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目的构建重组表达质粒pcDNA3.1-Her2-hsp70,并筛选稳定表达Her2蛋白的细胞株。方法采用RT-PCR法扩增人乳腺癌SKBR-3细胞中Her2和hsp70基因,插入质粒pcDNA3.1中,构建重组表达质粒pcDNA3.1-Her2、pcDNA3.1-hsp70,经双酶切后,回收hsp70基因,连接入质粒pcDNA3.1-Her2的N-末端,构建重组表达质粒pcDNA3.1-Her2-hsp70。将质粒pcDNA3.1和pcDNA3.1-Her2-hsp70分别转染小鼠乳腺癌4T-1细胞,采用Western blot法检测Her2蛋白的表达。结果重组表达质粒pcDNA3.1-Her2、pcDNA3.1-hsp70及pcDNA3.1-Her2-hsp70经双酶切鉴定,证明构建;转染质粒pcDNA3.1-Her2-hsp70的4T-1细胞中可见相对分子质量约65 000的Her2蛋白的表达。结论已成功构建了重组质粒pcDNA3.1-Her2、pcDNA3.1-hsp70及pcDNA3.1-Her2-hsp70,并获得了稳定表达Her2蛋白的小鼠乳腺癌4T-1细胞,为进一步探讨佐剂辅助异种抗原产生的抗肿瘤免疫效应奠定了基础。
Objective To construct recombinant plasmid pcDNA3.1-Her2-hsp70 and screen the cell lines stably expressing Her2 protein. Methods Her2 and hsp70 genes were amplified by RT-PCR from human breast cancer SKBR-3 cells and inserted into pcDNA3.1 to construct recombinant expression plasmids pcDNA3.1-Her2 and pcDNA3.1-hsp70. After double enzyme digestion, The hsp70 gene was recovered and ligated into the N-terminus of the plasmid pcDNA3.1-Her2 to construct the recombinant expression plasmid pcDNA3.1-Her2-hsp70. The pcDNA3.1 and pcDNA3.1-Her2-hsp70 were transfected into mouse breast cancer 4T-1 cells respectively, and the expression of Her2 protein was detected by Western blot. Results The recombinant plasmids pcDNA3.1-Her2, pcDNA3.1-hsp70 and pcDNA3.1-Her2-hsp70 were identified by double enzyme digestion. The results showed that the constructed recombinant plasmid pcDNA3.1-Her2-hsp70 transfected with pcDNA3.1-Her2- The molecular weight of about 65 000 Her2 protein expression. Conclusions Recombinant plasmids pcDNA3.1-Her2, pcDNA3.1-hsp70 and pcDNA3.1-Her2-hsp70 have been successfully constructed and 4T-1 cells of mouse breast cancer stably expressing Her2 protein have been obtained. To further explore the adjuvant-assisted The anti-tumor immune effect produced by xenoantigen laid the foundation.