pCDNA3.1/NY-ESO-1真核表达质粒的构建及在肝癌细胞系HepG2中稳定高表达

To construct the eukaryotic expression plasmid pCDNA3. 1 /NY-ESO-1 and to establish a NY-ESO-1 highly expressed HepG2 cell line

目的建立癌-睾丸抗原NY-ESO-1稳定表达的HepG2肝癌细胞系。方法设计NY-ESO-1的引物,PCR法从NY-ESO-1阳性表达的睾丸癌组织中扩增出目的片段,克隆至T载体,双酶切后定向连入pCDNA3.1载体,构建pCD-NA3.1/NY-ESO-1真核表达质粒。经酶切、PCR、测序检测其构建的正确性,脂质体转染法将重组质粒转入HepG2细胞,G418药物筛选出稳定转染的细胞系,RT-PCR法、间接免疫荧光法分别检测稳定转染HepG2细胞中NY-ESO-1基因、蛋白的表达水平。结果构建的pCDNA3.1/NY-ESO-1质粒经酶切、测序等检验表明质粒构建成功,筛选获得稳定高表达NY-ESO-1的HepG2细胞。结论构建了pCDNA3.1/NY-ESO-1真核表达质粒及稳定高表达NY-ESO-1的HepG2细胞株,为下一步以NY-ESO-1为靶标进行肝癌的抗原特异性免疫治疗研究奠定了实验基础。

Objective To establish the human hepatocarcinoma cell line（ HLA-A2 positive HepG2 cell line） with stabilized expression of NY-ESO-1（ New York esophageal squamous cell carcinoma 1） antigen（ cancer-testis antigen） . Methods NY-ESO-1 primers were designed and full length NY-ESO-1 gene was amplified from the tissue of testis carcinoma by RT-PCR（ reverse transcription-polymerase chain reaction） . The NY-ESO-1 gene was cloned into pMD18-T vector,subcloned into pCDNA3. 1 vector（ pCDNA3. 1 /NY-ESO-1） and finally confirmed by sequen-cing. Then the recombinant pCDNA3. 1 /NY-ESO-1 plasmid was transfected into the HepG2 cell line by LipofectamineTM 2000 and NY-ESO-1 highly expressed cells were selected with G418（ 400 μg/ml） . NY-ESO-1 gene and protein expression were detected by RT-PCR and indirect immunofluorescence methods. Results The recombinant pCDNA3. 1 /NY-ESO-1 plasmid was constructed and the HLA-A2 positive HepG2 cell line with highly NY-ESO-1 gene and protein expression was screened. Conclusion A NY-ESO-1 highly expressed HepG2 cell line is establish,which will be the perfect target of NY-ESO-1 based antigen special immunotherapy for hepatocellular carcinoma in the future.