Construction and eukaryotic expression of recombinant plasmid pEGFP-N1-SrV+encoding the variable region of the surface protein of Streptococcus mutans

Abstract

Objective To construct the recombinant plasmid pEGFP-NI-SrV ＋ and evaluate the expression of SrV ＋ in mammalian 293T cells. Methods sty ＋ , a gene encoding the variable region of the surface protein of the Streptococcus mutans OMZ175, was cloned chemically based on its reported nucleotide sequence. The eukaryotic expression plasmid, pEGFP-N1-SrV ＋ , was constructed by introducing the srv ＋ gene into the Kpn 1/Xho I site of pEGFP-Nt. The recombinant plasmid pEGFP-N1-SrV ＋ was transfected into 293T cells with lipofectamine and the expression level of SrV ＋ was evaluated. Results The eukaryotic expression plasmid pEGFP-N1-SrV ＋ was constructed successfully. GFP was observed by green fluorescent microscope, and a 72 x 103 protein was detected by Western blot. Real-time RT-PCR analysis revealed that the expression of the pEGFP-N1-SrV ＋ in 293T was excellent and significant compared the control group. Conclusion The recombinant plasmid pEGFP-N1-SrV ＋ was successfully constructed, which could encode the expression of SrV ＋ after transfeeted into the mammalian 293T Cells. Key words: Streptococcus mutans ; Variable region of surface proteins ; Eukaryotic expression vec- tor plasmid pEGFP-N1 ; 293T cells