A functional probiotic capable of secreting antibacterial peptides, its preparation method and application

Abstract

The present invention discloses a functional probiotic capable of secreting antibacterial peptides, a preparation method thereof, and an application thereof. The functional probiotic capable of secreting antibacterial peptides disclosed by the present invention can secrete a polypeptide with an amino acid sequence of Sequence 4. This antibacterial peptide has no antibacterial activity at low temperatures and restores antibacterial activity at 37°C. Oral administration of the functional probiotic of the present invention to weaned piglets daily can increase the survival rate of piglets by 14% and reduce the incidence of diarrhea by 12.5%; oral administration of the functional probiotic of the present invention to broilers daily can increase the weight by 4.2%, increase the survival rate by 1.7%, and reduce the feed-to-meat ratio by 7.9%. The functional probiotic and antibacterial peptide of the present invention can be used for the auxiliary breeding of livestock and poultry, effectively reducing the incidence of diseases, reducing production costs, and increasing the European benefit index. Therefore, the present invention completely breaks through the technical problem that antibacterial peptides are difficult to produce by fermentation, lays a foundation for the industrialization of antibacterial peptides, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to a functional probiotic capable of secreting antibacterial peptides, a preparation method thereof and an application thereof in the field of biotechnology. Background Art

[0002] With the rapid development of China's aquaculture industry, the prevention and control of livestock and poultry diseases have always been issues to be faced in the breeding process. In order to reduce the incidence of animals, a large number of antibiotics are used for the prevention and treatment of livestock and poultry diseases. China produces about 210,000 tons of antibiotic raw materials every year, and 97,000 tons of antibiotics are used in the livestock and poultry breeding industry. After long-term consumption by animals, antibiotics will accumulate in the animal body, thus bringing serious food safety problems and harming human health. Moreover, due to the abuse of antibiotics, the accelerated evolution of bacteria has led to the continuous emergence of "super bacteria" that can resist multiple antibiotics, seriously interfering with China's medical and health prevention and control system. In order to effectively improve the food safety level of the Chinese people, the Ministry of Agriculture issued Announcement No. 194 on July 1, 2020, prohibiting the use of antibiotics in the breeding of livestock and poultry. However, this has brought a new problem. Without the assistance of antibiotics, the incidence of livestock and poultry will increase, thereby increasing the breeding cost and ultimately leading to an increase in the price of livestock and poultry products. Therefore, it has become particularly urgent to develop antibacterial products that can replace antibiotics.

[0003] Antibacterial peptides are widely distributed in various organisms in nature. They are the first line of defense for organisms to resist the invasion of pathogenic bacteria and play an important role in the innate immune response. Antibacterial peptides have strong thermal stability and broad-spectrum bactericidal effects, and are currently the most promising products to replace antibiotics. Although antibacterial peptides have good safety and antibacterial ability, it has been difficult to achieve their large-scale production. Researchers initially tried to prepare antibacterial peptides using the Escherichia coli expression system, but all ended in failure. The reason is that antibacterial peptides will directly kill the engineered host bacteria used for expression, resulting in the inability of the bacteria to reproduce. Later, some researchers tried to express antibacterial peptides using insect cells. Although active antibacterial peptides can be expressed in insect cells, due to the high cost, industrialization cannot be achieved.

[0004] Bacillus subtilis is a Gram-positive bacterium widely present in soil and plants. It does not secrete exotoxins or endotoxins and belongs to food-grade safe microorganisms. Since it is an intestinal probiotic, it has been widely used in human microecological preparations and animal feed additives. Bacillus subtilis can secrete proteins efficiently extracellularly. Based on this characteristic, researchers transferred foreign genes into Bacillus subtilis and achieved the efficient secretion and expression of multiple target proteins, such as amylase, xylanase, polysaccharide monooxygenase, etc. In addition, due to the good safety and the characteristic of efficiently expressing foreign genes of Bacillus subtilis, some researchers further tried to transfer antimicrobial peptide genes into Bacillus subtilis, hoping to achieve the expression of antimicrobial peptides and thus obtain functional probiotics with antibacterial ability. However, in actual operation, it was found that due to the antibacterial characteristics of antimicrobial peptides, they are toxic to the host bacteria, resulting in the difficult growth of Bacillus subtilis containing antimicrobial peptide genes. Even if the culture time is extended, the small amount of bacterial liquid obtained is an ineffective bacterial strain that has lost the antimicrobial peptide gene. Therefore, the technical route of expressing antimicrobial peptides using Bacillus subtilis has never been able to achieve a breakthrough. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a functional probiotic with antibacterial activity, its preparation method and application.

