双亚基共表达重组人白细胞介素12真核质粒pEGFP-C1_IL-12的构建及表达

Construction and Expression of a Human Interleukin 12 Double-submit Co-expressing Eukarya Plasmid pEGFP-C1_IL-12

目的构建携带增强型绿色荧光蛋白(EGFP)和人白细胞介素12双亚基目的基因(hIL-12,P35、P40)的真核表达质粒pEGFP-C1_IL-12,为进一步研究基因的定位、表达及hIL-12的抗肿瘤作用奠定基础。方法提取脂多糖(LPS)诱导后的人脐带血淋巴细胞总RNA,RT-PCR法扩增人白细胞介素12的双亚基P35和P40的全长cDNA,采用重叠PCR法连接两个亚基,双酶切双亚基片段及载体pEGFP-C1,T4DNA连接酶连接,重组质粒转化大肠埃希菌,挑取阳性克隆,PCR、双酶切及测序鉴定证实质粒构建成功。借助脂质体将重组质粒转染人肝癌细胞系HepG2,通过荧光显微镜检测报告基因的表达,RT-PCR及Western blot法检测目的基因的表达。结果PCR及测序鉴定证实重组质粒pEGFP-C1_IL-12构建成功,RT-PCR及Western blot法证实重组质粒在HepG2细胞内正常表达。结论携带报告基因(GFP)和双亚基目的基因(hIL-12,P35、P40)的重组真核质粒pEGFP-C1_IL-12构建成功,并能在人肝癌细胞系HepG2中表达。

Objective To construct recombinant eukaryotic expression plasmid pEGFP-C1_IL-12 containing report gene （EGPF） and recombinant human interleukin 12 double-submit （P40 and P35）, and to establish foundation for further research of the gene allocation and expression. Methods Total RNA was extracted from human umbilical cord blood lymphocytes, from which the total cDNA of human interleukin 12 double-submit was amplified by RT-PCR. The linkaged double-submit connected by overlap PCR was inserted into the MCS of vector pEGFP-C1 to construct a recombinant plasmid, which was identified by PCR and sequencing analysis. Then the recombinant plasmid was transfected into HepG2 cells by techniques of lipidosome transfection and its expression was detected by fluorescent microscopy, RT-PCR and Western blot. Results Identification of pEGFP-C1_IL-12 by PCR and sequencing analysis showed that the length, inserted location and direction was correct, and the expression of report gene and target gene was observed by fluorescent microscopy, RT-PCR and Western blot. Conclusion The recombinant plasmid pEGFP-C1_IL-12 containing report gene and double-submit targeted gene was constructed successful- ly, which could be expressed in human hepatoma carcinoma cell line HepG2 cells.