重组质粒pcDNA3.1-Her2-hsp70的构建及其表达

Construction and expression of recombinant plasmid pcDNA3. 1-Her2-hsp70

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 strain for stable ex- pression of Her2 protein. Methods Her2 and hspTO genes were amplified by RT-PCR from human breast cancer SKBR-3 cells and inserted into plasmid pcDNA3. 1. The constructed reeombinant plasmid pcDNA3. 1-Her2 and pcDNA3. 1-hspTO were identified by restriction analysis, and hsp70 gene was recovered and linked to the N-terminus of plasmid pcDNA3. 1- Her2. Mouse breast cancer 4T-1 cells were transfeeted with the constructed plasmid pcDNA3. 1-Her2-hsp70 and control plasmid pcDNA3. 1 respectively, and determined for expression of Her2 protein by Western blot. Results Restriction analysis proved that recombinant plasmid pcDNA3. 1-Her2, peDNA3. 1-hspTO and pcDND3. 1-Her2-hspTO were con- strutted correcdy. The expression of Her2 protein, with a relative molecular mass of about 65 000, was observed in the 4T-1 cells transfected with recombinant p/asmid pcDNA3. 1-Her2-hsp70. Conclusion Recombinant plasmids pcDNA3. 1 / Her2, pcDNA3. 1/hsp70 and pcDNA3. 1/her2-hsp70 were constructed successfully, and the 4T-1 cells for stable ex- pression of Her2 protein was established, which laid a foundation of further study on anti-tumor immune effect induced by heteroantigen assisted by adjuvant.