[0006] The present invention first provides a protein named Cecropin-mut, which is A1) or A2):

[0007] A1) A protein with an amino acid sequence that is Sequence 4;

[0008] A2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1).

[0009] In order to facilitate the purification of the protein in A1), a tag as shown in the following table can be connected to the amino terminus or carboxyl terminus of the protein composed of the amino acid sequence shown in Sequence 4 in the sequence listing.

[0010] Table: Sequences of Tags

[0011] Label Residue Sequence Poly-Arg 5 - 6 (usually 5) RRRRR Poly-His 2 - 10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0012] The present invention also provides biomaterials related to Cecropin-mut, and the biomaterials are any one of the following B1) to B5):

[0013] B1) A nucleic acid molecule encoding Cecropin-mut;

[0014] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0015] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0016] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0017] B5) A cell line containing the nucleic acid molecule described in B1), or a cell line containing the expression cassette described in B2).

[0018] Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0019] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the Cecropin-mut protein of the present invention by using known methods, such as directed evolution and point mutation methods. Those nucleotides that have been artificially modified and have 75% or higher identity with the nucleotide sequence of the Cecropin-mut protein isolated from the present invention, as long as they encode the Cecropin-mut protein and have the function of the Cecropin-mut protein, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0020] Among the above biological materials, the nucleic acid molecule described in B1) can be any of the following b11), b12), b13) or b14):

[0021] b11) A cDNA molecule or DNA molecule whose coding sequence is the cDNA molecule or DNA molecule of Sequence 4 in the Sequence Listing;

[0022] b12) The DNA molecule shown in Sequence 4 of the Sequence Listing;

[0023] b13) A cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined by b11) or b12) and encodes Cecropin-mut;

[0024] b14) A cDNA molecule or DNA molecule that hybridizes with the nucleotide sequence defined by b11), b12) or b13) under stringent conditions and encodes Cecropin-mut;

[0025] The recombinant vector described in B3) is a recombinant expression vector capable of expressing Cecropin-mut obtained by introducing the nucleic acid molecule encoding Cecropin-mut into an expression vector.

[0026] B4) The recombinant microorganism is a recombinant microorganism capable of expressing Cecropin-mut obtained by introducing a nucleic acid molecule encoding Cecropin-mut into a starting microorganism.

[0027] B4) The recombinant microorganism can be obtained by introducing the recombinant vector described in B3) into the starting microorganism.

[0028] As used herein, the term "identity" refers to sequence similarity to a native nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity to the nucleotide sequence of the protein consisting of the amino acid sequence shown in Coding Sequence 4 of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0029] In the above biological materials, the stringent conditions may be as follows: hybridization at 50 °C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4 and 1 mM EDTA, and washing in 2×SSC, 0.1% SDS at 50 °C; it may also be: hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M NaPO4 and 1 mM EDTA, and washing in 1×SSC, 0.1% SDS at 50 °C; it may also be: hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M NaPO4 and 1 mM EDTA, and washing in 0.5×SSC, 0.1% SDS at 50 °C; it may also be: hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M NaPO4 and 1 mM EDTA, and washing in 0.1×SSC, 0.1% SDS at 50 °C; it may also be: hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M NaPO4 and 1 mM EDTA, and washing in 0.1×SSC, 0.1% SDS at 65 °C; it may also be: hybridization in a solution of 6×SSC, 0.5% SDS at 65 °C, and then washing the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS; it may also be: hybridization and washing the membrane twice in a solution of 2×SSC, 0.1% SDS at 68 °C, 5 min each time, and then hybridization and washing the membrane twice in a solution of 0.5×SSC, 0.1% SDS at 68 °C, 15 min each time; it may also be: hybridization and washing the membrane under the conditions of 65 °C in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS.

