Cloning,expression,purification and bioassay of recombinant human platelet factor 4

AIM: To obtain human platelet factor 4 (hPF4) with biological activity by means of genetic engineering.METHODS: DNA coding sequence for hPF4 was obtained by PCR and cloned into plasmid pRSET to construct fusion expressed vector pRSET-PF4.After pRSET-PF4 was transformed to E.coli DH5a,the bacteria were induced by IPTG.The expressed hPF4 fused protein was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE.The activity of rhPF4 was detected by choriallantoic membrane assay (CAM).RESULTS: The DNA sequencing showed that the expression vector pRSET-PF4 was constructed successfully.After induced by IPTG,the target protein accounted for 11% of the total bacterial protein and partly was in a soluble form.The purity of the fused protein was up to 84% after Ni-NTA affinity chromatography.The result of activity analysis by CAM showed that rhPF4 could inhibit the formation of new vessels in chick embryos.CONCLUSION: The recombinant hPF4 fused protein with biological activity is obtained.