[Expression and bioactivity effects to Hela of recombinant toxin protein rLj-RGD3 from Lampetra japonica].
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 micromol/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.
Zhang
Piqiao
P
Institute of Halobios Function Gene and Protein Science, Liaoning Normal University, Dalian 116029, China.
Wang
Jihong
J
Liu
Xin
X
Chu
Dan
D
Li
Qingwei
Q
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D005260:Female; D005397:Fish Venoms / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007798:Lampreys / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D012469:Salivary Glands / Q000737:chemistry
D000970:Antineoplastic Agents; D005397:Fish Venoms; D009842:Oligopeptides; C574387:RGD3 protein, Lampetra japonica; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid