Construction Of Non-antibiotic Vector GFP As Tagging Marker And Its Application

Along with the research of microbiology developed deeply, the researchers needed badly a cell marker that could be detected directly in the living tissue or animal alimentary tract at anytime.Then it could track the molecule occurrence of receptor;.Green fluorescent protein(GFP) owned much superiority and got more and more attention compared with the other proteinum gene that reported in situ, real time of the research in microbion physiology biochemistry.This research was applyed GFP to report gene and constructed the non-antibiotic expression vector pW425t-GFP, It was transferred into E.coli X13.We successely got GFP expressed in the E.coli under non-antibiotic circumstances.After continuous cultare 10 generation, the GFP was expressed stabliy in the recombinant germ. At last, BALB/c mice were Vaccinated orally by the above-mentioned recombinant pW425t-GFP,then its distribution could be observed in the alimentary tract.The concrete research content is as follow:First of all,it was needed to design a pair of primers on the basis of the GFP gene of vector PGFPuv base on the plasmid vector as templet.Applying the technique of PCR and amplifing 938 bp which included the whole GFP gene of LacZ promoter,the PCR production was cloned and transferred into pGEM-T-Vector, then it was inverted to recipient germ JM109 through T-A cloning technology. The method of T-A cloning technique, restriction enzyme correspond restriction endonuclease,PCR, sequencing and so on were used to identify the recombinant masculine cloning plasmid pGEM-T-GFP. The nucleotide sequence could be analyzed by DNASTAR software, the consequence manifest that this gene has 100% homology with the GFP gene order in the Genebangk' record.Using the gene recombinant in vitro technique replace the gene of ThyA/illotycin dipl-antibiotic resistance expression vector pW425et to the GFP gene that contain the strong promoter, constructed non-antibiotic resistance tagging expression vector pW425t-GFP, The methods just as extract plasmid, restriction enzyme correspond restriction endonuclease,PCR and so on can be used to construct recombinant masculine cloning plasmid pW425t-GFP.The expression vector was analysed through SDS-PAGE to detect the expressed quantity of GFP gene. Undergoing the observation in the fluorescence microscope ,the consequence manifest this expression vector was expressed GFP steadily,and sent out green fluorescence steadily. Continuous cultare 10 generation under non-antibiotic selective pressure circumstances, GFP could be expressed steadily in the recombination germ.Took above-mentioned recombination germ of GFP which could be expressed steadily to mice orally,and observed the distribution of this recombinant germ in the alimentary tract. After 4 weeks age, BALB/c mice were vaccinated with recombinant germ orally,got the gastrointestinal contents and inoculated culture and counted number at 2,6,12,24,48,72h,meanwhile got gaster, jejunum,ileum and appendix to cryostat section,then cryostat section wasput under the fluorescence microscope to observe the distribution of the recombinant germ. The consequence manifest the recombinant germ pW425t-GFP could be detected in the gastrointestinal after 6h vaccination. The bacterial number was the most at 42h,and the recombinant germ still existed at a certain quantity number (about10~4CFU/g). Manifest this recombinant germ could permanent planting in intestinal mucosa surface long time. pW425t-GFP can be observed limpidly under the fluorescence microscope .It resided in the enteric chief content, minority of those bacterium adherenced on each enteric mucosa, even a few bacterium could enter the enteric mucosa proper layer.This research provide rationale and experimental model for the next tagging vector.