新基因hSSB1逆转录病毒载体的构建、鉴定和稳定株的筛选

 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。

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

hSSB1 is a key signaling moleculue that participates in DNA damage response.In this study,the cDNA of hSSB1 gene amplified by RT-PCR was inserted into the retroviral vector pBABE.The recombinant positive plasmid clone was identified by endonuclease digestion,PCR amplification and sequencing analysis.The retroviral expression vector pBABE-hSSB1 and PIK packaging plasmid were cotransfected 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.