Construction Of CacyBP Gene Subcellular Localization Lentivirus Vectors And Packaging Of The Lentivirus

Objective Construction of CacyBP gene subcellular localization lentivirus vectors andpackaging of the lentivirus.Methods The cDNA template from human colon cancer cell line HCT-116wasprepared in our laboratory. According to CacyBP gene sequences, primers were designed andsynthesized. CacyBP gene was amplified by PCR and cloned into the subcellular localizationvectors: pCMV/myc/nuc (nuclear localization vector), pCMV/myc/cyto (cytoplasmiclocalization vector). Both the two plasmids were transformed into E.coli DH5α competentcells in LB medium plate screening with ampicillin. The recombinant plasmids wereidentified by PCR/dual-endonuclease and sequencing. pCMV/myc/nuc-CacyBP,pCMV/myc/cyto-CacyBP, pCMV/myc/nuc-GFP as templates, CacyBP-NLS and CacyBPgene fragments with EcoR I/Nhe I restriction sites were amplified by PCR; GFP-NLS genefragment with Age I/EcoR I restriction sites was amplified by PCR. They were cloned intothe lentivirus vectors and then the recombinant vectors were obtained: LV-GFP-CacyBP-NLS,LV-GFP-CacyBP, LV-GFP-NLS. The recombinant plasmids were identified bydual-endonuclease and sequencing. The recombinant lentivirus vectors and the packagingplasmids were co-transfected into293T cell and packaged into replication-defective lentivirusparticles. Then the lentivirus titers were detected by qPCR.Results1. The size of CacyBP gene amplified from the cDNA template of HCT-116by PCR was684bp, the constructed recombinant plasmids (pCMV/myc/nuc-CacyBP,pCMV/myc/cyto-CacyBP) were identified by PCR/dual-endonuclease and the size of product was consistented with the expected value, and sequences analysis showed that the cloned genewas the CacyBP gene.2. The titers of the lentivirus are listed below: LV-GFP-CacyBP-NLS2E+8TU/ml,LV-GFP-CacyBP2E+9TU/ml, LV-GFP-NLS2E+8TU/ml.Conclusions1. The subcellular localization lentivirus vectors were successfully constructed.2. The recombinant lentivirus vectors and the packaging plasmids were successfullypackaged into lentivirus particles with high titer.