The Expression Of GFP In The Chicken's Lactobacilli And Distribution Of The Recombinant Bacterium After Oral Administration In The Small Intestine Of Chicken

In order to explore the distribution of Lactobacilli in the gastrointestinal(GI) tract and the interaction with immune system of host, six strains isolated from chicken's gastrointestinal tract were chosen as the expression carrier of GFP (green fluorescent protein). The six lactobacilli strains were Lactobacillus fructosus C2, Lactobacillus delbrueckii subsp.delbrueckii C12, Lactobacillus intestinalis D2, Lactobacillus amylophilus D14, Lactobacillus delbrueckii subsp. lactis D17 , Lactobacillus acidophilus K9. Firstly, recombinant plasmid encoding the gfp gene were constructed and then electroporated into lactobacilli. The GFP were expressed in the Lactobacillus fructosus C2 (C2-GFP) and Lactobacillus delbrueckii subsp. lactis D17(D17-GFP) successfully.The recombinant strains could express the GFP steadily after passaging for 10 generations under no selection pressure of ampicillin. Orally administrate chicken with the recombinant lactobacillus delbrueckii subsp. lactis D17 (D17-GFP) and observe the distribution of the D17-GFP in the intestine. The whole experiment can be divided into two parts:The first section: The plasmid encoding the gfp gene was constructed and electroporated into lactobacilli which were isolated from the gastrointestinal(GI) tract of chicken. According to the gene sequence of plasmid pRV85, a pair of primer was design: P1 and P2. The gene pldhL-gfp encoding promoter and green fluorescent protein was amplified from plasmid pRV85. Then the sequence digested by Apa I and Cla I were inserted into a shuttle plasmid pLEM415 to get a hybrid plasmid.The resulting plasmid pLEM415::gfp was introduced into E.coli DH5a. pLEM415::gfp was extracted from DH5a and then was electroporated into several lactobacilli successfully. PCR and enzyme validation experiments of the pLEM415 ::gfp conformed the validity of the results.The Results showed that the gfp gene had tramsformed into the lactobacillus successfully.The transformants were fluorescent with green light when viewed with the fluorescence microscope. The GFP has been expressed in the lactobacilli steadily .The recombinant lactobacillus could be ued in vivo to observe the distribution and movement.