Title: Construction of recombinant bacteriophage T7 delivering eukaryotic expression box
Abstract: To construct a recombinant T7 phage delivering eukaryotic expression box and to detect the EGFP( enhanced green fluorecent protein) tag protein expression in vivo and in vitro,respectively,a 400-bp fragment( L) located at the upstream of 578 bp of T7 genome and a 200-bp fragment( R) located at the downstream were used as homologous arms.Eukaryotic expression box encoding EGFP protein( EEB) was inserted into left and right arm to construct homologous recombinant plasmid vector p UC-L-EEB-R. Plasmid vector was transformed into bacterium BL21 where homologous recombinant took place during the T7 phage propagation. EGFP protein expression was identified by phage genome transfection and phage particle immunization. PCR and restriction enzyme digestion analysis demonstrated that eukaryotic expression box was inserted into T7 phage genome correctly. Visible EGFP protein expression was detected both in vivo and in vitro. In mice liver and spleen,mRNA transcription of EGFP gene was detected by RT-PCR as well. These results indicate that homologous recombinanation is capable to construct recombinant T7 phage which could deliver eukaryotic expression box and achieve protein expression.
Publication Year: 2015
Publication Date: 2015-01-01
Language: en
Type: article
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