CN114831216B - Application of a Bacillus subtilis AMEP412 protein in regulating animal immunity - Google Patents

Abstract

The invention relates to a Bacillus subtilis AMEP412 protein, the amino acid sequence of which is shown in SEQ ID NO:1. The present invention also relates to the application of the above-mentioned Bacillus subtilis AMEP412 protein in regulating animal immunity. The invention discovers the new function of the Bacillus subtilis AMEP412 protein in animal immune regulation; the protein can activate the expression of serum-related immune molecules, reduce the incidence of animals, and improve the growth performance of animals; the invention accumulates new materials for animal feed additives.

Description

Application of a Bacillus subtilis AMEP412 protein in regulating animal immunity

technical field

The invention belongs to the field of animal feed and relates to an application of AMEP412 protein derived from Bacillus subtilis in regulating animal immunity.

Background technique

Antibiotics are widely used as feed additives in animal feed to reduce the occurrence of animal diseases. However, the long-term use of antibiotics will cause many problems such as bacterial resistance and antibiotic residues in animal food. New alternatives are urgently needed to reduce the use of antibiotics.

At present, there are some substitutes for antibiotics in the field of feed additives, mainly probiotics represented by Bacillus and Lactobacillus, and Chinese herbal medicines represented by Coptidis Rhizoma, Purslane, Scutellaria baicalensis and Dihuang. The two have different mechanisms of action. Probiotics mainly rely on inhibiting pathogenic bacteria, while Chinese herbal medicines mainly rely on regulating the immunity of animals to achieve their effects. But both of these have common defects: probiotics mainly rely on the secondary metabolites they secrete to function, but after entering the digestive tract and intestinal tract of animals, probiotics are no longer in the best growth environment, and the ability to produce secondary metabolites is greatly inhibited, resulting in too low amounts of active ingredients to fully function. The active ingredients in Chinese herbal medicine are not only contained in a small amount, but also cannot be fully released due to incomplete decomposition of plant tissues by the animal digestive system, which limits their function.

To sum up, ideal antibiotic substitutes should not be limited to tissue and cell levels, and direct intake of active substances at the molecular level is more direct and effective.

In previous studies, we isolated and identified AMEP412 protein from Bacillus subtilis BU412, which has the biological activity of antimicrobial peptides and has the potential to be a substitute for antibiotics, which can directly inhibit pathogenic bacteria in animals. However, the effect of AMEP412 protein on the immune regulation of animal body has not been reported.

In the present invention, we aim to provide a new application of AMEP412 protein in animal immune regulation.

Contents of the invention

The purpose of the present invention is to provide a new function of the Bacillus subtilis protein AMEP412, which mainly relates to the function of the protein on the immune regulation of animal organisms.

The present invention is realized through the following technical solutions:

A Bacillus subtilis AMEP412 protein, the amino acid sequence of which is shown in SEQ ID NO:1.

Further, the application of the AMEP412 protein in regulating animal immunity.

Further, the immune regulation function is to improve the immune index of animal serum.

Further, the immune regulation function is to improve animal disease resistance.

Further, the immune regulation function is to improve animal growth performance.

Positive effects of adopting the above-mentioned technical scheme: the present invention discovers the new function of the Bacillus subtilis AMEP412 protein in animal immune regulation; the protein can activate the expression of serum-related immune molecules, reduce the incidence of animals, and improve the growth performance of animals; the present invention has accumulated new materials for animal feed additives.

Detailed ways

The present invention is further described by the following examples. It should be understood that these examples are only for the purpose of illustration, and in no way limit the scope of the present invention.

Sources of biological material in the present invention:

1. The Bacillus subtilis BU412 used was preserved in the China Center for Type Culture Collection on March 30, 2016, and the preservation number is CCTCC M 2016142.

Example 1

Effects of feeding Bacillus subtilis AMEP412 protein to chickens on expression of serum-related immune molecules.

30-day-old young chickens were selected as treatment objects and divided into a control group (CK) and a treatment group (AMEP), with 15 chickens in each group, and a total of 3 repetitions. The control group was fed commercial feed, and the treatment group added 0.1 g of AMEP412 protein per kilogram of feed on the basis of the control. When the experimental chickens grew to 90 days old, the blood was collected by brachial vein puncture in a non-heparinized tube, and the serum was separated by centrifugation for subsequent analysis.

Immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), interleukin 1β (IL-1β), interleukin 2 (IL-2), and interleukin 6 (IL-6) were determined using kits.

The test results are shown in Table 1. The results showed that feeding Bacillus subtilis AMEP412 protein significantly increased the serum levels of immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM) and interleukin 2 (IL-2). However, the changes of interleukin 1β (IL-1β) and interleukin 6 (IL-6) were not significantly different. The above results indicated that AMEP412 protein feeding significantly improved the serum immunity level of chickens.

Table 1 Effect of Bacillus subtilis AMEP412 protein feeding on expression of immune molecules in chicken serum

index CK AMEP Immunoglobulin G (IgG) g/L 10.12±0.51b 13.11±0.27a Immunoglobulin A (IgA) g/L 3.46±0.25b 3.90±0.08 a Immunoglobulin M (IgM) g/L 1.38±0.18b 1.52±0.27 a Interleukin 1β (IL-1β) ng/L 22.32±1.85 21.83±0.88 Interleukin 2 (IL-2) ng/L 242.36±5.86b 289.63±11.28 a Interleukin 6 (IL-6) ng/L 102.23±7.88 101.74±10.51

Example 2

Effects of feeding pigs with Bacillus subtilis AMEP412 protein on expression of serum-related immune molecules.

Healthy weaned piglets with an average body weight of 7 kg were selected as treatment objects, and were divided into control group (CK) and treatment group (AMEP), with 10 piglets in each group, with 3 repetitions in total. The control group was fed commercial feed, and the treatment group added 0.1 g of AMEP412 protein per kilogram of feed on the basis of the control. After feeding for 30 days, blood was collected by brachial vein puncture in non-heparinized tubes, and the serum was separated by centrifugation for subsequent analysis.

Immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), interleukin 1β (IL-1β), interleukin 2 (IL-2), and interleukin 6 (IL-6) were determined using kits.

The test results are shown in Table 2. The results showed that AMEP412 protein feeding significantly increased the serum levels of immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), interleukin 2 (IL-2) and interleukin 6 (IL-6). However, the change of interleukin 1β (IL-1β) was not significantly different. The above results indicated that AMEP412 protein feeding significantly improved the serum immunity level of piglets.

Table 2 Effect of Bacillus subtilis AMEP412 protein feeding on the expression of immune molecules in pig serum

index CK AMEP Immunoglobulin G (IgG) g/L 8.15±1.31b 9.56±0.45 a Immunoglobulin A (IgA) g/L 2.18±0.11b 2.55±0.10 a Immunoglobulin M (IgM) g/L 1.32±0.11b 1.86±0.16 a Interleukin 1β (IL-1β) ng/L 28.53±2.37 29.04±1.68 Interleukin 2 (IL-2) ng/L 465.08±16.56b 489.93±22.33 a Interleukin 6 (IL-6) ng/L 95.32±16.21b 103.45±12.24 a

Example 3

Effect of Bacillus subtilis AMEP412 protein feeding on chicken disease.

The grouping pretreatment situation of experimental chicken is the same as embodiment 1.

The morbidity, diarrhea, and death of chickens in each treatment group from the age of 30 days to the age of 90 days were recorded.

After statistical analysis, the results are shown in Table 3. Feeding AMEP412 protein significantly reduced the morbidity, diarrhea and mortality of chickens. Among them, the morbidity rate decreased by 42.9%, the diarrhea rate decreased by 62.5%, and the fatality rate decreased by 63.6%. The above results showed that feeding AMEP412 protein significantly improved the disease resistance of chickens, and then improved the disease of chickens.

Table 3 The effect of Bacillus subtilis AMEP412 protein feeding on chicken morbidity

index CK AMEP Incidence % 4.19±0.43a 2.39±0.15b Diarrhea rate% 3.33±0.12 a 1.25±0.09b Fatality % 2.2±0.13 a 0.8±0.06b

Example 4

Effects of Bacillus subtilis AMEP412 protein feeding on disease status in pigs.

The grouping pretreatment of experimental piglets is the same as in Example 2.

During the experiment, the incidence, diarrhea, and death of piglets in each treatment group were recorded.

