摘要
目的构建细胞因子融合蛋白IP10-EGFRvⅢScFv,建立并检测稳定表达融合蛋白的细胞株NIH3T3。方法通过RT-PCR法扩增小鼠IP10基因,全基因人工合成EGFRvⅢScFv,两段目的基因依次克隆于表达载体pcDNA3.1(-)PCMV启动子下游,二者以编码(Gly4Ser)3的柔性接头序列(linker)相连。将构建好的pcDNA3.1(-)IP10-EGFRvⅢScFv转染NIH3T3细胞,并用G418筛选出阳性克隆,采用实时定量PCR和Western blot法检测克隆细胞目的基因和蛋白的表达量,并观察转染后的NIH3T3细胞体外生长状况。结果成功构建了真核表达载体pcDNA3.1(-)IP10-EGFRvⅢScFv,经过浓度为200mg/L的G418工作液筛选出了稳定高表达IP10-EGFRvⅢScFv的细胞系NIH3T3,且其生长状态并未受转染基因的影响。通过荧光检测、实时定量PCR、Western blot法检测到转染72h后目的基因和蛋白表达量最高,和稳定克隆株相比,表达量并无明显差异(P>0.05)。结论构建的新型细胞因子融合蛋白可以在NIH3T3细胞内获得稳定表达,为后期融合蛋白的提取及功能研究打下基础。
Objective To construct cytokine fusion protein IP10-EGFRvⅢScFv,then establish and evaluate NIH3T3 cells stably expressing the fusion protein.Methods The mouse IP10 gene was amplified by using RT-PCR,and entire genome of EGFRvⅢScFv was synthesized artificially.Then,we introduced the sequences encoding a flexible linker of（Gly4Ser）3 to connect mouse IP10 and EGFRvⅢScFv genes and subcloned sequentially into downstream of promoter of CMV in expression vector pcDNA3.1（-）.Mouse NIH3T3 cells were transfected with IP10-EGFRvⅢScFv and positive clones were screened in the presence of G418.The expression level of IP10-EGFRvⅢScFv was examined by real-time PCR and Western blot.Also,growth kinetics of IP10-EGFRvⅢScFv-NIH3T3 cells was observed in vitro.Results Expression vector of pcDNA3.1（-）IP10-EGFRvⅢScFv was successfully constructed and NIN3T3 stable cell line with high expression of IP10-EGFRvⅢScFv was screened out by G418 with the concentration of 200 mg/L.Simultaneously the growth of IP10-EGFRvⅢScFv-NIH3T3 cells was not influenced by the transfected gene in vitro.By using fluorescence,real-time PCR and Western blot,the expression of IP10-EG-FRvⅢScFv mRNA and protein reached the peak on the 72nd h after transient transfection,but there was no significant difference in comparison to stable expression clones（P0.05）.Conclusion A novel cytokine fusion protein can be expressed stably in NIH3T3 cells,which may lay a foundation for the fusion protein extraction and functional studies.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2012年第1期1-6,共6页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.30973077)
