| Animal diarrhea disease is a kind of disease with acute or chronic diarrhea caused by bacterial infection,viral infection or other factors as the main characteristics.The majority occurred in young animals,seriously threated the healthy development of animal husbandry.And enterotoxigenic Escherichia coli is a major causative factor in the pathogenesis of diarrhea in animals,ETEC can produce one or several enterotoxins,including heat labile enterotoxin(Heat-liable toxin,LT)and resistance to heat labile enterotoxin(Heat-stable toxin,ST),leading the water and electrolyte flow to the intestinal lumen through influencing the function of intestinal cells,resulting in similar cholera watery diarrhea.ST is a kind of small molecule protein,often used for vaccine development because of its good immunogenicity.LT is often used as an adjuvant for mucosal immunity by virtue of large molecular weight and strong immunogenicity.Probioties,also called bygone or biological agents,is made by special processing,using probiotics and its growth promoting substances which is beneficial and harmless to the host by the use of micro ecology principle.Livestock and poultry probiotics has gradually become the research focus of the society,because it is different from the other chemicals with some advantages such as non-toxic side effects(not chemicals),drug residue detection without residue,of antibiotics and other drugs without tolerance,etc At present,probiotics have been applied in various fields such as feed,agriculture,medicine and health care,food and some other fields.Yeast cell surface display technology is a kind of eukaryotic expression system which can guide the foreign target protein and then anchor in the yeast cell surface through the signal peptide.Saccharomyces cerevisiae is a commonly used display carrier.This technology has the characteristics of small expression error,presentation proteins variety,large number of molecules,easy screening,purifying,activity determination and so on.At present,this technology has been applied in many research fields,and has broad application prospects.In recent years,the excessive use of antibiotics has resulted in high concentration of veterinary drug residues to the animal body and the surrounding environment,destroyed the ecological environment of water and soil etc.With the emergence of resistant strains,traditional antibiotics are difficult to reach the expected effect.So design probiotics with good safety and high immunogenicity for the new specific target has become a research hotspot at present.This topic has successfully established enterotoxigenic Escherichia coli fusion protein in yeast surface display technology,experimental study show that the Saccharomyces cerevisiae gene engineering bacteria constructed not only has micro ecological regulating action,also can effectively improve the mucosal immune function of organism,has protective effect to the ETEC virulent attack.1、Establishment of the surface display system of Saccharomyces cerevisiae.First of all,clone the three kinds of enterotoxin produced by ETEC,est A,est B and elt AB.By using site directed mutagenesis method,the transformation of est A and elt AB genes connected with a seven amino acid linker sequence.PYD1-est A-elt AB-est B recombinant plasmid was successfully constructed by means of double enzyme digestion,connection and transformation,the EBY100/p YD1-est A-elt AB-est B saccharomyces cerevisiae genetic engineering bacteria was successful build in shock transformation way.Immunofluorescence assay and scanning electron microscopy assay showed that est A-elt AB-est B fusion protein was successfully displayed on the surface of Saccharomyces cerevisiae.2、Effects of Saccharomyces cerevisiae gene engineering bacteria on the intestinal epithelial cells in rats.Pick up 90 SPF SD rats,four to six weeks,half male and half female.The rats randomly divided into blank control group,genetic engineering bacteria and EBY-100 yeast group,there were 30 rats in each group.EBY100 yeast and gene engineering bacteria group were lavaged concentration of 107 CFU/m L,1 time a day,each time 2m L,blank control group was gave the same volume of physiological saline,This process lasts for 21 days.And each group was lavaged H10407 e.coli bacteria liquid,108 CFU/m L,once per day,4m L per time.The tissue samples were taken after the lavage process,7d、14d、21d and 3ds’ challenge experiment.(1)Genetic engineering bacteria influence on small intestinal villus.The duodenal width of yeast group and genetic engineering bacteria group was significantly higher than that of blank control group(P<0.01),and there was no significant difference between two group(P<0.05).The duodenum and jejunum villi width of yeast genetic engineering bacteria group were significantly higher than that of blank control group(P<0.01),V/C value of duodenum was obviously higher than that of EBY100 yeast group,the result showed a very significant difference(P < 0.01),the Ileum and jejunum crypt depth were significantly lower than in blank control group(P<0.05),the length and width of the villi became larger,and the decrease of the crypt depth were the performance of the small intestine to enhance the ability of nutrient digestion and absorption,which further proved the growth promoting effect of Saccharomyces cerevisiae gene engineering bacteria on small intestinal villi.From the result of challenge experiment,EBY100 saccharomyces cerevisiae group,the the length of duodenum,jejunum and ileum and the V/C values became significantly decrease(P<0.01),crypt depth was obviously rising trend,the jejunum crypt depth value became a biggest change in the past of filling the stomach,and it showed significant differences(P<0.01).The length and V/C value of Eby-100 yeast group significantly decreased(P<0.01),the jejunum crypt depth significantly increased(P<0.01).the jejunum and duodenum villus length in Yeast genetic engineering bacteria group had no significant change(P>0.05);the villus width and crypt depth each length showed no significant change(P>0.05);V/C value of the duodenum was no significant difference than that before challenge experiment(P>0.05).The ETEC challenge experiment results show that,the yeast genetic engineering bacteria for small intestinal villus had stronger protective effect than EBY100 yeast.