[0030] The above identity of 75% or more than 75% may be 80%, 85%, 90% or 95% or more identity.

[0031] In the above application, the expression cassette containing the nucleic acid molecule encoding the Cecropin-mut protein (Cecropin-mut gene expression cassette) described in B2) refers to DNA that can express the Cecropin-mut protein in a host cell. This DNA may not only include a promoter that initiates the transcription of the Cecropin-mut gene but also a terminator that terminates the transcription of the Cecropin-mut gene. Further, the expression cassette may also include an enhancer sequence.

[0032] An existing expression vector can be used to construct a recombinant vector containing the Cecropin-mut gene expression cassette.

[0033] In the above biological material, the vector can be a plasmid, cosmid, phage, or viral vector. Specifically, the plasmid can be a Bacillus subtilis secretion expression vector, such as pWB980.

[0034] Specifically, the recombinant vector described in B3) can be pWB980 / Cecropin. pWB980 / Cecropin is a recombinant vector obtained by replacing the DNA fragment between the HindIII and NheI recognition sequences of the pWB980 vector with the DNA fragment shown in Sequence 3.

[0035] In the above biological material, the microorganism can be yeast, bacteria, algae, or fungi. Among them, the bacteria can be Bacillus subtilis, such as Bacillus subtilis strains WB600 or WB800.

[0036] Specifically, the recombinant microorganism described in B4) can be WB600 / pWB980 / Cecropin. WB600 / pWB980 / Cecropin is a recombinant strain obtained by introducing pWB980 / Cecropin into Bacillus subtilis strain WB600.

[0037] In the above biological material, the cell line does not include propagation materials.

[0038] The present invention also provides a method for preparing a recombinant microorganism. The method includes: introducing an expression cassette containing a nucleic acid molecule encoding Cecropin-mut or a recombinant expression vector containing the expression cassette into a starting microorganism to obtain a recombinant microorganism.

[0039] The microorganism can be Bacillus subtilis, such as Bacillus subtilis strains WB600 or WB800.

[0040] The present invention also provides a method for preparing Cecropin-mut. The method includes: culturing the recombinant microorganism obtained by using the method for preparing the recombinant microorganism to obtain Cecropin-mut.

[0041] Any of the following applications of Cecropin-mut or the said biological material also falls within the scope of protection of the present invention:

[0042] X1) Raising animals;

[0043] X2) Improving the survival rate of animals;

[0044] X3) Enhancing the immunity of animals;

[0045] X4) Reducing animal diarrhea;

[0046] X5) Promoting animal growth;

[0047] X6) Lowering the feed conversion ratio of animals;

[0048] X7) Improving the European efficiency index of animals;

[0049] X8) Preparing products for raising animals;

[0050] X9) Preparing products for improving the survival rate of animals;

[0051] X10) Preparing products for enhancing the immunity of animals;

[0052] X11) Preparing products for reducing animal diarrhea;

[0053] X12) Preparing products for promoting animal growth;

[0054] X13) Preparing products for lowering the feed conversion ratio of animals;

[0055] X14) Preparing products for improving the European efficiency index of animals.

[0056] In the above applications, the said animals can be mammals, poultry (such as chickens), or livestock (such as pigs).

[0057] The present invention also provides a method for raising animals, which includes: adding Cecropin-mut or the said biological material as a feed additive to animal feed to obtain a mixed feed, and using the mixed feed to feed animals for raising animals.

[0058] In the above method, the said animals can be mammals, poultry (such as chickens), or livestock (such as pigs).

[0059] An animal feed containing Cecropin-mut or the said biological material also falls within the scope of protection of the present invention.

