| This study was designed to obtain recombinant Lactobacillus acidophillus. It takes advantage of the homologous recombination technology to integrating the exogenous gene pig epidermal growth factor (EGF) and green fluorescent protein (GFP) into the Lactob--acillus acidophilus with pSET4s. We can obtain the recombinant Lactobacillus acidophilus which can stably express EGF and GFP by resistance screening and identification, and then the research of biological characteristics of the recombinant Lactobacillus acidophilus is also essential.Experiment One:The analysis about the feasibility of the Plasmid pSET4s used to constuct the recombinant Lactobacilus acidophilus.(1) The condition of pSET4s electroporate into the Lactobacillus acidophilus. Lactobacillus acidophilus was static cultured at 37℃ until the OD600 value was about 0.6 and glycine LB culture medium of the final concentration was 1.0% can effectively improve the conversion efficiency.(2) Feasibility analysis about pSET4s used in the gene recombination of recombinant Lactobacillus acidophilus.Analyze the plasmid stability under conditions of 28℃ and the loss rate under conditions of 37℃ by plate count. We can find that the plasmid can exist stably in acidophilus at 28℃.When the cultured temperature was raised to 37℃, the plasmid would lost completely until 16 h, we would determined the pSET4s could be used to the gene recombinant of Lactobacillus acidophilus after the experiment.Experiment Two:The construction of the recombinant Lacto--bacillus acidophilus which coexpressing the GFP and EGF(1) The construction and identification about the recom--binant plasmidAnalysis the Lactobacillus acidophilus genome, choose two sites of the Lactobacillus acidophilus as the recombinant inte--gration site, one is a strong promoter expression sites, the other site is an inducible expression site, we could obtain the acido--philus homology upstream arms P1, P3 and the downstream homology arms P2, P4 by PCR amplificate. At the same time, using nested PCR to the splice of the EGF gene and GFP gene has been sequenced correctly, the correcttly splicing fragment pplice with the upstream and downstream homology arms and linearized vector pSET4s. We could conclude the recombinant plasmid was cons--tructed correctly by restriction enzyme digestion and PCR, and the recombinant plasmid have been correctly constructed were named as pSET4s-P1-EGF+GFP-P2 and pSET4s-P3-EGF+GFP-P4.(2) The construction and identification of the recombinant Lactobacillus acidophilusElectrotranform the recombinant expression plasmid pSET4s-P1-EGF+GFP-P2 and pSET4s-P3-EGF+GFP-P4 into the Lactobaci--llus acidophilus MG6243, we can obtain the recombinant Lacto--bacillus acidophilus by double resistance and temperature screen--ing, with the recombinant Lactobacillus acidophilus bacteria as a template, we can amplify the positive recombinant Lactobacillus acidophilus, and correctly constructed recombinant Lactobacillus acidophilus were called after ΔMG6243-1 and ΔMG6243-2.Experiment Three:The biological characteristics of the recombinant Lactobacillus acidophilus(1) Expression of the EGF and GFP gene in recombinant Lactobacillus acidophilusAnalysis by Coomassie blue and Western blot after polyacry--lamide gel electrophoresis with whole-cell protein, we could find that the recombinant bacteria with significant differences in protein bands compared with wild mushrooms, and the molecular size is consistent with the theoretical; we could find the recom--binant Lactobacillus acidophilus under the fluorescence micro--scope to exude the green fluorescence by fluorescence microscopy, it indicated that the GFP in recombinant bacteria could stably expressed, the result of the ELISA testing showed that the recombinant bacteria capable of stably express EGF, the concen-tration of EGF can be achieved about 3.1 ng/mL. These results showed that the exogenous proteins could stably express in the recombinant Lactobacillus acidophilus. Under blue excitation, we could see the obvious green fluorescence by fluorescence micro--scopy observation, indicating the recombinant Lactobacillus acid--ophilus which can express GFP gene successfully constructed, at the same time we should detect the expression of GFP gene in the recombinant bacteria by Coomassie blue staining and Western blot analysis.