Study On Cloning Expression And Biological Activity Of VEGF Gene From Sika Deer

Deer antler is the male sika deer or red deer with densely hairy.The tender angle deer antler did not grow into hard angle. Deer antler is also the only organ can be completely regenerated in mammals. People gradually discovered that deer antler had nalways been the a valuable traditional medicine and had high medicinal value. With the deeper study, deer antler cell growth factor has been known as one of the main effective factor of regulating antler regeneration and development. In recent years, the research of cell growth factor has become a hot spot and vascular endothelial growth factor(VEGF) has been proved to exist in the antler tip tissue. The velvet tissue in rapid growth, vascular is more and more vigorous. Vascular endothelial growth factor is a promoting and repair protein, and it specific effect on vascular endothelial cells, promote the proliferation, increases vascular permeability, improve blood circulation, plays an important regulation role in the development process of vascular form and growth. Therefore, the study will make a preliminary study on the function of VEGF gene in sika deer and provide a theoretical basis for further exploration of sika deer VEGF gene to the regulation mechanism of antler growth. The study also provide the reference frame for the antler cell factor in the rapid development of regenerative medicine and tumor medicine.We cloned the sika deer VFGF gene by the PCR technique,then purified and recovered the target fragments, finally inserted the gene into the cloning vector of pMD-18 T and transformed into host cells DH5É‘. We picked out the positive bacteria identified by PCR reaction and double enzyme cutting reaction and sequenced. The expression of VEGF protein in skin layer, mesenchymal layer and cartilage layer was detected by the immuneohistochemistry in deer antler tip tissue. The proncleus expression recombinant plasmid pET-30a-VEGF was constructed, and transformed into host cells Rosetta, and high efficiency transformed plasmid elected and it shaking culture. Recombinant plasmid pET-30a-VEGF induced by IPTG, and it detected by SDS-PAGE electrophoresis, then the detection of recombinant protein VEGF antigen activity by Western blotting. Finally target protein purified by nickel affinity column and made objective protein refolded, and recombination protein VEGF which had the effect on Mouse fibroblast(NIH3T3) cell was detected by MTT method.The result of experiment showed that VEGF sequnence cloned was 519 bp. The cow, ovis, pig nucleotide sequence homology were 98.78%, 97.56%, 96.95%.The amino acid sequence homology were 98.79%, 97.78%, 96.36%. The result of the enzyme immunohistochemistry showed that there was the expression of VEGF protein in mesenchymal layer, the skin layer and cartilage layer, but did not have difference.The result of MTT showed that the recombinant protein VEGFhad the effect on proliferation on NIH3T3 cells.