[0060] The present invention has obtained an improved antibacterial peptide Cecropin-mut that has no antibacterial activity below 28°C and resumes antibacterial activity at 37°C. Compared with traditional antibacterial peptides, the Bacillus subtilis strain containing the gene of this improved antibacterial peptide can achieve high-density fermentation culture below 28°C and efficiently express the antibacterial peptide at the same time. Since the expressed antibacterial peptide has no antibacterial activity at low temperature, it will not inhibit the reproduction of the bacterial strain. After the fermentation is completed, the fermentation broth is restored to 37°C, and the antibacterial peptide resumes its antibacterial activity, thereby enabling high-density fermentation, which is suitable for large-scale production. Oral administration of this probiotic to weaned piglets every day can increase the survival rate of piglets by 14% and reduce the incidence of diarrhea by 12.5%; oral administration of this probiotic to broiler chickens every day can increase the weight by 4.2%, increase the survival rate by 1.7%, and reduce the feed-to-meat ratio by 7.9%. This probiotic can be used for the auxiliary breeding of livestock and poultry, effectively reducing the incidence of diseases, reducing production costs, and improving the European benefit index. Therefore, the present invention has completely broken through the technical problem that antibacterial peptides are difficult to ferment and produce, laid a foundation for the industrialization of antibacterial peptides, and has good application prospects.

[0061] The following further describes the present invention in detail in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way. Specific embodiments

[0062] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified. In the following quantitative tests, three repeated experiments are set, and the results are averaged. In the following embodiments, unless otherwise specified, the first position of each nucleotide sequence in the sequence list is the 5'-terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3'-terminal nucleotide of the corresponding DNA / RNA.

[0063] The PWB980 plasmid and the Bacillus subtilis WB600 strain in the following embodiments are recorded in the literature "Expression of xylanase xynZF 318 in Bacillus subtilis WB600 and optimization of fermentation conditions, Science and Technology of Food Industry, 2020".

[0064] Example 1: Directed evolution screening of temperature-sensitive antibacterial peptides

[0065] 1. Optimize the antibacterial peptide gene Cecropin of black soldier fly and introduce random mutations to obtain a random mutation gene containing the antibacterial peptide of black soldier fly.

[0066] The wild - type black soldier fly antimicrobial peptide gene after gene optimization is as shown in Sequence 1 of the sequence listing, encoding the protein shown in Sequence 2 of the sequence listing.

[0067] 2. Insert the black soldier fly antimicrobial peptide mutant gene obtained in step 1 between the HindIII and NheI restriction enzyme cleavage sites of the Bacillus subtilis secretion expression vector pWB980 (Beijing Tian'enze Gene Technology Co., Ltd., product number: 60908 - 6945y) (that is, replace the DNA fragment between the HindIII and NheI recognition sequences of pWB980 with the black soldier fly antimicrobial peptide mutant gene) to obtain an expression vector containing the black soldier fly antimicrobial peptide mutant gene, named pWB980 / Cecropin.

[0068] 3. Electro - transform the expression vector containing the black soldier fly antimicrobial peptide mutant gene obtained in step 2 into Bacillus subtilis WB600 (Beijing Tian'enze Gene Technology Co., Ltd., product number: 12 - 210y). Electro - transformation conditions: 2.5 milliseconds, 1.5 kV. Spread the electro - transformed bacterial liquid on an LB plate containing 100 micrograms / ml kanamycin (North China Pharmaceutical); culture at 28 °C for 36 hours.

[0069] 4. Pick single colonies and spot - inoculate them in parallel on two antibiotic - free LB plates, that is, inoculate single colonies on two plates respectively, and the colony positions on the two plates correspond one by one;

[0070] 5. Place the two spot - inoculated plates at 28 °C and 37 °C respectively and culture for 24 hours;

[0071] 6. Select the colonies where the bacteria proliferate at 28 °C but do not proliferate at 37 °C, which are the colonies expressing temperature - sensitive antimicrobial peptides;

[0072] 7. Inoculate the colonies expressing temperature - sensitive antimicrobial peptides into 0.5 ml of LB liquid medium and culture at 28 °C for 36 hours;

