摘要目的 构建重组慢病毒载体PLV-MAGE--A3-GFP,并检测其在人肺癌细胞系A549的表达情况.方法 利用DNA重组技术将MAGE-A3基因克隆到带GFP的慢病毒表达载体质粒PLV中,经限制性酶切和测序鉴定重组载体.将重组慢病毒载体PLV- MAGE- A3 -GFP与包装质粒pCMV-dR 8.91及pCMV-VSV-G共转染人胚肾上皮细胞株293TAB,包装重组慢病毒PL V-MAGE-A3-GFP,并感染人肺癌细胞株A549,用Western blot法检测MAGE-A3蛋白的表达情况.结果 限制性内切酶酶切和DNA测序分析证实,构建了重组慢病毒载体PLV-MAGE-A3-GFP,免疫荧光显微镜下观察显示慢病毒感染A549细胞后,MAGE-A3基因能够在细胞内正确地转录、翻译并稳定表达MAGE-A3基因.结论 成功构建了慢病毒载体PLV-MAGE-A3-GFP,包装的病毒能够成功感染人肺癌细胞系A549,并使MAGE-A3基因得以稳定表达,为进一步对非小细胞肺癌进行免疫治疗提供了适合的稳定转染的载体.

abstractsObjective To construct the recombinant lentiviral vector containing human MAGE-A3 gene and measure the expression level of MAGE-A3 in lung cancer cell line of A549.Methods The MAGE-A3 fragment was cloned into the lentiviral vector PLV with GFP by recombinant DNA technology.The resultant lentivirus were confirmed by PCR restriction enzyme digestion and DNA sequencing.Recombinant lentivirus PLV-MAGE-A3-GFP were produced from 293TAB by transient calcium-phosphate co-transfection with pCMV-dR8.91 and pCMV-VSV-G.A549 cells were infected by PLV-MAGE-A3-GFP lentivirus and the expression of MAGE-A3 was confirmed by Western blot.Results The recombinant lentiviral vector carried the MAGE-A3 gene was successfully constructed.Western blot analysis revealed that the MAGE-A3 gene could be correct transcripted and translated in A549 cell which infected by PLV-MAGE-A3-GFP.Conclusions The recombinant lentiviral vector PLV-MAGE-A3-GFP is constructed successfully,it can be used to transfect lung carcinoma cells.MAGE-A3 can be stablely expressed and promote the proliferation of A549 cell.