(2) Stability analysis of the recombinant Lactobacillus acidophilusThe recombinant Lactobacillus acidophilus subcultured for 10 generations, the result of PCR amplified the target gene fragment showed that recombinant bacteria which were passaged for 10 generations were still able to amplify the target gene bands, and wild mushroom not, while the fluorescence microscope of recombinant bacteria found that the recombinant bacteria which were passaged for 10 generations were still have fluorescence, the results showed that exogenous gene could be stably inherited in the recombinant bacteria.(3) The biological characteristics of the recombinant Lactobacillus acidophilusThe characteristics of the recombinant Lactobacillus acido--philus, inculuding the growth characteristics, acid resistance and cholate tolerance were deemined through vitro test, the result showed:There was no significant difference between Lactobacillus acidophilus and recombinant Lactobacillus acidophilus, the growth characterials also did not change significantly. Lactobacillus acidophilus and recombinant Lactobacillus acidophilus have been tested with good ability and certain bile acid tolerance, while there were some degree difference.Experiment Four:Research on the function of recombinant Lactobacillus acidophilus to intestinal in mice(1) A total 50 female mice which is 6-8 weeks and about 20 g wereThe results showed that:Compared with the control group, Lac--tobacillus acidophilus group reduced significantly the crypt depth in ileum (P<0.05), increased the ratio of villus height to crypt depth in ileum (P<0.05); compared with the control group, both the Recombinant Lactobacillus acidophilus AMG6243-1 and Reco--mbinant Lactobacillus acidophilus AMG6243-1 inducibly express-ed with IPTG group increased the villus height in duodenum significantly (P<0.05), significantly increased the the ratio of villus height to crypt depth in duodenum(P<0.05)and jeju--um(P<0.05), reduced the crypt depth in duodenum (P<0.05) and ileum (P<0.05), Recombinant Lactobacillus acidophilus ΔMG6243-1 inducibly expressed with IPTG group increased the villus height in ileum significantly (P<0.05) ileum; while recombinant Lactobacillus acidhplius ΔMG--6243-2 group increased the villus height in duodenum and jejunum significantly (P<0.05), significantly increased the ratio of villus height to crypt depth in duodenum (P<0.05) and jejunum (P<0.05), significantly decreased crypt depth in duodenum (P<0.05) and jejunum (P<0.05)(2) A total 60 female mice which is 6-8 weeks and about 20 g wereThe results showed that:Compared with the control group, LPS challenge reduced the villus height in duodenum and jejunum (P<0.05), increased the ratio of villus height to crypt depth in duodenum, jejunum and ileum (P<0.05), compared with LPS group, Lactobacillus acidophilus MG6243 group significantly increased the the ratio of villus height to crypt depth in ileum (P<0.05), Compared with LPS group, the ratio of villus height to crypt depth in duodenum and ileum of ΔMG6243-1 group significantly increased; fed with ΔMG6243-1 induced expressed with IPTG, the villus height in duodenum, jejunum and ileum jejunum villus height increased (P<0.05), increased the ratio of villus height to crypt depth in duodenum,jejunum and ileum (P<0.05), increased the villus height in jejunum (P<0.05), reduced the the ratio of villus height to crypt depth in jejunum (P<0.05), increased the the ratio of villus height to crypt depth in duodenum (P<0.05)Conclusion:We have constructed triumphantly Recombinant Lactobacillus acidophilus which stably inheritant and express the exogenous gene EGF and GFP. There were no significant difference in growth characteristics, and there were no significant differernce between thse three bacterial strains while pH was 2.0、2.5 or 4.0, the same to the situation that the cholate was 0.2 percent and 0.5 percent or raised to 0.8 percent; while the acid resistance and bile salt resistance of the Recombinant Lactobacillus acidophilus ΔMG6243-2 was significantly lower than the Recombinant Lactobacillus acidophilus ΔMG6243-1 and the Lactobacillus acidophilus MG6243 in the condition which pH was 3.0 and the cholate was 1.0 percent. Fedding mice the Recombinant bacteria, the result of animal experiments found that the Recombinant Lactobacillus acidophilus which havs prebiotic effect could obviously promote the intestinal growth of the experimental animals to maintain intestinal health effects, and to some extent, the Recombinant Lactobacillus acidophilus can effectively alleviate the growth inhibition in intestinal by LPS challenge. |
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