[0073] 8. Add the bacterial liquid that can proliferate at 28 °C into the sample wells of the plate coated with Escherichia coli DH5α and culture at 37 °C for 15 hours; Dilute the bacterial liquid with an inhibition zone by 10 times and further confirm the inhibition zone;

[0074] 9. Extract plasmids from the confirmed positive strains, sequence them, and analyze the antimicrobial peptide - encoding nucleic acid sequences;

[0075] 10. After multiple rounds of screening, finally, 7 strains expressing temperature - sensitive antimicrobial peptides that can effectively proliferate at 28 °C and proliferate inefficiently at 37 °C are obtained from about 300,000 single colonies. After sequencing and comparison, compared with the amino acid sequence of the antimicrobial peptide protein, there are 4 point mutations in the amino acid sequence of the mutant protein (K4 - H, V 10 - L, Q 30 - N, L 35-V). It was named Cecropin-mut, and its sequence is shown in Sequence 4 of the Sequence Listing, which is encoded by the DNA sequence shown in Sequence 3 of the Sequence Listing. The strain expressing Cecropin-mut was designated as WB600 / pWB980 / Cecropin. WB600 / pWB980 / Cecropin contains pWB980 / Cecropin-mut, and pWB980 / Cecropin-mut is a recombinant vector obtained by replacing the DNA fragment between the HindIII and NheI recognition sequences of the Bacillus subtilis secretion expression vector pWB980 with the DNA fragment shown in Sequence 3.

[0076] Example 2. Identification of temperature-sensitive antibacterial peptides

[0077] 1. The strain (WB600 / pWB980 / Cecropin) expressing Cecropin-mut obtained in Example 1 was cultured in LB medium at 28 °C for 72 hours, centrifuged at 4000 g for 30 minutes, the supernatant was collected, the culture supernatant was filtered through a filter with a pore size of 0.22 μm, and the supernatant was ultrafiltered and concentrated with an ultrafiltration tube with a molecular weight cut-off of 10 kD, and the upper concentrated polypeptide solution was collected.

[0078] 2. The collected supernatant was passed through a gel filtration chromatography column (Sephadex G-25F, GElife), and 50 mM phosphate buffer (pH 7.4) was allowed to flow through the column at a flow rate of 1 ml / min, and the target polypeptide was collected. The concentration of the polypeptide was calibrated using a UV spectrophotometer.

[0079] 3. The wild-type Cecropin polypeptide was chemically synthesized by Beijing Kanglonghua Cheng Co., Ltd., and its polypeptide sequence is the same as Sequence 2, and its DNA coding sequence is Sequence 1.

[0080] 4. Gradient dilute the wild-type Cecropin synthetic polypeptide and the screened improved Cecropin-mut respectively, add them into the spotting wells of the plate coated with Escherichia coli DH5α. At the same time, set ampicillin (North China Pharmaceutical) as the positive control, and culture them at 28°C or 37°C for 24 hours respectively, and observe the inhibition zones. Define the lowest concentration that can form an inhibition zone as the minimum inhibitory concentration. The results show that at 28°C, both the wild-type Cecropin synthetic polypeptide and ampicillin form inhibition zones, while the screened improved polypeptide Cecropin-mut only shows extremely weak antibacterial effects; at 37°C, the minimum inhibitory concentrations of both the wild-type Cecropin synthetic polypeptide and the improved polypeptide Cecropin-mut are 9 μg / mL, and the minimum inhibitory concentration of ampicillin is 35 μg / mL. These results can prove that the screened improved polypeptide Cecropin-mut has temperature-sensitive characteristics, its antibacterial ability is inhibited at 28°C, and its antibacterial ability can be restored at 37°C. Moreover, the antibacterial peptide obtained by expression with Bacillus subtilis has the same inactivating efficacy as the antibacterial peptide obtained by chemical synthesis, and the minimum inhibitory concentration is lower than that of ampicillin.

