Bacillus flagellin FlgE as animal intestinal TLR5 specific ligand and application of bacillus flagellin FlgE in replacement of feed antibiotics

Abstract

The invention relates to bacillus flagellin FlgE serving as a specific ligand of animal intestinal tracts TLR5 and application of the bacillus flagellin FlgE in replacement of feed antibiotics, and belongs to the technical field of microbial engineering, FlgE interacts with the animal intestinal tracts TLR5 to promote colonization of probiotics in the animal intestinal tracts and competitively inhibit colonization and growth of harmful bacteria in the animal intestinal tracts, the animal is a pig or a chicken. According to the invention, in-vitro interaction between bacillus flagellin FlgE and pig source TLR5 as well as between bacillus flagellin FlgE and chicken source TLR5 is verified, and a novel TLR5 specific ligand protein is found, so that a novel thought and a novel direction are provided for researching a colonization mechanism of probiotic bacillus in intestinal tracts; related test results also provide theoretical and practical basis for development of safer and more efficient animal feed antibiotic substitutes based on probiotic bacillus.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial engineering, and particularly relates to a Bacillus flagellin FlgE as a specific ligand of TLR5 in animal intestinal tracts and application thereof in replacing antibiotics for feed. BACKGROUND

[0002] A flagellum is a filamentous appendage on the surface of a bacterium, and is a main organ for movement of the bacterium. The structure of the flagellum is relatively conservative, and is mainly composed of a flagellar basal body, a flagellar filament, and a flagellar hook. The flagellar hook is a universal joint structure composed of flagellar hook protein FlgE, and serves to connect the flagellar basal body on the cell membrane and the flagellar filament outside the cell.

[0003] Although researches show that the bacterial flagellar protein FlgE is a specific ligand of TLR5, and the interaction between the bacterial flagellar protein FlgE and TLR5 plays a crucial role in bacterial adhesion and colonization, nutritional metabolism of animal intestinal tracts, and immune regulation processes. However, the interaction characteristics of different flagellar proteins of the same bacterium or flagellar proteins from different bacteria with TLR5 are different. Bacillus is a new type of biocontrol bacteria with potential application prospect. At present, there are few reports on the interaction and application of the flagellar protein FlgE of Bacillus and TLR5. Therefore, the application verifies the Bacillus flagellar protein FlgE as a specific ligand of TLR5 in animal intestinal tracts and application thereof in replacing antibiotics for feed. SUMMARY

[0004] The application aims to provide the Bacillus flagellar protein FlgE as a specific ligand of TLR5 in animal intestinal tracts and application thereof in replacing antibiotics for feed.

[0005] The application achieves the above-mentioned purpose through the following technical solutions. The application provides the Bacillus flagellar protein FlgE as a specific ligand of TLR5 in animal intestinal tracts and application thereof in replacing antibiotics for feed.

[0006] As a further optimization scheme of the application, the FlgE interacts with TLR5 in animal intestinal tracts, promotes colonization of probiotics in the animal intestinal tracts, and competitively inhibits colonization and growth of harmful bacteria in the animal intestinal tracts.

[0007] As a further optimization scheme of the application, the animal is a chicken or a pig.

[0008] The application further provides application of the Bacillus flagellar protein FlgE as a chicken feed additive. The chicken feed additive is a compound probiotic agent containing the FlgE and chicken intestinal tract probiotics, or is a chicken intestinal tract recombinant probiotic capable of secreting and expressing the FlgE.

[0009] The application also provides a use of the Bacillus flagellin FlgE as a pig feed additive, wherein the pig feed additive is a compound probiotic agent containing FlgE and pig intestinal tract probiotics, or is a pig intestinal tract recombinant probiotic capable of secreting and expressing FlgE.

