新基因hSSB1逆转录病毒载体的构建、鉴定和稳定株的筛选

Construction and Identification of hSSB1 Retrovirus Expressing Vector and Screening of Stable Transfected Cells

hSSB1(Human Single-strand DNA-binding protein)是参与细胞DNA损伤应答的一个重要信号分子。根据GenBank提供的hSSB1基因序列扩增其cDNA序列,插入到pBABE逆转录病毒载体中,连接后的质粒转化后经过双酶切,PCR扩增及测序来鉴定pBABE-hSSB1阳性克隆。将阳性表达的pBABE-hSSB1和包装质粒转染到HEK293T细胞中,产生病毒液。将包装好的病毒感染细胞并用嘌呤霉素(puromycin)筛选稳定表达hSSB1的细胞株。重组pBABE-hSSB1质粒经双酶切,PCR扩增鉴定及DNA测序分析等方法证实克隆成功。Western blotting检测发现转染重组质粒pBABE-hSSB1的细胞株中hSSB1蛋白的表达水平高于对照组。该研究成功构建了针对hSSB1基因的逆转录病毒载体(pBABE-hSSB1),并得到了稳定高表达hSSB1的细胞株,为深入研究其功能奠定了基础。

hSSB1 is a key signaling the eDNA of hSSB1 gene amplified moleculue that participates in by RT-PCR was inserted into combinant positive plasmid clone was identified by endonuclease quencing analysis. The retroviral expression vector pBABE-hSSB1 DNA damage response. In this study, the retroviral vector pBABE. The re- digestion, PCR amplification and se- and PIK packaging plasmid were co-transfected into 293T cells to produce the retrovirus. The packaging retrovirus was then infected into cancer cells and the over-expression cells were selected with puromycin, pBABE-hSSB1 positive clones have been validated to be correct by restriction endonuclease, PCR amplification and DNA sequencing analysis. The protein level of hSSB1 in pBABE-hSSB1 transfected cancer cell line was significantly up- regulated as validated by Western blotting. Our data indicate that the recombinant plasmid of pBABE- hSSB1 was successfully constructed and transfected stably into cancer cells. It established a favorable foundation for further functional study.