[0081] Example 3. Preparation of functional probiotics

[0082] I. Immunization protocol

[0083] Culture the Bacillus subtilis WB600 / pWB980 / Cecropin of Example 1 in LB liquid medium at 28°C for 12 - 15 hours, inoculate the fermentation medium (20 g / L of sucrose, 5 g / L of ammonium sulfate, 50 g / L of wheat bran, 2.5 g / L of trisodium citrate, 0.3 g / L of dipotassium hydrogen phosphate, 0.2 g / L of magnesium sulfate, 0.02 g / L of ferric sulfate, PH 7.2) according to a ratio of 1:100, and add the antifoaming agent according to a ratio of 1:2000. Adjust the PH value with ammonia water and ferment at 28°C for 35 hours. Collect the bacterial cells and dry the bacterial cells with a spray dryer to obtain dry bacterial powder. Calibrate the bacterial powder to confirm that the viable bacteria content per gram of bacterial powder is not less than 10 11 individuals.

[0084] Example 4. Functional probiotics improve the survival rate of piglets

[0085] In a certain pig farm in Liaoning Province, piglet diarrhea often occurred, resulting in a survival rate of 78% for piglets. 2,541 weaned piglets at 30 days old with an average weight of 5 kg were randomly divided into two groups. There were 1,015 piglets in the experimental group and 1,526 piglets in the control group. Functional probiotics were added to the feed of the weaned piglets in the experimental group, with 100 grams of the dried bacterial powder of Example 3 added per ton of feed, and it was used continuously for 1 month. Among the 1,015 weaned piglets (experimental group), 117 cases of diarrhea occurred, and finally 977 survived, with a survival rate of 96.3% and a daily weight gain of 0.51 kg. Among the 1,526 weaned piglets (control group) that did not use the dried bacterial powder of Example 3, 266 cases showed diarrhea symptoms, and finally 1,256 survived, with a survival rate of 82.3% and a daily weight gain of 0.45 kg. The only difference in the feed fed to the experimental group and the control group was that the feed of the experimental group contained the dried bacterial powder of Example 3, while the control group did not contain the dried bacterial powder of Example 3.

[0086] Unpaired t-test analysis was performed on the data, and P < 0.05, indicating a significant difference in the results between the control group and the experimental group. As shown in Table 1, the use of the dried bacterial powder of Example 3 increased the survival rate of piglets by 14%, effectively reduced the occurrence of diarrhea, and the diarrhea incidence rate decreased by 12.5%. The dried bacterial powder of Example 3 could also increase the daily weight gain of piglets. The dried bacterial powder of Example 3 can be used as a functional probiotic to improve the survival rate and weight of piglets and reduce the diarrhea rate.

[0087] Table 1: Technical indicators of weaned piglets

[0088]

[0089]

[0090] Example 5. Functional probiotics effectively reduce the feed conversion ratio of broilers

[0091] In a certain broiler farm in Shandong, from breeding to slaughter, 10,000 white - feather broilers were fed with the dried bacterial powder of Example 3, with 100 grams added per ton of feed, as the probiotic group. Another 20,000 broilers that did not add the dried bacterial powder of Example 3 were used as the control group. Before the experiment, the average weight of 7 - day - old chicks in both groups was 40 grams, and the two groups were randomly divided. Before slaughter, the weight, survival rate, feed conversion ratio, and European efficiency index of the two groups of broilers were analyzed. The results are shown in Table 2. The average weight of the probiotic group increased by 4.2%, the survival rate increased by 1.7%, the feed conversion ratio decreased by 7.9%, and the overall European efficiency index was significantly improved. In addition, the overall diarrhea rate during the entire experiment was significantly lower than that of the control group, and the feces were more formed. Unpaired t - test analysis was performed on all the data, and P < 0.05, indicating a significant difference in the results between the control group and the experimental group.

