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目的:构建真核表达载体pIRES2-EGFP-TIM2,并在小鼠肝癌细胞系H22中进行表达。方法:用RT-PCR法扩增得到TIM2基因,构建重组真核表达载体pIRES2-EGFP-TIM2,并进行BamHI及BglⅡ双酶切鉴定和测序。通过脂质体法转染H22细胞,用RT-PCR法检测H22细胞中TIM2mRNA的表达。结果:构建了真核表达载体pIRES2-EGFP-TIM2,用脂质体法转染H22细胞后,用荧光显微镜观察和RT-PCR法检测,可见细胞内有EGFP及TIM2mRNA的表达。结论:成功地构建重组真核表达载体pIRES2-EGFP-TIM2,并在小鼠H22细胞中表达,为进一步研究TIM2在肿瘤生物治疗中的应用奠定了基础。
OBJECTIVE: To construct eukaryotic expression vector pIRES2-EGFP-TIM2 and express it in mouse hepatoma cell line H22. Methods: The TIM2 gene was amplified by RT-PCR and the recombinant eukaryotic expression vector pIRES2-EGFP-TIM2 was constructed. The recombinant plasmid was identified by BamHI and BglII restriction enzymes. H22 cells were transfected by liposome and the expression of TIM2 mRNA in H22 cells was detected by RT-PCR. Results: The eukaryotic expression vector pIRES2-EGFP-TIM2 was constructed and transfected into H22 cells by lipofectamine. The expression of EGFP and TIM2 mRNA was detected by fluorescence microscope and RT-PCR. Conclusion: The recombinant eukaryotic expression vector pIRES2-EGFP-TIM2 was successfully constructed and expressed in mouse H22 cells, which laid the foundation for further study on the application of TIM2 in tumor biotherapy.