Molecular Clonging, Expression And Some Biological Functions Of Lampetrin1, A RGD-motif Peptide From Lampetra Japonica

RGD peptides are small molecular weight proteins which contain RGD sequence. As a competitive antagonist, it can interfere the combination between the extracellular matrix and cells integration by their specific RGD sequence[1].Moreover, RGD peptides also can inhibit platelet aggregation, angiogenesis and tumor cell proliferation [1-2] by closing integrin signal transduction pathway.A cDNA library was constructed from the oralgland of Lampetra japonica. 1281 virtual ESTs[3] were founded after random selection of clones for sequencing, Bioinformatical analysis showed three RGD peptide genes and three open reading frames which can translate RGD motif.Lampetrin1 is a recombinant protein of RGD-peptide from Lampetra japonica . In this study, total RNA was extracted from salivary gland and was reversely transcribed to cDNA. By amplifying of cDNA, we obtained Lampetrin1's gene with 423bp. Sequencing indicated that Lampetrin1 was non-homology with other RGD-peptide from other species. Lampetrin1 contains motif- RGD. After cloning, transforming, and expression of Lampetrin1 gene, the Lampetrin1 was purified. The soluble homogeneous protein was obtained and could significantly inhibit angiogenesis of 7-days-old chick embryos and aggregation of rabbit platelet induced by ADP. The result shows that lamptrin1 has typical RGD-peptides'functions.Lampetrin1 could inhibit proliferation of SMMC-7721 cells, the ID50 value of Lampetrin1 was 2.64μmol/L. The reason maybe is Lampetrin1 can bind with integrin which on cell surface to prevent the downstream signal transduction pathway, accordingly induce apoptosis.Lampetrin1 treated SMMC-7721 cells were stained with Hoechst 33258, a fluorescent DNA-binding dye, to identify the apoptotic nuclei. This result also demonstrated that Lampetrin1 at the concentration of 1.37μmol/L could induce apoptosis in SMMC-7721 cells. Extracting DNA, by means of agarose gel electrophoresis and ethidium bromide staining, we can see typical DNA ladder in apoptosis cell lines.An important biological feature of malignancy is the invasion and the transfer to adjacent normal tissue. Imitation the three steps of tumor cell in-break ECM, we found that Lampetrin1 can inhibit the SMMC-7721 cells adhesion to matrigel, migration through transwell and invasion through matrigel in vitro. This result shows that Lampetrin1 can restrain SMMC-7721 cells invading in ECM, then restrain their metastasis. Lampetrin1 was obtained by gene engineering and was confirmed to have the typical functions of RGD peptides. Lampetrin1 can be made antitumor agents in the future and its potential clinical value could be developed.Meanwhile we prepared the polyclonal antibodies of recombinant protein Lampetrin1; This work make a good foundation for the filtration and confirmation of Lampetrin1 gene and protein.The completion of this project, we will be the first one who found and confirmed a new gene which has RGD peptides function from Lampetra japonica. That added new member from the different species for RGD peptides family, laid the theoretical foundation for the research on functional genomics and proteomics of Lampetra japonica. We also provide detailed scientific basis for anti-thrombotic,anti-cancer gene drug development.