新抑癌基因PIGl1高表达HepG2细胞株的建立

目的构建人PIGll逆转录病毒载体，建立高表达PIGll基因的HepG2细胞，为进一步研究PIGll基因在肝癌细胞中的功能提供一个理想的试验平台。方法将已有的peDNA3，1／NT—GFP—PIGll质粒用PCR法扩增出PIGll片段，构建含人PIGllcDNA的重组质粒pLXSN—PIGll。重组载体经PcR鉴定和DNA测序分析。将该重组质粒用lipofeetamine^TM 2000转染包装细胞PA317，获得DLXSN—PIGll逆转录病毒。用病毒感染HepG2细胞，获得PIGll高表达的HepG2细胞株。结果获得384bp人PIGll基因，测序证明PIGlleDNA编码序列与Genbank中报道的一致，转染PA317细胞后用病毒上清感染HepG2细胞，经RT—PcR检测发现HepG2细胞中PIGllmRNA表达明显升高。结论成功构建了高表达PIGll基因的HepG2细胞株，为进一步研究其生物学行为奠定了基础。

Recombinant PIG11 Gene Retroviral Vector Construction and Its High Expression in HepG2 Cells

Objective To construct the human PIGll gene retroviral vector and obtain a HepG2 cell line high expressing PIG1 lgene by infection, which can be used for the further research on the function of PIGll gene in liver cancer cell. Methods PIG11 gene was amplified by PCR from expression vector pcDNA3. 1/NT- GFP - PIG11, this gene fragment insert was put into retroviral vector pLXSN, then this new vector pLXSN- PIGll was identified by PCR and DNA sequencing analysis. This vector was transfected into packing cell PA317 mediate by lipofectamine^TM2000, which can induce retrovirus release. Then we use this retrovims infect HepG2 cell to achieve PIGll gene high expression HepG2 cell line. Results 384 bp PIGll gene fragment was obtained, which is the same as PIG11 cDNA in genbank report. RT - PCR was used to detect infected HepG2 cells, which indentified PIG11 rnRNA was up - regulated. Conclusion The retroviral vector pLXSN- PIG11 was successfully constructed and infected into HepG2 cells, new HepG2 cell line was obtained, which highly express PIGll gene.