叶酸受体α真核表达载体的构建及在HEK293细胞中表达的鉴定

Construction and Identification of Eukaryotic Expression Vector Encoding Folate Receptor α

从高表达叶酸受体α的卵巢癌细胞株SKOV3中提取总RNA,RT-PCR扩增FRα基因片段,使用HindⅢ和EcoRⅠ双酶切后克隆到真核细胞表达载体pcDNA3.1中,对其进行酶切及测序鉴定;用脂质体介导法将重组质粒转染入人胚肾细胞HEK293中,并利用RT-PCR、Western blot及细胞免疫荧光染色检测细胞内FRα基因的表达情况。通过RT-PCR扩增得到750 bp左右的FRα基因片段,并构建了真核表达重组质粒,经酶切、测序鉴定正确。转染HEK293细胞后,RT-PCR检测到细胞中FRαmRNA的存在,Western blot、细胞免疫荧光证明FRα蛋白在细胞中的表达。成功构建了FRα基因的真核表达重组质粒,该质粒可在真核细胞中表达FRα蛋白,这些实验结果为卵巢癌基因疫苗的研究奠定了基础。

Total RNA was isolated from human ovarian cancer cell line SKOV3. The DNA fragment encoding folatc receptor ot was amplified using RT-PCR and the gene was cloned into the eukaryotic vector pcDNA3.1 between the two restriction enzyme cutting sites Hind m and EcoR I . The recombinant plasmid was identified by restriction endonuclease digestion and DNA sequencing. HEK293 cells was transfected with the pcDNA3.1-FRα plasmid using lipofectamine superFectin 11. The expression of the FRet in HEK293 cells was tested by RT-PCR,western blot and immunofluorescence staining. A 750 bp DNA fragment was amplified by RT- PCR from SKOV3 cells. The eukaryotic expression vector pcDNA3.1-FRet was successfully constructed and transfected into HEK293 ceils. The expression of FRet in HEK293 ceils was confirmed by RT-PCR,western blot and immunofluoreseence staining. The recom- binant eukaryotic expression plasmid pcDNA3, 1-FRet was constructed successfully and FRet expression was confirmed. The construc- ted pcDNA3.1-FRα recombinant vector will be used as DNA vaccination in ovarian cancer immunotherapeutic study.