肝癌相关抗原HAb18G真核表达载体的构建及表达

The construction and expression of eukaryotic expression vector containing hepatoma associated antigen HAb18G

目的在真核细胞中高效表达肝癌相关抗原HAb18G。方法构建含有肝癌相关抗原HAb18G基因的真核表达载体 ,并经限制性酶切及部分序列分析证明插入是否正确。用阳离子脂质体介导转染COS 7细胞和CHO细胞 ,并分别进行瞬时及稳定表达 ;通过间接免疫荧光染色和流式细胞仪检测蛋白的表达情况。结果成功地构建了真核表达载体 pcDNA 3/HAb18G ,并经限制性酶切及部分序列分析证明基因插入正确。间接免疫荧光染色证实 ,HAb18G高效表达于转染细胞的胞膜上。流式细胞仪分析显示 ,COS 7细胞的转染率约为7.5 % ,平均荧光强度为7.36 ,从CHO细胞获得的单个阳性克隆的平均荧光强度为2.17。结论以上结果为大量获得表达的HAb18G蛋白分子用于其生物学功能的研究奠定了基础。

Aim To express hepatoma associated antigen HAb18G in eukaryotic cell efficiently. Methods After constructing a eukaryotic expression vector pcDNA 3/HAb18G for the expression of hepatoma associated antigen HAb18G,it was checked whether gene was inserted correctly by partial nucleotide sequencing and restriction endonuclease digestion. Then vector pcDNA 3/HAb18G was transfected into COS 7 cells and CHO cells respectively by mixing with lipofectamine 2000 reagent to obtain transient and stable expression. The expression of HAb18G was detected by indirect immunofluorescence staining and flow cytometry assay. Results A eukaryotic expression vector pcDNA 3/HAb18G containing gene coding hepatoma associated antigen HAb18G has been successfully constructed, and its open reading frame was confirmed correctly by partial nucleotide sequencing and restriction endonuclease digestion. It was confirmed that HAb18G was expressed efficiently on the membranes of transfected cells by indirect immunofluorescence staining and flowcytometric analysis. In addition, the following results were revealed by FACS assay: positive cos 7 cells were about 7.5 percent of total cells with mean immunofluorescence intensity 7.36, while the immunofluorescence intensity of single cloned positive CHO cell was 2.17. Conclusion These results can be used for getting a large amout of expression protein and studying its biological functions.