Objective To construct and identify recombinant human umbilical vein endothelial cell line EA. hy926 overexpressing triple mutant hypoxia-inducible factor 1α(HIF-1α). Methods Primers design was conducted according to triple mutant HIF-1α sequence of plasmid pcDNA3. 1 +-HIF1-Ala402-Ala564-Ala803,and the gene segment was linked to the entry vector after amplification by PCR. The recombinant lentiviral vector with triple mutant HIF-1α was constructed by combining the entry vector carrying target gene,plasmid pT590 and pENTR_L4_PCMV_R1,then was transformed into DH5α competent cells. The positive clones were collected and sequenced for identification. The recombinant lentiviral vector with triple mutant HIF-1α and the auxiliary packaging plasmid were cotransfected into HEK293T cells,then the fluid containing virus was collected. The EA. hy926 cells were divided into Lenti-HIF-1α group infected by recombinant lentivirus overexpressing HIF-1α,Lenti-negative group infected by negative control lentivirus,and MOCK group added with medium. The cell lines overexpressing HIF-1α were screened with 1 μg/ml puromycin after corresponding intervention,then the efficiency of viral infection was observed,and the expression of HIF-1α protein was analyzed by Western Blot assay. Results The result of sequencing and identification indicated that recombinant plasmid with triple mutant HIF-1α was successfully synthesized. High-expression of enhanced green fluorescent protein(EGFP) was observed in the HEK293T cells under fluorescence microscope. After infection by recombinant lentivirus,high-expression of EGFP was observed in the EA. hy926 cells under fluorescence microscope. The HIF-1α protein was stably expressed in the cells of Lenti-HIF-1α group and was higher than that in the cells of the Lenti-negative group or the MOCK group. Conclusion The recombinant lentiviral vector overexpressing triple mutant HIF-1α was successfully constructed,and the human umbilical vein endothelial cell line stably overexpressing HIF-1α was obtained.

Objective To construct the recombinant adenovirus vectors overexpressing angiopoietin-2(Ang-2) in mice.Methods Mouse Ang-2 coding sequence was chemically synthesized and then was linked to the shuttle plasmid after amplification by PCR and identification.The recombinant plasmid carrying target gene was transformed into DH5α competent cells.The positive transformants were analyzed by electrophoresis and sequenced for identification.Shuttle plasmid carrying target gene and helper packaging plasmid were cotransfected into HEK293 cells.HEK293 cells were repeatedly frozen and thawed to obtain recombinant adenovirus.The titer of recombinant adenovirus was detected by end-point dilution method.The recombinant adenovirus was transfected into HEK293 cells,then Ang-2 protein was extracted and its expression was analyzed by Western blot.Results A characteristic band about 1.5 kp was observed in the electrophoresis for PCR products.The gene sequencing showed that the sequence is identical to the Ang-2 gene sequence from GeneBank.The titer of adenovirus was 1×108.8 pfu/ml after packaging by HEK293 cells.Western blot analysis showed a characteristic band with the size of 57 kD,which indicated that the recombinant adenovirus could express Ang-2 protein.Conclusion The recombinant adenovirus vector of Ang-2 gene can be successfully constructed.

Objective To construct and identify recombinant adenovirus vectors carrying mouse angiopoietin-1( Ang-1) gene.Methods The Ang-1 gene sequence was made by chemosynthesis and amplified by PCR,and then subcloned into shuttle plasmid. The recombinant plasmid was transformed into competent cell,the transformants was identified by PCR electrophoresis analysis and DNA sequencing. The adenovirus shuttle plasmid carrying exogenous gene and auxiliary packaging plasmid carrying most of the adenovirus genome were cotransfected into HEK293 cells. The recombinant adenovirus was obtained by repeated freezing and thawing. The recombinant adenovirus titer was detected by endpoint dilution assay. The 293 T cells were transfected with the recombinant adenovirus,and then the supernatants were harvested following the 48-hour transfection for a Western Blot analysis on the expression of Ang-1 protein. Results The PCR electrophoresis analysis showed that there was a characteristic band( approximately 1. 5 kbp in size). The DNA sequencing showed that the Ang-1 gene sequence was in accordance with the Gen Bank sequence. The recombinant adenovirus titer was about 1 × 109 pfu / ml after HEK293 packaging. The Western Blot analysis on the supernatants following 293 T cell transfection showed that there was a characteristic band of 58 k D,indicating the recombinant Ang-1 protein of the correct size was obtained after transfection. Conclusion The recombinant adenovirus vector carrying Ang-1 gene has been constructed successfully,which could provide the experimental evidences for further research on the effect of Ang-1 protein on hematopoietic stem cell mobilization and angiogenesis.