Construction Of Bicistronic Expression Vector Containing HPV Type 6b L1 Gene And Its Expression In Mammalian Cells

Objectives To construct bicistronic expression vector containing HPV type 6b L1gene and to detect its expression in mammalian cells; to prepare NIH3T3 cells stablymaintaining HPV6b L1 genome by transfection and selection methods, which mighthave great potential for genetic engineering vaccine against condylome acuminatumand provide useful dates in stucying the biology characteristic of HPV L1 protein.Methods The gene fragment of HPV6b L1 has been digested with BamH I and SacI and cloned into the vector pIRES2-EGFP. Then the recombinant expression vectorwas transformed into coli DH5α, which was identified by BamH I and Sac I digestion.The positive vector was sequenced to get late gene L1 recombinant sequence. Thenthe recombinant was transfected into NIH3T3 cells by techniques of gene transfecti-on. The vector expressed the gene HPV6b L1 in NIH3T3 cells, which was detectedby fluoroscopy and RT-PCR.Results Identification of pIRES2-HPV6bL1-EGFP by enzyme digestion and seque-ncing showed HPV6b L1 which was inserted into the recombinant was correct and theexpression of EGFP in transfected cell was observed.Conclusions The pIRES2-HPV6bL1-EGFP expression vector makes it easy toassess the expression of HPV6b L1 protein and EGFP protein and to sift thetransfected cells. It is quite potential in the future research for the development ofgenetic engineering vaccine.