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目的:构建小鼠富含半胱氨酸分泌蛋白-1(Crisp-1)真核表达载体,并观察其在COS-7细胞中的表达。方法:从小鼠附睾组织中提取总RNA,RT-PCR扩增Crisp-1基因开放阅读框(ORF),将目的基因克隆至真核表达载体pcDNA3.1上,经脂质体介导转染COS-7细胞,并利用间接免疫荧光和RT-PCR检测其在COS-7细胞中的表达。结果:酶切电泳鉴定结果显示Crisp-1基因克隆入载体中,测序结果证实克隆的基因序列与基因库(GeneBank)中的Crisp-1序列相符。脂质体转染后,间接免疫荧光和RT-PCR结果均表明重组质粒pcDNA3.1-Crisp-1能在COS-7细胞中表达。结论:成功获得了能在COS-7细胞中表达的小鼠Crisp-1DNA疫苗的真核表达质粒pcDNA3.1-Crisp-1,为后续研究其生物学活性及免疫避孕效应奠定了基础。
OBJECTIVE: To construct a mouse eukaryotic expression vector encoding the secreted protein-1 (Crisp-1) and to observe its expression in COS-7 cells. Methods: Total RNA was extracted from mouse epididymal tissues. The ORF of Crisp-1 gene was amplified by RT-PCR. The target gene was cloned into eukaryotic expression vector pcDNA3.1 and transfected into COS by liposome -7 cells, and their expression in COS-7 cells was detected by indirect immunofluorescence and RT-PCR. Results: The results of restriction endonuclease digestion showed that the Crisp-1 gene was cloned into the vector and the sequencing confirmed that the cloned gene sequence was consistent with the sequence of Crisp-1 in GeneBank. Indirect immunofluorescence and RT-PCR results showed that the recombinant plasmid pcDNA3.1-Crisp-1 could be expressed in COS-7 cells after lipofection. CONCLUSION: The eukaryotic expression plasmid pcDNA3.1-Crisp-1 of mouse Crisp-1 DNA vaccine that can be expressed in COS-7 cells was successfully obtained, which laid the foundation for further study on its biological activity and immune contraceptive effects.