[0010] The application has the following beneficial effects: The application verifies that the Bacillus flagellin FlgE has in-vitro interaction with chicken-derived TLR5 and pig-derived TLR5, discovers a new TLR5 specific ligand protein, and further verifies that the Bacillus flagellin FlgE can help the intestinal tract probiotics of chickens or pigs to colonize in the corresponding animal intestinal tract, and can inhibit the growth of harmful bacteria in the animal intestinal tract, and further improve the growth performance, immune performance and slaughter performance of the animals, thereby laying a solid theoretical foundation for the development of the Bacillus probiotics as a feed additive and in the field of animal feeding. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Figure 3 is a result graph for GST pull-down verification of the interaction between the recombinant proteins GST-TLR5 and HIS-FlgE, wherein a represents GST antibody incubation, b represents HIS antibody incubation, and M represents protein molecular weight Marker (11-180 kD); Figure 2 Figure 4 is a result graph for immunoprecipitation, wherein L1 represents an immunoprecipitation complex using anti-pTLR5 antibody, L2 represents FLgE protein, and L3 represents an immunoprecipitation complex using IgG. DETAILED DESCRIPTION

[0012] The following further describes the application, and it is necessary to point out here that the following detailed description is only used to further illustrate the application, and cannot be understood as a limitation on the protection scope of the application, and the skilled in the art can make some non-essential improvements and adjustments to the application according to the above application content.

[0013] I. Experimental materials (1) Escherichia coli DH5a, BL21(DE3), Rosetta-gami B(DE3), gami strain(DE3) and hygromycin are commercially available bacterial strains; (2) Vectors: pET-32a and pGEX-4T-1 are commercially available plasmids; (3) The primer sequences were all synthesized by Shengong Bioengineering (Shanghai) Co., Ltd., and the primer sequences are shown in the following table (" " is a restriction enzyme site): ; The methods used in the present application are conventional methods known to those skilled in the art, and the reagents and other materials used are commercially available products, unless otherwise specified.

[0014] Experimental method 1. Preparation of purified flgE protein According to the nucleotide sequence of the Bacillus flagellin flgE gene shown in SEQ ID NO. 1, the flgE gene was amplified from the genomic DNA of Bacillus according to the genomic DNA extraction instructions of Kang Weishiji Company, the flgE gene was connected to the pMD19-T vector, and sequencing verification was performed, and the cloning vector pMD19-flgE was constructed; the flgE gene can also be artificially synthesized according to SEQ ID NO. 1 and connected to the pMD19-T vector.

[0015] The cloning vector pMD19-flgE was used as a template, and the flgE gene was amplified using primers (flgE-orf-f, flgE-orf-r) added with restriction enzyme sites EcoR I and Xho I, and the amplified product was recovered by gel; The amplified product was connected to the multiple cloning site of the pET-32a vector, and the expression vector pET32a-flgE was constructed. The expression vector pET32a-flgE was transformed into the competent cells of Escherichia coli BL21 for expression and purification, and the purified flgE protein (amino acid sequence as shown in SEQ ID NO. 2) was obtained.

[0016] 2. Interaction research and application of FlgE and chicken-derived TLR5 2.1. Preparation of purified chicken-derived TLR5 protein The amino acid sequence of ChTLR5 (accession number: GenBank: ABW07794.1) was obtained from NCBI, and ChTLR5 was subjected to signal peptide prediction by online software SignalIP-5.0. The results showed that ChTLR5 has a signal peptide sequence, and the signal peptide sequence is: MMLHQRLIIVFGIALAGDICA; Total RNA was extracted from the chicken cecum and reverse transcribed into cDNA. Using primers (chTLR5ER-f, chTLR5ER-r) and the chicken cecum cDNA as a template, the chTLR5ER gene was cloned. The signal peptide sequence on the chTLR5ER gene sequence was removed for codon optimization. The optimized codon was sent to Qingke Biotechnology for gene synthesis. The optimized sequence was named chTLR5ER-opti (sequence shown in SEQ ID NO.3).

[0017] The recombinant expression vector pGEX-chTLR5ER-opti was constructed using the chTLR5ER-opti gene sequence and transformed into Escherichia coli DH5α. After successful sequencing and enzyme digestion verification, the recombinant plasmid was extracted and transformed into the Escherichia coli expression strain Rosetta-gamiB(DE3). After IPTG induction of expression, the GST-TLR5ER recombinant protein was purified using GST-tagged magnetic beads. The second elution protein without obvious impurities was selected as the purified GST-TLR5ER recombinant protein.

