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目的:构建人乳腺珠蛋白(hMAM)与人热休克蛋白70(hHSP70)融合基因的真核表达载体pcDNA3.1-hMAM-HSP70,并观察重组载体在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,经酶切测序分析后,与人HSP70基因克隆入真核表达载体pcDNA3.1,构建了融合基因真核表达载体pcDNA3.1-hMAM-HSP70。将重组载体电穿孔法转染入COS-7细胞,间接免疫荧光法检测目的基因的表达。结果:成功扩增了人HSP70基因与hMAM基因,酶切测序结果表明,重组载体含有hMAM与人HSP70的融合基因,在COS-7细胞中可检测到hMAM表达。结论:hMAM与人HSP70融合基因的真核表达载体构建成功,为进一步进行DNA肿瘤疫苗研究提供了实验依据。
OBJECTIVE: To construct eukaryotic expression vector pcDNA3.1-hMAM-HSP70 fused with human mammary gland globin (hMAM) and human heat shock protein 70 (hHSP70) and to observe the expression of recombinant vector in COS-7 cells. Vaccine research laid the foundation. Methods: The hMAM gene was amplified by PCR and cloned into eukaryotic expression vector pcDNA3.1 with human HSP70 gene after digestion and sequencing. The fusion gene eukaryotic expression vector pcDNA3.1-hMAM-HSP70 was constructed. The recombinant vector was transfected into COS-7 cells by electroporation, and the expression of the target gene was detected by indirect immunofluorescence. Results: Human HSP70 gene and hMAM gene were successfully amplified. The results of restriction enzyme digestion showed that hMAM gene was expressed in COS-7 cells. Conclusion: The eukaryotic expression vector of fusion gene hMAM and human HSP70 was successfully constructed, which provided the experimental basis for further research on DNA tumor vaccine.