[0092] Table 2: Technical indicators of broilers

[0093] Group Slaughter age (days) Body weight (kg) Feed conversion ratio Survival rate (%) European efficiency index Diarrhea rate (%) Probiotic group 40 2.95 1.52 99.28 482 2.35 Control group 40 2.83 1.65 97.62 416 4.67

[0094] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below. Sequence Listing <110>Dalian Huatai Biotechnology Co., Ltd. <120>A functional probiotic capable of secreting antibacterial peptides, its preparation method and application <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 141 <212> DNA <213> Artificial sequence <400> 1 ggttggtgga aacgcgtttt caaaccggtg gaaaaactgg gtcagcgcgt tcgtgatgct 60 ggtattcagg gcctggaaat tgcacagcag ggcgccaatg ttctggcaac cgctcgcggt 120 ggcccgccgc agcagggcta a 141 <210> 2 <211> 46 <212> PRT <213> Artificial sequence <400> 2 Gly Trp Trp Lys Arg Val Phe Lys Pro Val Glu Lys Leu Gly Gln Arg 1 5 10 15 Val Arg Asp Ala Gly Ile Gln Gly Leu Glu Ile Ala Gln Gln Gly Ala 20 25 30 Asn Val Leu Ala Thr Ala Arg Gly Gly Pro Pro Gln Gln Gly 35 40 45 <210> 3 <211> 141 <212> DNA <213> Artificial sequence <400> 3 ggttggtggc atcgtgtttt taaaccgctg gaaaaactgg gccagcgcgt ccgtgatgct 60 ggtattcagg gtctggaaat cgcccagaat ggtgcaaacg ttgtggcgac cgctcgtggc 120 ggtccgccgc agcagggtta a 141 <210> 4 <211> 46 <212> PRT <213> Artificial sequence <400> 4 Gly Trp Trp His Arg Val Phe Lys Pro Leu Glu Lys Leu Gly Gln Arg 1 5 10 15 Val Arg Asp Ala Gly Ile Gln Gly Leu Glu Ile Ala Gln Asn Gly Ala 20 25 30 Asn Val Val Ala Thr Ala Arg Gly Gly Pro Pro Gln Gln Gly 35 40 45

Claims

1. An antibacterial peptide, which is A1) or A2): A1) A protein with an amino acid sequence of Sequence 4; A2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1).

2. A biological material related to the protein described in Claim 1, which is any one of the following B1) to B5): B1) A nucleic acid molecule encoding the protein described in Claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A cell line containing the nucleic acid molecule described in B1), or a cell line containing the expression cassette described in B2).

3. The biological material according to Claim 2, wherein: B1) The nucleic acid molecule is as follows in b11) or b12): b11) The coding sequence is the cDNA molecule or DNA molecule of Sequence 3 in the Sequence Listing; b12) The DNA molecule shown in Sequence 3 of the Sequence Listing; B3) The recombinant vector is a recombinant expression vector capable of expressing the protein recited in Claim 1, which is obtained by introducing the nucleic acid molecule encoding the protein recited in Claim 1 into an expression vector; B4) The recombinant microorganism is a recombinant microorganism capable of expressing the protein recited in Claim 1, which is obtained by introducing the nucleic acid molecule encoding the protein recited in Claim 1 into a starting microorganism.

4. The biological material according to Claim 2 or 3, wherein: The microorganism is Bacillus subtilis.

5. A method for preparing a recombinant microorganism, comprising: An expression cassette containing the nucleic acid molecule encoding the protein recited in Claim 1 or a recombinant expression vector containing the expression cassette is introduced into a starting microorganism to obtain a recombinant microorganism.

6. A method for preparing the protein described in Claim 1, comprising: The recombinant microorganism obtained by the method recited in Claim 5 is cultured to obtain the protein recited in Claim 1.

7. Any one of the following applications of the protein described in Claim 1 or the biological material described in any one of Claims 2 - 4: X1) Preparing animal feeding products; X2) Preparing products for improving animal survival rate; X3) Preparing products for improving animal immunity; X4) Preparing products for reducing animal diarrhea; X5) Preparing products for promoting animal growth; X6) Preparing products for reducing the feed-to-meat ratio of animals; X7) Preparing products for improving the European efficiency index of animals.

8. The application according to Claim 7, wherein: The animal is a mammal.

9. The application according to Claim 7, wherein:The animal is a poultry or livestock.

10. An animal feed containing the protein described in claim 1 or the biological material described in any one of claims 2-4.

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