重组七鳃鳗细胞毒素蛋白rLj-RGD3表达及对Hela细胞活性的影响

Expression and bioactivity effects to Hela of recombinant toxin protein rLj-RGD3 from Lampetra japonica

摘要:

为研究重组七鳃鳗细胞毒素蛋白rLj-RGD3的抗肿瘤活性, 确定其生物学地位及意义, 本研究提取成体七鳃鳗(Lampetra japonica)口腔腺组织总RNA, 经RT-PCR获得cDNA序列长度为357 bp的目的基因片段, 将其克隆于 pET23b载体, 获得带有组氨酸标签的分子量为15 kD的基因重组蛋白rLj-RGD3的高效可溶性表达，通过组氨酸亲和层析纯化蛋白。采用MTT法检测不同浓度rLj-RGD3对bFGF诱导下的Hela细胞增殖的抑制作用, 结果表明rLj-RGD3 能显著抑制Hela细胞的增殖, IC50为2.6 mmol/L。rLj-RGD3作用后的Hela细胞经Hoechst染色及DNA ladder检测结果显示, 细胞均发生凋亡。rLj-RGD3对Hela细胞黏附玻连蛋白(VN)作用的实验结果为有效抑制。采用Transwell细胞培养板对bFGF诱导下的Hela细胞迁移实验表明, rLj-RGD3能够抑制Hela细胞的迁移, 且抑制率达60%。采用人工基质膜基质Matrigel及Transwell模仿体内环境研究rLj-RGD3对Hela细胞浸润行为实验显示, 以bFGF为趋化剂的Hela细胞穿透Matrigel的浸润行为明显受到抑制。以上研究表明, rLj-RGD3具有典型的RGD毒素蛋白的功能, 有望应用于抗肿瘤基因工程药物开发, 具有重要的生物学意义。

Abstract:

Lj-RGD3 was a toxin from the saliva gland of Lampetra japonica. To study the anti-tumor function of rLj-RGD3 and confirm its biological status and significance, we extracted total RNA from the saliva gland and amplified the cDNA of Lj-RGD3 by RT-PCR. The cDNA of Lj-RGD3 was 357 bp long and encoded a polypeptide composed of 118 amino acids including 2 cysteines, 17 histidines and 3 RGD (Arg-Gly-Asp) motifs. We cloned the cDNA into the plasmid pET23b, and expressed the recombinant protein rLj-RGD3 in Escherichia coli BL21. Fusion rLj-RGD3 with the C-terminal his-tag was a 15 kD soluble protein. Using the His-Bind affinity chromatography, we purified rLj-RGD3. Furthermore, we determined the biological activities of rLj-RGD3. To examine the ability of rLj-RGD3 inhibiting Hela cells proliferation, we used MTT assay. The results showed that, rLj-RGD3 inhibited bFGF induced proliferation of Hela cells in a dose-dependent manner, the IC50 value was 2.6 μmol/L. Hoechst staining assay revealed that, the nuclei of the cells treated with rLj-RGD3 were stained much brighter than that of untreated cells due to chromatin condensation. Furthermore, the DNA ladder patterns from the cells treated with rLj-RGD3 were also observed. These results demonstrated that rLj-RGD3 could induce apoptosis of Hela cells. Cell adhesion, migration and invasion are critical processes in tumor metastasis. rLj-RGD3 significantly inhibited adhesion of Hela cells to vironectin in a dose-dependent manner. In order to determine the effect of rLj-RGD3 on Hela cells migration toward bFGF, we used Transwell containing insert filter. rLj-RGD3 showed a significant inhibition on Hela cells migration, the inhibition rate was 60%. In the invasion assay, the Matrigel and Transwell were used to imitate environment in vivo. The results of invasion assay revealed that, rLj-RGD3 significantly inhibited bFGF induced invasion of Hela cells. Taken together, these results revealed that rLj-RGD3 had typical functions of RGD toxin protein and will be valuable in developing anti-tumor recombinant medicine.