Construction and expression of the recombinant lentiviral vector containing human PPFP gene

Abstract

Objective To construct the recombinant lentiviral vector containing human PPFP gene,to observe its expression in 293T cells. Methods The PPFP gene was isolated and amplified by PCR technique from PEGFP-C-PPFP plasmid which was constructed before. Then the gene was subcloned into the expression plasmid of lentiviral vector,pGC-FU (containing Flag gene), to generate the lentiviral expression vector, pGC-FU-PPFP. The correct PPFPgene was confirmed by PCR, endoenzyme digestion, se-quencing, analysis and contrast. Recombinant lentiviruses which containing PPFP gene and Flag gene were produced by 293T cells following the co-transfection of pGC-FU-PPFP and packaging plasmids-pHelper 1.0 and pHelper 2.0. The titer of virus was measured after collecting and concentrating the viral supernatant.In order to certify the expression of PPFP gene, PPFP-Flag fusion protein expressions in 293T were detec-ted by Western blot analysis. Results A PPFP gene fragment of 2591bp was obtained by PCR. The cor-rect lentiviral expression recombinant vector, pGC-FU-PPFP was confirmed by PCR, endoenzyme digestion,sequencing,analysis and contrast. After the pGC-FU-PPFP and packaging plasmids were transfected into 293T cells, the titer of the acquired recombinant lentivirus reached 3.5×10~+7 TU/ml;after PPFP was de-tected by Western blot analysis and the result showed its molecular weight was 90KDr, it could be con-finned the PPFP gene expressed in 293T cells. Conclusion The recombinant lentiviral vector pGC-FU-PPFP was constructed successfully,and the lentiviral overexpression system is also estabhshed. Key words: PPFP gene; Lentivirus vector; Follicular thyroid carcinoma