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

Sheng Wu Gong Cheng Xue Bao. 2009 May;25(5):686-94.
[Article in Chinese]

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antineoplastic Agents / pharmacology*
  • Apoptosis / drug effects
  • Female
  • Fish Venoms / biosynthesis
  • Fish Venoms / genetics*
  • Fish Venoms / isolation & purification
  • Fish Venoms / pharmacology
  • HeLa Cells
  • Humans
  • Lampreys / metabolism*
  • Molecular Sequence Data
  • Oligopeptides / biosynthesis
  • Oligopeptides / genetics*
  • Oligopeptides / isolation & purification
  • Oligopeptides / pharmacology
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / pharmacology*
  • Salivary Glands / chemistry

Substances

  • Antineoplastic Agents
  • Fish Venoms
  • Oligopeptides
  • RGD3 protein, Lampetra japonica
  • Recombinant Proteins
  • arginyl-glycyl-aspartic acid