小鼠内皮抑制素真核质粒的构建表达及其体外活性

Construction and Expression of Mouse Endostatin Eukaryotic Plasmid and Its Bioactivity in vitro

目的构建小鼠内皮抑制素(endostatin)真核表达质粒,进行分泌性表达,研究其体外生物学活性,以进一步用于肿瘤的基因治疗。方法人工合成小鼠Igκ轻链信号肽序列,RT-PCR获得小鼠内皮抑制素编码序列,一起克隆入真核表达载体pcDNA3.1中,对其进行酶切鉴定和序列分析。重组质粒经脂质体介导转染受体细胞BHK-21,ELISA检测细胞培养上清中内皮抑制素的含量。用此上清作用于经bFGF刺激的ECV304内皮细胞和HepG2肝癌细胞,MTT法检测细胞的增殖。结果经酶切鉴定及DNA测序分析,Igκ信号肽序列及内皮抑制素编码序列成功克隆到目的位点上,其转染细胞上清中可以检测到分泌出的内皮抑制素,含量为(65.5±10.9)ng/106细胞;并且可以抑制ECV304内皮细胞的增殖,抑制率约为29.2%,而对HepG2肝癌细胞增殖没有影响。结论成功构建了小鼠内皮抑制素真核质粒,其分泌性表达产物可以抑制内皮细胞的增殖,具有较好的生物学活性。

Objective To construct and express mouse endostatin eukaryotic plasmid and study the bioactivity in vitro for further research on tumor gene therapy. Methods DNA sequence of mouse Igκ signal peptide was synthesized and inserted into eukaryotic expressing plasmid pcDNA3.1 with mouse endostatin coding sequence amplified by RT-PCR. The recombinant was confirmed by restriction enzyme digestion and DNA sequencing. Endostatin was detected by ELISA from the supernatant of recipient cell BHK-21 transfected with the recombinant mediated by liposome. And the supernatant was used to culture ECV304 with bFGF and hepatoma cell line HepG2. The proliferation of latter was assayed by MTT. Results Restriction enzyme digestion and DNA sequencing showed that Igκ signal peptide sequence and endostatin coding sequence were correctly inserted into the target site of vector. Free endostatin of （65.5±10.9） ng/10^6 cells could be detected in the supernatant of the transfected BHK-21, which could inhibit the proliferation of ECV304 by 29.20/00 and had no influence on HepG2 cells. Conclusion Mouse endostatin eukaryotic plasmid was constructed successfully and the secreted endosatin had an inhibitory activity on the proliferation of endothelial cells.