| Intestinal flora is vital to the health of animal,because it has a variety of physiological functions.But when flora was imbalance,it can lead to disease.In this case,antibiotics were used to solve this problem.However antibiotics produced a lot of negative effects for the extensive use and abuse of antibiotics.Lactobacillus plantaricin is a polypeptide or a precursor polypeptide with antibacterial activity,which was produced through lactic acid bacteria in the metabolic.Lactobacillus plantaricin was used as a feed additive of antibiotics in livestock and poultry production,because it was non-toxic,no negative effects,no residue,no pollution to the environment.However it has brought great trouble to scale production of lactobacillus plantaricin,because Lactobacillus plantaricin was difficult to be separated and purificated.Therefore genetic engineering technology was used to express Lactobacillus plantaricin in vitro in the study.Then the bacteriostasis of Lactobacillus plantaricin was verified.Finally Lactobacillus plantaricin K was fed to broilers to explore it medate the intestinal microbial flora and immune function.The details are as follows:1.Prokaryotic expression of four Lactobacillus plantaricin.Methods:Based on the coding sequence of Lactobacillus plantaricin E?F?J?K,four pairs of specific primers were designed and was identified by PCR.The recombinant prokaryotic expression plasmid was constructed by ligating PCR production with prokaryotic expression vector pET-32a?+?.The recombinant plasmids was verified with PCR,restriction enzyme and sequencing.The recombinant plasmids were transformed into the E.coli BL21?DE3?competent cells to induce expression.The optimization induction conditions was selected by SDS-PAGE.And the recombinant protein was identified by Western blot.The recombinant protein was purified by Ni-NTA affinity chromatography.The Results:The results of PCR,restriction enzyme and sequencing showed that four recombinant prokaryotic expression plasmids pET-32a?+?-plnE,pET-3 2a?+?-plnF,pET-3 2a?+?-plnJ and pET-3 2a?+?-plnK were constructed successfully.They were transformed into the E.coli BL21?DE3?competent cells,and the SDS-PAGE results showed that the four recombinant proteins were induced by IPTG,which were 26.2ku,25.7ku,26.0ku and 26.2ku,respectively.it was consistent with the expected results.The results of induction of recombinant protein showed that the induction conditions of pET-32a?+?-plnE protein were 12h,37? and 1.0mmoL/L,the induction conditions of pET-3 2a?+?-plnF protein were 12h,37? and 0.6mmoL/L,the induction conditions of pET-32a?+?-plnJ protein were 12h,37? and 1.2mmoL/L,the induction conditions of pET-32a?+?-plnK protein was 12h,37? and 0.4mmoL/L.Western blot results showed that the four recombinant Lactobacillus plantaricins were correctly expressed.The results of purification of Ni-NTA affinity chromatography showed that pET-32a?+?-plnE protein,pET-32a?+?-plnJ protein,pET-32a?+?-plnK protein were incubated with 280mM imidazole,And the pET-32a?+?-plnF protein was incubated with 200mM imidazole concentration.2.Bacteriostatic experiment of Lactobacillus plantaricin in vitro.Methods:Staphylococcus aureus,Escherichia coli,Salmonella and Pasteurella were used as indicators.Oxford cup method was used to detect the antibacterial effect of four recombinant Lactobacillus plantaricins at different concentrations.Add 1uL,10uL,50uL and 100uL Lactobacillus plantaricin?0.50mg/mL?to find the best antibacterial concentration.Finally,the antibacterial effect of plnE+plnF,plnJ+plnK,plnE,plnF,plnJ and plnK was analyzed respectivly.The Results:when the amount was 50uL?final concentration of 2.50×10-3mg/mL?,four recombinant Lactobacillus plantaricins have best antibacterial effect.The antibacterial effect of Lactobacillus plantaricin E and F alone was showed the same results as the combination of Lactobacillus plantaricin E+F,the antibacterial effect of Lactobacillus plantaricin J and K alone also was showed the same results as the combination of Lactobacillus plantaricin J+K.From the antibacterial test results of Oxford Cup,it showed that it was obvious inhibition zone with the concentration of 2.50×10-3mg/mL of four Lactobacillus plantaricins respectively.So it was indicated that the four recombinant Lactobacillus plantaricins all have broad spectrum antibacterial activity.3.Effects of Lactobacillus plantaricin K on intestinal flora in yellow broiler.Methods:300 4-day-old healthy yellow broilers were randomly divided into 5 treatment groups,5 replicates per treatment group,12 replicates each.I group was blank control group,? group was acid control group,? group was 5.00×10-3mg/mL Lactobacillus plantaricin K group,? group was 2.50×10-3mg/mL Lactobacillus plantaricin K group,V group was 1.25×10-3mg/mL Lactobacillus plantaricin K group.The test period was 28 days.The yellow broilers were weighed after 1 week,2 weeks and 4 weeks respectively.The chicken duodenum was collected and the content of SIgA was determined by ELISA in each treatment group.The expression levels of immune cytokines?MDA5,IFN-?,IFN-?,TLR3,IFITM10,IFITM3 gene?were measured by fluorescence quantitative PCR in duodenum mucosa.Finally,the contents of cecal in yellow broiler was analysied by Illumine-Hiseq of 16S rDNA V3-V4.The Results:After fed with plantaricin K two weeks,the ADG,ADFI and the content of SIgA in duodenal mucosa were significantly higher in group ? than that in group I?P<0.05?,and the mRNA expression of MDA5,IFN-a,IFN-?,TLR3,IFITM10 and IFITM3 were significantly higher than that in group I?P<0.01?.After fed with plantaricin K,Bifidobacterium,and the abundance of RuminococcaceaeUCG013 in group ? and V were significantly higher than in that group ??P<0.05?.The abundance of Lactobacillus in group ? and ? were higher than that in group ??P>0.05?.For two weeks,the abundance of Streptococcus in group V was significantly higher than that in group ??P<0.05?,the abundance of Lactobacillus in group ? and ? was higher than that group I?P>0.05?.For four weeks,the abundance of Lactobacillus in group ? and V was higher than that in group ??P>0.05?.In conclusion,recombinant plasmids of pET-32a?+?-plnE,plnF,plnJ and plnK were successfully constructed.Four recombinant Lactobacillus plantaricins had broad-spectrum antibacterial activity.Feeding 2.50×10-3mg/mL Lactobacillus plantaricin K for two weeks it could increase the content of SIgA in duodenal mucosa,stimulate the expression of immune cytokines,and enhances the abundance of Acidobacteria,Tenericutes,Bacillaceae and Lactobacillus in broilers,which promoted broiler growth. |
PDF Full Text Request