[0018] 2.2 Interaction Research A recombinant HIS-FlgE protein with a His tag at the N-terminus was artificially synthesized. A pull-down experiment was performed using purified GST-TLR5ER recombinant protein as the bait protein and HIS-FlgE recombinant protein as the prey protein, followed by Western blot identification. Figure 1 (a) shows the identification results of the control group (CK), experimental group (Exp), GST protein, GST-TLR5ER recombinant protein, and HIS-FlgE recombinant protein after incubation with GST antibody. The results show that the target band with a molecular weight of 95kD was detected in the experimental group and the GST-TLR5ER recombinant protein group. Figure 1 (b) The identification results of the control group (CK, GST+GSTMagBead+HIS-FlgE), experimental group (Exp, GST-TLR5ER+GSTMagBead+HIS-FlgE), GST protein, GST-TLR5ER recombinant protein, and HIS-FlgE recombinant protein after incubation with HIS antibody. The results showed that a target band with a molecular weight of 48kD was detected in the experimental group and the HIS-FlgE recombinant protein group, indicating that the HIS antibody successfully enriched the bait protein GST-TLR5ER, and the bait protein GST-TLR5ER pulled down the prey protein HIS-FlgE.

[0019] The above results indicate that GST-TLR5ER and HIS-FlgE interact in vitro.

[0020] 2.3 Application of flgE protein in chicken feed additives Using genetic engineering technology to synthesize and purify the expression of flgE protein, while using aureomycin as a positive control, flgE protein and aureomycin are added to the basic diet of broiler chickens respectively, to explore the possibility of using flgE protein as a chicken feed additive product development.

[0021] Take 300 cage chickens, and randomly divide them into negative control group, positive control group I and II, experimental group I and II, a total of 5 test groups, each group with 20 chickens; Each group from 1 day old, respectively, feed the basic diet, add 30ppm, 60ppm aureomycin basic diet, add 0.4%, 0.8% flgE protein basic diet, according to the daily management requirements of chicken farms, using natural light plus artificial light, 16h of light per day, maintain natural ventilation in the breeding area, feed twice a day and record the feed intake during the test period, for 21 days; The components and their mass percentages of the basic diet are: corn 56%, soybean meal 30%, fish meal 5%, egg powder 5%, lysine 0.2%, calcium hydrogen phosphate 1.5%, stone powder 1.5%, salt 0.3%, premix 0.5%.

[0022] Detect the growth performance of each group of broilers, and calculate the mortality rate, detect the proportion of harmful bacteria in the chicken intestine, the results are shown in the following table 1: Table 1: Effect of different feeds on growth performance and intestinal microbial flora of chickens ; From the above table, it can be seen that the interaction between Bacillus flagellin FlgE and chicken TLR5 protein can help probiotics colonize the intestine, so that feeding broilers with chicken feed containing Bacillus flagellin FlgE can significantly inhibit the growth of harmful bacteria in the intestinal microorganisms of chickens, thereby achieving the effect of improving the growth performance, immune performance and slaughter performance of broilers. The improvement effect of FlgE is comparable to that of aureomycin, but its use effect is safer than that of aureomycin, which may be due to the fact that the animal intestine is one of the sources of flgE.

[0023] 3. Interaction between FlgE and porcine TLR5 and its application 3.1 Preparation of purified porcine TLR5 protein According to the instructions of Thermo Scientific Fast Pure RNA extraction kit, total RNA of porcine jejunum was extracted and reverse transcribed into cDNA, and pTLR5 gene (sequence as shown in SEQ ID NO. 4) was amplified using porcine jejunum cDNA as template; The primers (pTLR5-f, pTLR5-r) with added enzyme cutting sites BamH I and Xho I were designed, and the amplified pTLR5 gene fragment and the plasmid PGEX-4T-1 were subjected to double enzyme cutting of BamH I and Xho I, respectively, and then connected by T4 ligase to construct the recombinant plasmid pGEX-4T-1-pTLR5. The recombinant plasmid pGEX-4T-1-pTLR5 verified to be correct by enzyme cutting was transformed into the gami strain (DE3) E. coli competent cells, and the pGEX-4T-1-pTLR5 protein was obtained after IPTG induction expression. The protein was subjected to ultrasonic treatment at 12000 rpm for 30 min, and the supernatant was collected. Part of the supernatant was used for interaction experiment, and the other part was used for antibody production.

