Construction Of Recombinant Baculovirus Carrier Of Ac-egfp And Ac-hgf Regulated By Dose Of DOX And Timeliness

Objective：Connection of Tet-on system with egfp or hgf to constructa new kind of recombinant baculovirus. Different concentrations of DOXregulation of the corresponding degree of egfp(or hgf) expression.Methods：PCR amplification of the egfp and the hgf gene. Restrictionenzyme of BamHI and XbaI cut DNA fragment containing egfp and hgf, areconnected in PMD19-T vector and then transfect DH5α, the plasmids areextracted, and then identified after digested by restriction endo-nuclease. Digestion of PMD19-T vector, the target gene connect pTRE-tightafter recovery and then transfect DH5α, the plasmids are extracted,after digested by restriction endo-nuclease (named pTRE-egfp and pTRE-hgf) and then identified; Regain about2kb part after XhoI and HindIIIdigest pTet-on-advanced and then connect pFastBac1(XhoI and HindIIIdigest), transfect DH5α, the plasmids are extracted, and then identified(named pFast-Tet); XhoI digest recombinant plasmid pFast-Tet, pTRE-egfpand pTRE-hgf. Connect pFast-Tet after the recovery of the target frag-ments,and transfect DH5α,plasmids are extracted, and then digested byrestriction endonuclease (named pFast-Tet-egfp and pFast-Tet-hgf).pFast-Tet-egfp,pFast-Tet-hgf transfect DH10Bac competent cells containing ofAcMNPV Bacmid and helper plasmid, sifted by IPTG,X-Gal,gentamicin, kana-mycin and tetracycline,and then extract the Bacmid DNA, identify the both(named Ac-egfp and Ac-hgf).Ac-egfp is transfected into MBSCs cells, anddifferent concentrations of doxycycline(final concentrations of DOX asfollows:0,200,500,1000ng/ml) to regulate expression of target gene.Results： egfp,hgf and Tet-on system could be successfullyconstructed in a baculovirus vector, identifications are correct. Andhigh trans-fection efficiency is reached in bone marrow mesenchymal stemcells after transformation, egfp and hgf can be expressed at high levels with high concentrations of DOX.With low concentrations ofDOX,expressions of egfp and hgf gradually decreased.Conclusion：The present study demonstrate that the introductionof Tet-on system to construct a new recombinant baculovirus vectorcontaining egfp or hgf in the same baculovirus could be done. Recombinantbaculovirus Improve reporter gene expression and the space.DOX can bevarying degrees regulation of expression of egfp and hgf, and both arein low background expression without DOX.