FUS1和IL-12双基因真核共表达质粒的构建及对A549细胞促凋亡作用研究

Construction and Identification of Eukaryotic Co-expression Plasmid Carrying FUS1 and Recombinant Human IL-12 Genes and Its Effection on Apoptosis of A549 Cells

目的构建FUS1和人IL-12双基因真核共表达质粒并在肺癌A549细胞中检测基因表达及诱导细胞凋亡作用。方法采用RT-PCR技术从人肺成纤维细胞MRC-5中逆转录扩增全长FUS1基因,通过PCR技术从pORF-hIL-12质粒中扩增含双亚基的全长IL-12基因。FUS1和IL-12分别经酶切后定向插入真核细胞双顺反子载体pVITRO2的多克隆位点中,酶切和测序鉴定双基因共表达质粒pVITRO2-FUS1-IL-12的构建。以pVITRO2-FUS1-IL-12转染肺癌A549细胞,通过RT-PCR、ELISA和Western blot方法检测目的基因的表达,以Hoechst33258染色及流式细胞术验证pVITRO2-FUS1-IL-12在体外的促凋亡作用。结果酶切和测序分析表明成功构建了双基因真核共表达质粒pVITRO2-FUS1-IL-12,在转染后的细胞中同时检测到了FUS1和IL-12的表达,两种基因在双基因表达载体上的表达量基本不受影响。双基因共表达质粒pVITRO2-FUS1-IL-12具有明显诱导肺癌细胞凋亡的作用。结论成功构建pVITRO2-FUS1-IL-12双基因真核共表达质粒,为肺癌的多基因联合治疗提供了又一新的思路和方法。

Objective To construct a eukaryotic co-expression plasmid containing FUS1 and IL-12, and to investigate the influence of the recombinant plasmid on the cultured human lung cancer cell line A549. Methods RT-PCR was applied to ampIify FUS1 from MRC-5 cell, the eDNA fragment of IL-12 was derived from pORF-hIL- 12 by PCR, FUS1 cDNA fragment and IL-12 cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-FUS1-IL-12. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequenceing and transfected into A549 cells. The expression of genes in pVITRO2-FUSI-IL-12 were identified by RT-PCR, ELISA and Western blot. The function of pVITRO2- FUS1-IL-12 inducing the apoptosis of lung cancer was identified by Hoechst33258 and flow cytometry. Results The restriction cndonuclease digestion and sequencing suggested that co-expression vector pVITRO2-FUS1-IL-12 was constructed successfully, the expression of FUS1 and IL-12 could be detected in A549 cells. The expressions of the two genes in pVITRO2 were not affected. The A549 cells transfected with pVITRO2-FUS1-IL-12 exhibited significant cell apoptosis. Conclusion The recombinant eukaryotie expression vector pVITRO2-FUS1-IL-12 was constructed and expressed in eukaryotic ceil successfully, which would contribute to a novel and effective treatment strategies for combined gene therapy for lung cancer.