Cloning and expression of human vasohibin۱ gene in E. coli

Background: Angiogenesis is an important process in various physiologic and pathologic states. The most significant stimulator of angiogenesis is vascular endothelial growth factor (VEGF). In contrast, vasohibin۱ acts as an angiogenesis inhibitor which specifically inhibits new vessels formation. The aim of the present study was cloning and expression of vasohibin۱ gene in E. coli as well as purification of recombinant vasohibin۱ protein. Methods: Total RNA was extracted from human umbilical vein endothelial cells and cDNA was synthesized by RT-PCR. cDNA was amplified using a specific designed primer set. The PCR product was evaluated by electrophoresis and then cloned in pET۲۸a expression vector which transformed into E.coli BL۲۱ (DE۳) as a host. IPTG is used as an expression inducer in media. Alternatively, PCR products were analyzed by sequencing and double digestion with EcoRI and HindIII restriction endonuclease. The expressed protein was purified by Ni-NTA column and confirmed by SDS Page and western blotting. Evaluation of gene inhibition was carried out through western blottting and RT-PCR. Results: No mutation or sequence variants were found in PCR products as a result of sequencing analysis. Moreover, the quantity and quality of expressed recombinant protein in the presence of IPTG with selected vector in E. coli was approximately high. VASH۱ significantly prevented the receptor expression. The quality and level of expressed protein in pET۲۸ expression vector indicated the efficacy of the applied system in vasohibin۱ production. Conclusions: The produced vasohibin۱ protein probably can be used as an angiogenesis inhibitor in further studies on retinopathies.Background: Angiogenesis is an important process in various physiologic and pathologic states. The most significant stimulator of angiogenesis is vascular endothelial growth factor (VEGF). In contrast, vasohibin۱ acts as an angiogenesis inhibitor which specifically inhibits new vessels formation. The aim of the present study was cloning and expression of vasohibin۱ gene in E. coli as well as purification of recombinant vasohibin۱ protein. Methods: Total RNA was extracted from human umbilical vein endothelial cells and cDNA was synthesized by RT-PCR. cDNA was amplified using a specific designed primer set. The PCR product was evaluated by electrophoresis and then cloned in pET۲۸a expression vector which transformed into E.coli BL۲۱ (DE۳) as a host. IPTG is used as an expression inducer in media. Alternatively, PCR products were analyzed by sequencing and double digestion with EcoRI and HindIII restriction endonuclease. The expressed protein was purified by Ni-NTA column and confirmed by SDS Page and western blotting. Evaluation of gene inhibition was carried out through western blottting and RT-PCR. Results: No mutation or sequence variants were found in PCR products as a result of sequencing analysis. Moreover, the quantity and quality of expressed recombinant protein in the presence of IPTG with selected vector in E. coli was approximately high. VASH۱ significantly prevented the receptor expression. The quality and level of expressed protein in pET۲۸ expression vector indicated the efficacy of the applied system in vasohibin۱ production. Conclusions: The produced vasohibin۱ protein probably can be used as an angiogenesis inhibitor in further studies on retinopathies.

Keywords:

Angiogenesis , Vasohibin۱ , Cloning , Gene expression.