| Most lactic acid bacteria(LAB) are commonly considered as safe probiotics. Some strains of lactic acid bacteria are found to have the ability to adhere and live in the gastro-intestinal tract, urinogenital system or mucosa with no contemporary immunogenicity. Importance is widely attached to the research of the LAB as live oral and mucosal vaccine. It is verified in different labs that the antigens presented by live LAB can induce immuno-responses. Compared with Lactococcus, Lactobacillus is the better choice of oral vaccine for some properties, including their immunogenicities, resistance to the circumstance of the alimentary tract and remaining in long period in gastro-intestinal tract, however, Lactococcus is more effective in selecting antigen to induce immunological response, while the positive result of Lactobacillus is limited. The reason of such difference is not clear at present and may relate to the low level of antigen expression in Lactobacillus, which suggests that it is necessary to select different strains of LAB and match different expression vectors as oral vaccine vehicles.Two strains of LAB, one coccus and one bacillus which are the normal residents in intestinal tract and which could be the host bacteria, were respectively isolated from LAB sanitarian products. The coccus is identified as Enterococcus fececalis belonging to Enterococcus category and the bacillus is identified as Lactobacillus plantarum belonging to Lactobacillus, by analysis of complete 16sRNA sequence and physiological and biochemical characteristics. Resistance experiment suggests that Enterococcus faecalis is sensitive or semi-sensitive to most drugs, and is semi-sensitive to vancomycin. One large and one small plasmid naturally exist in the Lactobacillus plantarum, while the Enterococcus faecalis doesn't carry any plasmid. We find that the transformation efficiency of the two strains is in direct proportion to the voltage. And the transformation efficiency of Enterococcus faecalis is obviously higher than that of Lactobacillus plantarium by pulse electroporation with different voltages, which suggests that the two strains of LAB have different usage in the research of genetic engineering of LAB.A small cryptical plasmid pEFR was isolated from Enterococcus faecium strain df101. The complete sequence analysis of the plasmid show that it consists of 3176 bps, which contains four putative ORFs. ORF1 encodes a putative protein and is highly similar to repA which functions in replication. There is typical -10 region, -35 region and RBS sequence on the upstream of ORF1 and on the upstream of the -35 region there are four tandem repeats of 22 bps sequence, and similar sequence wasfound on the upstream of the replicase genes of pMBBl, pVS40, pLU510. The replication mechanism of pEFR is classified to the 6-replication type by southern blot. The functional replication DNA sequence of pEFR was identified by deletion and insertion experiments, and nisin-inducible expression vector, pWY6992 and pWY7130, were constructed based on the DNA-sequence. GFP can be expressed in the host Enterococcus facecalis and Lactobacillus plantarum we isolated using the expression vectors we constructed The expression level depends on the Nisin concentration to some extent. We are now working on the expression and oral vaccination of the hepatitis B virus surface antigen and nucleic antigen by using the expression system in LAB.There were two plasmids in the isolated Lactobacillus plantarum. The negative result of elimination experiment by drugs and temperature suggests that the two plasmids were quite stable in the host. Complete sequence analysis of the two plasmids reveal that the small plasmid pLP2000 with 2061 nucleotide encodes a putative 37 KDa replicase. And in the downstream region of the replicase gene, it existed multi-tandem repeat of 17 bps. The plasmid pLP9000 with 9253 nucleotide encodes 9 ORFs. ORF1 encodes a putative Magnesium transporter and ORF2 encodes a putative integrase-Phage integrase, while the relative reading f...
|
PDF Full Text Request