(2)Fecal p H test.During the experiment of 0 to 21 d,the p H of blank group were higher than that of the experimental group,and starting from the lavage 14 days,yeast group and genetic engineering bacteria group and were significantly lower than the blank control group(P<0.01),there were most significant difference at the 21 st day(P<0.01).After ETEC challenge experiment,the p H of blank control group significantly increased.(P<0.05);the p H of EBY100 yeast group had no significant change(P>0.05);Yeast genetic engineering bacteria group also had no significant change(P>0.05).The above results showed that the yeast genetic engineering bacteria played a role in maintaining the balance of p H.(3)Analysis of changes in intestinal flora of rats.From the beginning of the first 14 days,the number of intestinal flora in each experimental group began to increase significantly,showing a very significant difference at the 21 st day(P<0.01).Among them,in addition to the enterococcus,the growth rate of lactobacillus、Bacillus bifidus、clostridium and bacteroid group were the largest in the EBY-100 yeast.After ETEC,The growth rate of intestinal flora in the yeast gene engineering bacteria group was significantly higher than that in the EBY100 yeast group(P<0.01),This result further proves that the genetic engineering bacteria of Saccharomyces cerevisiae can regulate the intestinal flora.3、The experimental animal immunity of yeast and genetic engineering bacteria(1)Effects of yeast gene engineering bacteria on the content of SIg A in the intestinal mucosa of rats.The secretion of SIg A in the yeast engineering bacteria group was the highest at the 14 th day,and the EBY100 yeast group and the control group were significantly different(P<0.01);The yeast engineering bacteria group and Eby-100 yeast group were significantly higher than those in the blank group and there was no significant difference between the two groups at the 21 st day(P>0.05).The secretion of SIg A in the control group and the experimental group was significantly lower than that of the control group after ETEC attack(P<0.01),But the decrease of yeast gene engineering bacteria group was the smallest,which showed that the yeast genetic engineering bacteria increased the immune function of small intestinal mucosa.(2)Effects of yeast gene engineering bacteria on the cytokines in the intestinal mucosa of rats.At the beginning of the experiment,there were no significant changes in IL-2,IL-4 and IFN-γ.From the beginning of the 14 th day,EBY100 yeast group and gene engineering bacteria group,the levels of each cell factor increased significantly(P<0.05);at the 21 st day,it showed a very significant upward trend(P<0.01).The secretion of the three factors of EBY100 yeast group and gene engineering bacteria group was significantly higher than that of the 21 st day blank control group after ETEC.Among them,the gene engineering bacteria group was the highest secretion of IL-2,IL-4,the lowest secretion of IFN-γ,the above results indicated that the yeast gene engineering bacteria can effectively improve the secretion of IL-2,IL-4 and IFN-γ in intestinal mucosa,thereby improving the immunity of the body.(3)Effects of yeast gene engineering bacteria on immune organs of rats.The coefficients of thymus and spleen were increased in the blank control group and the experimental group of two groups at the 14 th day,the growth rate of the yeast group was the smallest,and was significantly lower than that of the genetic engineering bacteria.After challenging of ETEC,the blank group and the yeast group were decreased,while the yeast gene engineering bacteria group had no significant change,even slight rise,and the blank control group was significantly different(P<0.05).The results showed that the yeast gene engineering bacteria could promote the development of thymus and spleen.(4)Effect of yeast gene engineering bacteria on serological indexes of rats.The content of ALT、 AST、TG、TBILI、DBILI、GLU、CHO、ALB and TP in serum can be found in the serum.Before the experiment of the 21 st day,the test results of the indexes of each experiment group had no significant change,but after challenging the ETEC、ALB、TP and GLU values of each group showed an upward trend,but did not reach the significant level.Among them,the yeast gene engineering bacteria group was the smallest,the results showed that the yeast gene engineering bacteria did not cause liver damage and other indicators of blood changes.To sum up,the following conclusions are drawn:1、The enterotoxins of Escherichia coli in yeast surface display technology was Successfully established,animal experiments proved that the construction of engineering bacteria of rat to safety,non-toxic side effects.2、Saccharomyces cerevisiae gene engineering bacteria can effectively reduce the p H value of the rat intestinal ETEC,can maintain the acid-base balance in the intestinal tract.3、Engineering bacteria can adjust the dynamic balance of intestinal microecology of rat,intestinal bacteria population was significantly upward trend;ETEC challenged engineering bacteria play a probiotic effect of yeast and the intestinal bacteria population continues to achieve steady growth.4、Engineering bacteria can improve the intestinal mucosal immunity,increase the secretion of SIg A,IL-2,IL-4 and IFN-gamma;when the strong toxin ETEC invasion,can effectively protect the small intestinal mucosa from infringement. |
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