GM-CSF基因修饰的卵巢癌细胞NuTu-19/GM-CSF的构建

Construction of ovarian cancer cell NuTu-19/GM-CSF genetically modified by GM-CSF

目的构建能分泌表达粒细胞-单核细胞集落刺激因子(GM-CSF)的卵巢癌细胞NuTu-19/GM-CSF。方法将T-h GM-CSF重组质粒转化入感受态大肠杆菌中,筛选出耐药性单克隆菌落送测序。利用PCR技术扩增出GM-CSF目的基因片段,将PCR产物连入pGEM-T easy载体,转化入大肠杆菌中扩增,用SDS碱裂解法提取Teasy-GM-CSF重组质粒,用限制性内切酶酶切Teasy-GM-CSF重组载体,连入经相同酶酶切的pLXSN反转录病毒载体中。用磷酸钙沉淀法转染PA317包装细胞系进行病毒包装,用含有病毒的上清液感染卵巢癌NuTu-19细胞,经G418加压筛选感染成功的NuTu-19/GM-CSF细胞,用免疫细胞化学和ELISA法检测NuTu-19/GM-CSF分泌表达GM-CSF蛋白的情况。结果T-h GM-CSF重组质粒测序结果与GenBank Accession#:NM_000758基因序列对比后确定为人类GM-CSF基因编码序列;PCR产物酶切电泳在Mark 400-500 bp中有一条带,符合GM-CSF基因序列长度;重组Teasy-GM-CSF质粒用EcoRⅠ、BamHⅠ限制性内切酶酶切后,电泳鉴定在400-500 bp之间有一目的条带,重组反转录病毒质粒pLGM-CSFSN酶切电泳有目的条带;G418加压筛选感染后的NuTu-19细胞,14 d后慢慢形成单克隆继续生长;免疫细胞化学检测到感染成功的NuTu-19细胞膜表面有明显的棕褐色颗粒沉淀,ELISA法检测细胞培养上清液中GM-CSF的质量浓度为150 pg/mL。结论成功构建了能分泌表达人GM-CSF蛋白的卵巢癌细胞NuTu-19/GM-CSF,为以后研究卵巢癌免疫治疗提供必备、有效、可靠的工具基础。

Objective To construct NuTu-19/GM-CSF ovarian cancer cells which can secrete and express granulocyte-macrophage colony-stimulating factor（GM-CSF）. Methods The T-h GM-CSF recombinant plasmid was transformed into competent Top109 Bacillus colis and the positive colonies were cultivated in soft agar medium.The T-h GM-CSF recombinant plasmid was used as models to amplify GM-CSF gene fragment by PCR.The certain fragment was linked with the pGEM-T easy vector and transformed into Top109 bacillus coil.SDS alkaline lysis was used to get T easy-GM-CSF recombinant plasmid with pLXSN retrovirus vector cut by the same enzymes.The pseudotype virus was packed after transfecting PA317 cells using calcium phosphate precipitation.The supernate fluid which contained virus to infect NuTu-19 cells was saved.The positive cells were harvested through pressurizing selected by G418.The GM-CSF protein secreted by NuTu-19/GM-CSF cells was tested by immunocytochemistry and ELISA. Results T-h-GM-CSF included the human GM-CSF gene fragment which contained 435 bp and signal peptide coding region,compared with the sequence of Genebank Accession#： NM-000758.Production of PCR was between 400 bp and 500 bp in the agarose electrophoresis,and had the same length with hGM-CSF gene.There was an electrophoretic strap between 400 bp and 500 bp and the sequence of the cutting fragment was completely correct after cutting T easy-GM-CSF recombinant plasmid by Eco RⅠ and Bam HⅠ enzymes.It showed that the amplification of the GM-CSF gene fragment and the connection with T easy vector were successful.There was still the same strap between 400 bp and 500 bp in the agarose electrophoresis of pLGM-CSFSN after being cut by Eco RⅠ and Bam HⅠ enzymes.The infected NuTu-19/GM-CSF cells cultivated in medium containing G418 continued to grow in the forms of monoclone 14 days later.There were some visible granules on the surface of the cellular membrane.The amount of GM-CSF in suspent liquid of cells culture was 150 pg/mL tested by ELISA. Conclusion The construction of NuTu-19/GM-CSF which can secret and express GM-CSF protein is successful,and offers effective and necessary basis for future study on ovarian cancer immunotherapy.