Inhibition Effects Of Snake Venom Metalloproteinase Inhibitor BJ46a On Melanoma Cells Invasion And Metastasis

BJ46a is a potent inhibitor of the snake venom metalloproteinases (SVMPs)proteolytic activities. It is from the serum of the venomous snake Bothrops jararaca. Being a new member of the fetuin family, cystatin superfamily, BJ46a interacts with the metalloproteinases by the formation of a noncovalent complex which occurs between the metalloproteinase domain and the inhibitor. Both SVMPs and MMPs protein families belong to the superfamily metzincin of metalloproteinases, so BJ46a may inhibit tumor invasion and metastasis by the formation of a complex with MMPs. In this study, BJ46a gene was synthesized first, then baculovirus expression vector was constructed to produce BJ46a recombinant protein. Finally, transfection of BJ46a gene into mouse melanoma cell line was done. The effects of BJ46a on invasion and metastasis of melanoma cells at both the protein level and gene level were investigated.1. BJ46a gene synthesisThe twenty fragments of BJ46a gene from GenBank (AF294836) were synthesized. Slowly annealing PCR methods were conducted to obtain the full length of BJ46a gene. This gene was cloned into vector pET-42a (+) at SacI / XhoI site. The construct was confirmed by PCR ,enzyme digestion and sequence analysis. The gene constructed by this method was found to contain 6 sequence errors, which were subsequently corrected by site-directed mutagenesis. The successful synthesis of snake BJ46a gene and construct of pET-42a(+)/BJ46a plasmid were useful for further study at both protein level and gene level. 2. Inhibition effects of the recombinant BJ46a produced in baculovirus expression system on the invasion and metatasis of melanoma cellsBac to Bac baculovirus expression system was used to generate a recombinant baculovirus by site-specific transposition. First, the corrected snake BJ46a gene was cloned into the multiple cloning site of pFastBac HT C vector, and then the ligation reaction was transformed into DH5α-T1 competent E.coli , followed by being selected ampicillin-resistant transformants using LB agar plate. Positive transformants were verified by both PCR and restriction analysis. The pFastBac construct was isolated and transformed into DH10Bac competent cells which contain the bacmid to generate a recombinant bacmid DNA . After 48 hours, blue/white selection was applied. White colony was picked and restreaked, result showed pure white colonies. Recombinant DNA was analyzed by PCR. Thus, a recombinant pFastBac HT C vector and a recombinant shuttle vector (bacmid) of baculovirus were generated. Cellfectin reagent complexes contained BJ46a gene were then used to tranfect insect cells Sf9. Recombinant protein was produced by cells infected with high titer viral stock. SDS-PAGE and western-blot were used to detect the protein expression. Finally, recombinant fusion protein was purified by ProBond? at the point of maximal expression ( 4 days post-infection). The activity of MMPs could be reduced by purified BJ46a protein. The ability of tumor cell invasion was identified by transwell chamber in vitro and tumor invasion animal model in vivo. The ability of B16 cells treated with recombinant BJ46a to invade the reconstituted basement membrane decreased significantly along with obviously inhibited B16 cells treated with recombinant BJ46a injected into C57BL/6 mice via the tail lateral vein to form experimental pulmonary metastasis model . These results demonstrate that BJ46a might have a strong effect on the inhibition of the invasion and metatasis potential in vitro and in vivo.3. Inhibition effects of BJ46a stable transfection on melanoma cells invasion and metastasisRecombinant expression plasmid pcDNA3.1 HisC - BJ46a was constructed and transfected into the murine melanoma cell line B16 using fugene 6 gene transfer technique, BJ46a clones were then screened by G418 and identified via RT-PCR . The ability of tumor cell invasion and migration was identified by transwell chamber in vitro and tumor invasion animal model in vivo.The invasion decreased significantly compared to nontransfected cells or cells transfected with pcDNA3.1HisC along with obviously inhibited the experimental pulmonary metastasis,but the abilities of proliferation and adhesion and migration of Bl6/pcDNA3.1HisC-BJ46a cells had no change.The data showed that transfection of BJ46a gene into mouse melanoma cell line resulted in the inhibition of the invasion potential in vitro and in vivo.In conclusion,this study provides the first evidence that BJ46a could suppress tumor invasion and metastasis both in vitro and in vivo, and offers the theory foundation and prerequisite for development and application of anti-tumor invasion and metastasis drug.