野生型rLj-RGD3基因重组突变体rLj-112分子克隆及对HeLa细胞活性影响

为研究突变体rLj-112蛋白的抗肿瘤活性，人工合成七鳃鳗野生型rLj-RGD3蛋白和突变型rLj-112蛋白，通过比较两种蛋白质的抗增殖、迁移和促凋亡的活性，确定突变体rLj-112蛋白的生物学意义及地位。采用MTT方法检测不同浓度的rLj-112蛋白对HeLa细胞增殖的抑制作用。结果表明，rLj-112蛋白能显著抑制HeLa细胞的增殖，且IC50为4.3 μmol/L。使用Transwell细胞培养板对bFGF诱导的HeLa细胞迁移实验表明，rLj-112蛋白能抑制HeLa细胞的迁移。rLj-112蛋白作用后，HeLa细胞经Hoechst33258和AnnexinV-FITC染色结果显示，细胞均凋亡。流式细胞仪进一步证明，rLj-112蛋白能诱导HeLa细胞发生凋亡，且呈剂量依赖性。由此可见，与野生型rLj-RGD3蛋白比较，突变型rLj-112蛋白有较高的细胞毒性作用，具有抗肿瘤的功能，有望应用于抗肿瘤基因工程药物的开发，具有重要的生物学意义。

Wild-type RLj-RGD3 Genes Recombinant Mutants—Molecular Cloning and Bioactivity Effects to HeLa of RLj-112

To study the anti-tumor function of rLj-112 and confirm its biological status as well as significance, lampreys wild type rLj RGD3 protein and mutation type rLj 112were artificially synthesized by comparing the two proteins anti proliferation and migration, and promote apoptosis activity, confirm its biological status and significance. The ability of rLj-112 inhibiting HeLa cells proliferation was examined by MTT assay. The results showed that rLj-112 inhibited proliferation of HeLa cells in a dose-dependent manner. The IC50 value was 4.3 μmol/L. The effect of rLj-112 on HeLa cells migration toward bFGF was determined by Transwell containing insert filter. rLj-112 showed a significant inhibition on HeLa cells migration. Hoechst and AnnexinV-FITC staining assay revealed that the nuclei of the cells treated with rLj-112 were stained much brighter than that of untreated cells due to chromatin condensation. Flow cytometry instrument was further evidenced that rLj-112 protein can induce apoptosis of HeLa cells and present dose-dependent manner. The results of invasion assay revealed that mutant protein rLj-12 has higher cytotoxic effect and function of anti-tumor compared with wild type rLj GD3 protein. Taken together, these results revealed that rLj-112 will be valuable in developing anti-tumor recombinant medicine.