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目的:构建人乳腺珠蛋白(hMAM)基因重组质粒pcDNA3.1-hMAM,并观察重组质粒在COS-7细胞中的表达,为肿瘤DNA疫苗研究奠定基础。方法:利用PCR方法扩增hMAM基因,酶切测序后克隆入真核表达载体pcDNA3.1,构建真核表达载体pcDNA3.1-hMAM。将重组载体脂质体转染法转染入COS-7细胞,间接免疫荧光法和ELISA检测目的基因的表达。结果:成功扩增hMAM基因,酶切测序表明,重组质粒含有hMAM基因,在COS-7细胞中可检测到hMAM表达。结论:hMAM基因疫苗构建成功,为进一步进行DNA肿瘤疫苗研究提供了实验依据。
OBJECTIVE: To construct recombinant plasmid pcDNA3.1-hMAM of human mammary gland globin (hMAM) and to observe the expression of the recombinant plasmid in COS-7 cells, which lays the foundation for the study of tumor DNA vaccine. Methods: The hMAM gene was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.1 after digestion and sequencing. The eukaryotic expression vector pcDNA3.1-hMAM was constructed. The recombinant vector was transfected into COS-7 cells by lipofection method, and the expression of the target gene was detected by indirect immunofluorescence and ELISA. Results: The hMAM gene was successfully amplified. Sequencing by restriction enzyme digestion showed that hMAM gene was expressed in the recombinant plasmid and hMAM expression was detected in COS-7 cells. Conclusion: The successful construction of hMAM gene vaccine provided an experimental basis for further research on DNA vaccine.