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目的 :克隆小鼠白细胞介素 2 1(IL 2 1)基因 ,构建真核表达质粒 ,用以进行肿瘤的基因治疗。方法 :用RT PCR法 ,从ConA活化的小鼠T细胞中扩增IL 2 1cDNA ,克隆入哺乳动物细胞高效表达质粒 pcDNA3.1中 ,构建重组mIL 2 1真核表达质粒。重组体用载体上的通用引物和PCR下游引物为测序引物 ,鉴定克隆的正确性。将已鉴定的重组质粒用脂质体法转染Sp2 / 0细胞 ,用RT PCR法鉴定转染细胞中IL 2 1基因的表达 ,用MTT比色法检测表达的mIL 2 1诱导的NK细胞杀伤活性的增强。结果 :正确构建了重组真核表达质粒 pcD NA3.1/mIL 2 1,并在转染的细胞中检测出IL 2 1的表达 ,表达的mIL 2 1可在体外增强NK细胞的杀伤活性。结论 :成功地构建了重组真核表达质粒pcDNA3.1/mIL 2 1,为进一步在肿瘤动物模型中进行IL 2 1基因治疗及疗效观察奠定了基础
OBJECTIVE: To clone mouse interleukin-2 (IL-2) gene and construct eukaryotic expression plasmid for gene therapy of tumors. Methods: IL2 1 cDNA was amplified from ConA-activated T lymphocytes by RT-PCR and cloned into mammalian expression plasmid pcDNA3.1 to construct recombinant mIL 2 1 eukaryotic expression plasmid. The universal primer on the recombinant vector and the PCR primer were used as sequencing primers to confirm the correctness of the clone. The identified recombinant plasmids were transfected into Sp2 / 0 cells by lipofectamine. The expression of IL-2 gene in transfected cells was identified by RT-PCR and the expression of mIL-2-induced NK cell killing was detected by MTT assay Increased activity. Results: The recombinant eukaryotic expression plasmid pcDNA3.1 / mIL 2 1 was constructed correctly and the expression of IL 2 1 was detected in the transfected cells. The expressed mIL 2 1 could enhance the killing activity of NK cells in vitro. CONCLUSION: The recombinant eukaryotic expression plasmid pcDNA3.1 / mIL 2 1 has been successfully constructed, which lays the foundation for the further treatment of IL 2 1 gene in the tumor animal models and the observation of the curative effect