IL-12双亚基共表达载体的构建及其在人骨髓间充质干细胞中的表达

目的构建人IL-12（hIL-12）p40和p35双亚基真核共表达载体并转染人骨髓间充质干细胞（hMSC）。方法根据hIL-12p40和p35亚基全长cDNA序列分别设计合成引物行PCR扩增，将扩增所得p40和p35片段采用overlap PCR法拼接，获得的rhIL-12融合基因与pGEM—TEasy质粒连接，将鉴定正确的pGEM—T／rhIL-12重组质粒克隆至pcDNA3．1（＋）真核表达质粒中，构建pcDNA3．1（＋）／rhIL-12真核表达载体，并进行测序鉴定。将鉴定正确的pcDNA3．1（＋）／rhIL-12经脂质体介导转染hMSC，同时以转染pcDNA3．1（＋）空质粒作为对照组，在倒置显微镜下观察细胞生长形态；在转染后第4天采用蛋白质印迹法检测细胞培养上清液中rhIL-12融合基因的表达；另分别在转染后第2、4、6、8、10、12、14天，采用ELISA方法检测细胞培养上清液中rhIL-12融合基因的表达水平。结果PCR扩增结果显示特异性扩增出p40（1000bp）、p35（600bp）、rhIL-12（1600bp）片段，均与预期DNA表达片段大小一致。pcDNA3．1（＋）／rhIL-12测序显示克隆的rhlL-12基因序列与报告序列完全相同。倒置显微镜下观察，可见转染pcDNA3．1（＋）／rhIL-12的hMSC生长形态和生长速度与对照组hMSC相比均无明显差异。蛋白质印迹和ELISA检测显示，转染pcDNA3．1（＋）／rhIL-12的hMSC培养上清液中可见rhIL-12融合蛋白的持续表达；而对照组中均未检测到rhIL-12融合蛋白表达。结论成功构建了hIL-12p40和p35双亚基真核共表达载体pcDNA3．1（＋）／rhIL-12，为利用hIL-12进行非病毒载体抗肿瘤基因治疗奠定了基础。

Construction of vector co-expressing IL-12 double subunits and its expression in human mesenchymal stem cells

Objective To construct a vector co-expressing human IL-12 （hlL-12） 1340 and p35 subunits and to transfect it into human mesenchymal stern cells （hMSC）. Methods Full-length cDNAs of rhlL-12 p40 and p35 subunits was amplified by PCR. The amplified p40 and p35 fragments were used to establish rhIL-12 fusion gene by overlap PCR. The fusion gene was then inserted into the pGEM-T Easy plasmid. Afterwards, identified pGEM-T/rhIL-12 recombinant plasmid was cloned into eukaryotic expression plasmid pcDNA3.1（＋） to construct eukaryotic expression vector pcDNA3.1（＋）/rhIL-12. And the identified pcDNA3.1（＋）/rhlL-12 or pcDNA3.1（＋） blank vector （control） was transfected into hMSC mediated by liposome. Inverted microscopy was employed to observe the morphology of hMSC. After the transfection, the expression of rhlL- 12 fusion gene in supernatant was detected at day 4 by Western blotting; and the level of the expression was determined by ELISA at days 2, 4, 6, 8, 10, 12, and 14. Results By PCR full-length cDNAs of 1940 （1000 bp）, p35 （600 bp）, and rhlL-12 （1600 bp） were amplified specifically with expected length. The sequence of the cloned rhlL-12 gene was identical to the reported one. Inverted microscopy showed no significant difference in morphology and growth rate between the hMSC transfected with pcDNA3.1（＋）/rhlL-12 and the control group. Both Western blotting and ELISA revealed continuous expression of rhlL-12 fusion gene in the supernatant of the hMSC transfected with pcDNA3.1 （＋）/rhlL-12, while no such expression was detected in the control. Conclusion An eukaryotic vector co-expressing p40 and p35 subunits of hIL-12 has been constructed, such a vector can be used in further studies on non-virus vectors for anti-tumor gene therapy.