After statistical analysis, the results are shown in Table 4. Feeding AMEP412 protein significantly reduced the morbidity, diarrhea and mortality of piglets. Among them, the morbidity rate decreased by 56.9%, the diarrhea rate decreased by 56%, and the fatality rate decreased by 66.7%. The above results showed that AMEP412 protein feeding significantly improved the disease resistance of piglets, and then improved the disease of piglets.

Table 4 The effect of Bacillus subtilis AMEP412 protein feeding on pig morbidity

index CK AMEP Incidence % 5.45±0.65 a 2.35±0.35b Diarrhea rate% 14.13±0.12a 6.21±0.09b Fatality % 1.2±0.12 a 0.4±0.09b

Example 5

Effect of Bacillus subtilis AMEP412 protein feeding on growth performance of chickens.

The grouping pretreatment situation of experimental chicken is the same as embodiment 1.

When the young chickens grew to 90 days old, the initial weight and final weight of the chickens in each treatment group were investigated, and the weight gain was calculated. After statistical analysis, the results are shown in Table 5.

According to statistical analysis, AMEP412 protein feeding significantly improved the weight gain index of chickens, and the weight gain increased by 10.8%. The above results indicated that AMEP412 protein feeding significantly improved the growth performance of chickens, and then increased the weight gain index of chickens.

Table 5 Effect of Bacillus subtilis AMEP412 protein feeding on growth performance of chickens

index CK AMEP initial weight g 35.1±0.87 35.4±0.69 Last weight g 963.6±5.6b 1063.9±8.6 a weight gain g 928.5±4.8b 1028.5±7.9 a

Example 6

Effects of Bacillus subtilis AMEP412 protein feeding on pig growth performance.

The grouping pretreatment of experimental piglets is the same as in Example 2.

After feeding for 30 days, the initial weight and final weight of pigs in each treatment group were investigated, and the weight gain was calculated. After statistical analysis, the results are shown in Table 6.

According to statistical analysis, AMEP412 protein feeding significantly improved the weight gain index of piglets, and the weight gain increased by 32.3%. The above results indicated that AMEP412 protein feeding significantly improved the growth performance of piglets, and then increased the weight gain index of piglets.

Table 6 Effect of Bacillus subtilis AMEP412 protein feeding on growth performance of piglets

index CK AMEP initial weight kg 7.34±0.42 7.28±0.35 Last weight kg 12.79±0.35b 14.49 ± 0.36 a Weight gain kg 5.45±0.28b 7.21±0.38a

The present invention discovers the function of subtilis protein AMEP412 on animal immune regulation. AMEP412 improves animal disease resistance and promotes animal growth performance by activating immune-related molecules at serum levels. The invention provides new materials for animal feed additives.

sequence listing

<110> Heilongjiang Quansheng Biotechnology Co., Ltd.

Heilongjiang Bayi Agricultural University

<120> Application of a Bacillus subtilis AMEP412 protein in regulating animal immunity

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 76

<212> PRT

<213> Bacillus subtilis

<400> 1

Met Phe Gly Pro Ile Leu Lys Ala Leu Lys Ala Leu Val Ser Lys Val

1 5 10 15

Pro Trp Gly Lys Val Ala Ser Phe Leu Lys Trp Ala Gly Asn Leu Ala

20 25 30

Ala Ala Ala Ala Lys Tyr Ser Tyr Thr Ser Gly Lys Lys Ile Leu Ala

35 40 45

Tyr Ile Gln Lys His Pro Gly Lys Ile Val Asp Trp Phe Leu Lys Gly

50 55 60

Tyr Ser Val Tyr Asp Val Ile Lys Met Ile Leu Gly

65 70 75

Claims (3)

1. The bacillus subtilis AMEP412 protein with the amino acid sequence shown in SEQ ID NO. 1 is characterized in that: the bacillus subtilis AMEP412 protein is applied to improving serum immune indexes of chickens and piglets.

2. The bacillus subtilis AMEP412 protein with the amino acid sequence shown in SEQ ID NO. 1 is characterized in that: the bacillus subtilis AMEP412 protein is applied to promoting the growth of chickens and piglets.

3. The bacillus subtilis AMEP412 protein with the amino acid sequence shown in SEQ ID NO. 1 is characterized in that: the bacillus subtilis AMEP412 protein is applied to improving disease resistance of chickens and piglets.