Studies On Anguilla Anguilla Intestine Probiotics For The Prevention And Cure Of The Disease

To obtain probiotics for the prevention and cure of the disease caused by Aeromonas hydrophila, the antagonistic bacteria against pathogenic A. hydrophila were isolated from the intestine of Anguilla anguilla, Among them, 3 strains were chosed for further studies. Enhanced green fluorescent protein (GFP) gene was introduced to label the 3 strains of candidate probiotics, it was observed to express stably in strain Aa3-22. Labelled Aa3-22 was used to investigate its colonization on the intestine of A. Anguilla.It is described in chapterⅡthe screening of antagonistic bacteria against A. hydrophila from A. anguilla intestine. 643 strains were isolated from healthy Anguilla anguilla intestinal tract, 65 strains of antagonistic bacteria against A. hydrophila were identified by dot-inoculating method, 14 of 65 strains with strong antagonistic activity were chosen for antibacterial spectra, growth curves and co-agglutination assay. The results showed that: 2 isolated strains (Na3-38 and Nb3-15), had wider range of antibacterial spectra than others, 5 of 7 indicating bacteria were obviously inhibited by isolated strains of Aa3-22 and Mb3-7; 12 isolated strains had more advantage in R than A. hydrophila; the co-agglutination ratio was more than 10% between 11 isolated strains and A. hydrophila. The results indicate that the isolated strains of Aa3-22 and Mb3-7 will be used for developing probiotics based on their antibacterial spectrum, growth curve and the co-agglutination with A. hydrophila. Antibacterial spectrums and co-agglutinations of isolated strains of Na3-38 and Nb3-15 were obviously higher than other strains elucidates some mechanisms of inhibiting other bacteria growth by excreting ati-material and influencing the adhesion by binding to A. hydrophila.It is described in chapter III the construction of prokaryotic expression plasmid containing enhanced green fluorescent protein (EGFP) gene, which was used as a tag in eel intestinal candidate probiotics. The amplification EGFP gene primers were designed and synthesized, which including HindⅢand BamHⅠrestriction sites. The full-length EGFP cDNA was amplified by polymerase chain reaction(PCR)using pEGFP-C1 plasmid as template. Then the amplified EGFP cDNA was added single "A" at its 3'end by using the template-independent terminal transferase activity of Taq DNA polymerase when there was only dATP exsisted. The purified EGFP cDNA being added single "A" at its 3'end was then ligated with pMD18-T vector, and the ligated products were transformed into E. coli Top10. Single white clonies were picked to be propagated and identified by the blue filter, the correct recombinant plasmid was named as T-EGFP.The recombinant plasmid T-EGFP and expressive plasmid pET32a were digested by restrictive endonuclease BamHⅠand HindⅢrespectively, the EGFP cDNA fragment and linearized pET32a fragment were purified by Gel Recycling then connected by Ligase.The resultant ligation was transformed into E. coli BL21. The positive clone was propagated and the recombinant plasmid was extracted for DNA sequencing. The EGFP gene in the recombinant plasmid of the cDNA sequence is equal to the EGFP gene in the pEGFP-C1 plasmid. The correct recombinat plasmid was named after p-EGFP. Green fluorescent has been confirmed in fluorescence microscopy detection with isopropyl-β-D-galactoside (IPTG) inducing. The expressive plasmid was transformed into the candidate probiotics Aa3-22, Mb3-27 and Na3-38, the results showed that the expression only in the Aa3-22 strain is stable.It is described in chapter IV the colonization of candidate probiotic E-Aa3-22 on eel intestinal by the tracer of green fluorescent protein marker.. The tagged intestinal bacteria moved from foregut to hindgut gradually. In the time of 3h after oral administration, the number of E-Aa3-22 in foregut, midgut, and the content about 1×10~4 cfu/g but none in hindgut. Marked bacteria can inhibit the colonization of other bacteria, so the number of heterotrophic bacteria decline to a minimum value in foregut and midgut. At 24-36h after oral administered , the number of the marker bacteria in eel intestinal reach maximum 1.0×10~5 cfu/g because of colonization and propagation. The activity of digestive enzyme were significant difference (P<0.01) in different position of eel intestine. Taged bacteria had a slight inhibition of digestive enzymes.