Expression Of Human VascularEndothelial Growth Factor Receptor 3 Extracellular Domain 1-3

Objective:1 :To construct the prokaryotic expression vector of Human VEGFR3. 2:To express the fusion protein of Human VEGFR3.Method:We designed proper primer.The pMD18T-VEGFR3 which constructed previously by our laboratory was used as template and amplified with up stream primer containing EcoR I site and down stream primer containing BamH I site.After collection and purification,we subcloned this PCR product into pBV220 and transformed E coli DH 5 α . The positive bacteria was selected and induced at 42℃.The bacteria was broken by ultrasonication.The inclusion body containing target protein VEGFR3 was separated and washed. Also we constructed expression plasmid of VEGFR3 fusion protein with His Tag by adding encoding sequence of six consecutive histidine to down stream specific primer.The interesting fusion protein was identified by Western-blotting with His Tag antibody and purified by NTA-Ni²⁺ resion.Result:The pBV220-VEGFR3 expression plasmid was constructed successfully.The target protein VEGFR3 can express as inclusion body in Ecoli DH 5 α .Meanwhile we constructed VEGFR3 fusion expression system and through NTA-Ni²⁺ resion we obtained purified VEGFR3 fusion protein.Conclusion:Applying technology of genetic engineering,we constructed pBV220 plasmid.Also we obtained VEGFR3 fusion protein with high purity.