[0024] 3.2, Interaction study The anti-pTLR5 antibody was coupled with the protein A / G magnetic beads, and then the mixture of pGEX-4T-1-pTLR5 protein and purified FlgE protein was added to perform immunoprecipitation experiment to obtain a complex. The two groups of immunoprecipitated complexes obtained were subjected to Western blot analysis with anti-HIS antibody, and the results are shown in Figure 2 (L1: immunoprecipitated complex using anti-pTLR5 antibody, L2: FLgE protein, L3: immunoprecipitated complex using IgG), in which FlgE showed a unique band at 48KD, indicating that the HIS antibody immunoprecipitated FlgE specifically, indicating that there was an interaction between FlgE and pTLR5. Importantly, no protein was detected in the IgG control group, which confirmed that the interaction was specific and not due to non-specific binding of magnetic beads or antibodies.

[0025] The above results show that there is a specific interaction between FlgE and pTLR5.

[0026] 3.3, Application of FlgE protein in pig feed additives The FlgE protein was synthesized and purified in large quantities by genetic engineering technology, and the flavomycin was used as a positive control. The flgE protein and the flavomycin were added into the basic daily ration of pigs, respectively, to explore the possibility of developing FlgE protein as a pig feed additive product.

[0027] A negative control group, a positive control group I, a positive control group II, an experimental group I and an experimental group II are set up, each group has 3 repetitions, each group uses 6 pigs (initial weight 7.5±0.5 kg), and male and female pigs are half and half, and other grouping conditions are random, each group is fed with a basic diet, a basic diet added with 30 ppm and 60 ppm of flavomycin (final concentration in the basic diet), a basic diet added with 0.4% and 0.8% of flgE protein, according to the daily management requirements of a pig farm, natural light and artificial light are used, the light time is 12 hours per day, the natural ventilation of the breeding area is maintained, the pigs are fed 5 times per day and the feed intake is recorded during the test period, and the pigs are fed for 28 days; The components and mass percentages of the basic diet are as follows: corn 45%, bran 16%, soybean meal 20%, whey powder 10%, oil 5%, lysine 0.2%, calcium hydrogen phosphate 1.5%, stone powder 1.5%, salt 0.3%, premix 0.5%.

[0028] The growth performance of the pigs in each group is detected, the mortality is counted, the proportion of harmful bacteria in the intestinal tract is detected, and the results are shown in Table 2.

[0029] Table 2: Effect of different feeds on growth performance of pigs and intestinal microbial flora ; As can be seen from the above table, since the flagellin FlgE of Bacillus can interact with the TLR5 protein of pigs, therefore, the pig feed containing the flagellin FlgE of Bacillus can achieve the effect of inhibiting the growth of harmful bacteria in the intestinal microorganisms of pigs, so as to efficiently and safely improve the growth performance, immune performance and slaughter performance of pigs.

[0030] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as a limitation on the scope of the patent of the present application. It should be pointed out that, for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which all belong to the protection scope of the present application.

Claims

1. A Bacillus flagellin FlgE as a specific ligand of animal intestinal TLR5 and its application in replacing antibiotic in feed.

2. Use according to claim 1, characterized in that: The FlgE interacts with animal intestinal TLR5, promotes the colonization of probiotics in animal intestine, and competitively inhibits the colonization and growth of harmful bacteria in animal intestine.

3. Use according to claim 1, characterized in that: The animal is chicken or pig.

4. Use of Bacillus spore flagellin protein FlgE as a feed additive for chickens, characterized in that: The chicken feed additive is a compound probiotic agent containing FlgE and chicken intestinal probiotics, or a recombinant probiotic capable of secreting and expressing FlgE in chicken intestine.

5. Use of Bacillus spore flagellin protein FlgE as a feed additive for swine, characterized in that: The pig feed additive is a compound probiotic agent containing FlgE and pig intestinal probiotics, or a recombinant probiotic capable of secreting and expressing FlgE.

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