| Enterotoxigenic escherichia coil is one of the main pathogenic diseases causing diarrhea in animals,especially in young animals and seriously threated the healthy development of livestock breeding.ETEC can produce one or several enterotoxins,the heat-stable enterotoxin is the main factor of animals' diarrhea.In recent years,due to the rising price of drugs in diarrhea treatment and the emergence of drug resistant strains in the main pathogenic bacteria,a new type of probiotic for the prevention of diarrhea has become a hot spot of the current research.Saccharomyces cerevisiae is one of the most important probiotic in humans and animals and it is rich in many kinds of nutrients and beneficial to growth factors,it can improve the animal performance at the same time,promote intestinal mucosal development and immune function,improve intestinal flora.Besides,the yeast is a eukaryotic organism having the organelle and enzyme required for post-translational modification,thereby the yeast surface display technology can express the gene diversity through correctly folded protein accurately.This technology can display many kinds of protein and a large number of molecules,it is easy to operate in screening,purifying and activity determination.At present,this technology has been applied in many research fields,and has broad application prospects.The aim of this study is to demonstrate the fusion protein of Escherichia coli heat-stable enterotoxin(ST)on the surface of Saccharomyces cerevisiae,to obtain the ST yeast gene-engineering bacteria and explore its effects on rat intestinal flora,intestinal mucosal structure,and mucosal immunity.The study is to lay the foundation for the development of probiotics with specific immune function.The main study contents and results are as follows:1 ? Application of surface display technology to construct ST yeast gene-engineering bacteria.Applying Overlap PCR method to connect the site directed mutagenesis of estA and estB,and then the fusion gene fragment was translated to pYD1 plasmid to construct the surface display plasmid pYD1-estA-estB.The recombinant plasmid was electroporated into EBY100 Saccharomyces cerevisiae,then the ST yeast gene-engineering bacteria was successfully obtained by screening.Immunofluorescence assay showed that ST fusion protein was successfully displayed on the surface of Saccharomyces cerevisiae.2?Effects of ST yeast gene-engineering bacteria on intestinal flora in rats.Pick up 90 SPF grade,four to six weeks old SD rats,half male and half female,the rats randomly divided into control group,engineering bacteria group and yeast group.The rats from the control group was gavaged with saline,engineering bacteria group and yeast group were gavaged with engineering bacteria liquid and Saccharomyces cerevisiae liquid at concentration of 107 cfu/mL respectively,2mL per rat a day for 21 days,and each group was gavaged with Escherichia coli H10407 bacteria liquid with concentration of 108 cfu/mL,5mL per rat for 3 days.Collected the feces of rats in each group after gavaged 7,14,21 day and after gavaged Escherichia coli 3 days,then investigated the effects of Saccharomyces cerevisiae on intestinal microflora OTU(microbial operable unit)and 5 kinds of intestinal landmark bacteria: Bacteriodes,Clostridium,Enterococcus,Bifidobacterium,Lactobacillus' s number changes through T-RFLP and Real-time PCR,detected the pH of intestinal floras.The results showed that the OTU and the number of bacteria in engineering bacteria group and yeast group were higher than control group in different degrees,and the pH was significantly lower than control group.After the challenge,the value of OTU,Bacteriodes,Lactobacillus and Bifidobacterium were significantly higher than the other two groups(P < 0.01).Compared with gavaged with ETEC,engineering bacteria group had no significant difference in pH(P>0.05).The results showed that Saccharomyces cerevisiae genetic engineering bacteria could enrich the intestinal flora polymorphism,increase the number of important bacteria in the intestine and reduce the intestinal pH.After challenge,the engineering bacteria can effectively reduce the effect of ETEC on intestinal flora and important bacteria,maintaining intestinal pH stability.3?Effects of ST yeast gene-engineering bacteria on small intestinal villi in rats.By collecting the duodenum,jejunum,and ileum of the rats to make paraffin section and statistical analysis of each rat duodenum,jejunum and ileum's villus length,crypt depth,width,the ratio of villus length and crypt depth(V/C)changes.Compared with control group,villus length and V/C value of intestinal villus in engineering bacteria and yeast group were significantly higher.After gavaged with Escherichia coli,compared with the engineering bacteria group,the length and V/C value of duodenum and jejunum in the control group and the yeast group decreased in different degrees;the crypt depth of jejunum significantly increased(P<0.01);the V/C value of ileum significantly decreased(P<0.01).The results show that engineering bacteria can promote the development of small intestinal mucosa,enhance the absorption of intestinal mucosa and effectively resist the damage of ETEC to the small intestinal villi.4?Effects of ST yeast gene-engineering bacteria on intestinal mucosal immunity in rats.Using ELISA,Real-time PCR to study the content change of intestinal mucosa in secretory immunoglobulin A(sIgA),interleukin-2(IL-2),interleukin-4(IL-4),interferon-?(IFN-?),immunoglobulin G(IgG)in serum and detected the content of rat serological index: ALT?GLU?CHO?ALB?TP.During the 21 days,the results showed that the content of sIgA and cytokines in engineering bacteria group and yeast group were higher than control group,and there was no significant difference in serological index.The titer of IgG in serum reached 1:320 on 21 day.After gavaged with ETEC,the data of genetic engineering bacteria group changed the least,which showed that the ST yeast gene-engineering bacteria can improve the intestinal mucosal immunity,promote the secretion of cytokines,and it is safe,stimulate the body to produce an immune response,non-toxic and reduce the effect of ETEC on mucosal immunity.In summary,this study successfully constructed Saccharomyces cerevisiae gene-engineering bacteria which can display Escherichia coli ST,the results show that the gene-engineering bacteria not only has the good effect of probiotic bacteria,but also improve intestinal mucosal immunity,effectively prevent and protect the animal intestinal flora changes and intestine mucosa damage by the ETEC. |
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