Genetic engineering strain for displaying Escherichia coli enterotoxin by Saccharomycetes and application of genetic engineering strain

[0079] Construction of ST yeast genetically engineered bacteria:

[0080] 1. Primer design

[0081] Primers were designed using Primer5 software, as shown in Table 1:

[0082] Table 1 Primer Sequence

[0083]

[0084] 2. Cloning of the target gene

[0085] The genome of the resuscitated enterotoxigenic Escherichia coli H10407 strain was extracted, and the estA and estB genes were cloned. The specific reaction system is shown in Table 2.

[0086] Table 2 PCR reaction system

[0087]

[0088] PCR reaction conditions of estA gene: 94°C, 10min; 94°C, 30s, 48°C, 30s, 72°C, 20s, 30 cycles; 72°C, 10min.

[0089] PCR reaction conditions of estB gene: 94°C, 10min; 94°C, 30s, 53°C, 30s, 72°C, 1min 45s, 30 cycles; 72°C, 10min.

[0090] PCR products were subjected to agarose gel electrophoresis, recovered and purified. The results of PCR amplification of estA gene are shown in figure 1 , lane M is the DNA molecular weight standard, lanes 1 to 4 are the PCR amplification results

[0134] To verify the effect of ST yeast genetically engineered bacteria on intestinal flora:

[0135] Select 90 SPF grade SD rats aged 4-6 weeks, half male and half male. They were randomly divided into blank control group, engineering bacteria group and yeast group, and each group was fed with normal saline, 10 7 cfu / mL engineering bacteria liquid, 10 7 cfu / mL saccharomyces cerevisiae bacterial solution 2mL / rat, once a day, continuously fed for 21 days, and then fed 10 times to each group of rats for 3 consecutive days 8 cfu / mL Escherichia coli H10407 bacterial solution 5mL / monkey, take the required fecal samples on the 7th, 14th, 21st day of feeding and 3 days after the challenge, and use the terminal restriction fragment length polymorphism (T-RFLP) , Real-time fluorescent quantitative PCR (Real-time PCR), to study the effect of yeast genetically engineered bacteria on intestinal flora OTU (microbial operable unit) and 5 kinds of iconic bacteria in the intestine

[0157] To verify the effect of ST yeast genetically engineered bacteria on the structure of intestinal villi

[0158] The duodenum, jejunum, and ileum tissues of rats were collected to make slices, and the length, width, crypt depth, and villi length / crypt depth (V / C) changes. Animal grouping is the same as in Example 2. On the 7th, 14th, 21st day of feeding and 3 days after the challenge, 6 rats in each group were taken, and about 2 cm of each sample of duodenum, jejunum and ileum were collected. The location of small intestine collection: the duodenum was collected 2-3 cm away from the pyloric opening; the jejunum was collected in the middle of the jejunum; the ileum was collected 2-3 cm away from the ileocecal opening. After sampling, each section of small intestine was stored in 4% formaldehyde solution for more than 24 hours for later use.

[0159]The results showed that, compared with the blank control group, the length of each section of intestinal villi and the V