Preparation of a fusion antimicrobial peptide against major pathogenic bacteria of bovine mastitis

Abstract

The present invention discloses a method for preparing a fusion antimicrobial peptide for resisting the main pathogenic bacteria of bovine mastitis. By evaluating the antibacterial activity of multiple antimicrobial peptides obtained by synthesis, two antimicrobial peptides "RGLRRLGRKIAHGVKKYGPTVLRIIRIAG" and "LKRVWKRVFKLLKRYWRQLKKPVR" with the best antibacterial effect are screened out, and they are connected through Flexible Linker "GGGGS" to obtain a fusion antimicrobial peptide. A specific expression vector is constructed, and the fusion antimicrobial peptide AP-14829 is expressed by Pichia pastoris X33 and the activity is evaluated. The fusion antimicrobial peptide AP-14829 obtained by the present invention has a strong inhibitory effect on several main pathogenic bacteria of bovine mastitis, and the antibacterial effect is significantly better than that of a single antimicrobial peptide. In vitro hemolytic experiments have confirmed that AP-14829 has no obvious damaging effect on red blood cells. The polypeptide obtained by yeast expression is closer to the natural state than prokaryotic expression, so the activity is higher, thereby ensuring the normal exercise of the polypeptide function.

Description

Technical Field

[0001] The present invention relates to the preparation of a fusion antimicrobial peptide and a yeast expressing the fusion antimicrobial peptide. Background Art

[0002] Bovine mastitis is an inflammatory disease caused by a variety of pathogenic microorganisms, which is characterized by invasion of mammary tissue and long-term infection. It is divided into serous mastitis, fibrinous catarrhal mastitis, suppurative mastitis, hemorrhagic mastitis, gangrenous mastitis and latent mastitis. In recent years, my country's dairy industry has developed rapidly, with the number of dairy cows, raw milk production and dairy product production doubling, dairy product consumption steadily increasing, and the total import and export trade of dairy products increasing. The dairy industry has a long industrial chain and a high degree of industrial correlation. The healthy development of the dairy industry plays an important role in optimizing the rural industrial structure, increasing farmers' income, improving residents' dietary structure, and driving the development of related industries in the national economy. As the first of the three major diseases in the dairy industry, bovine mastitis affects the global dairy industry, causing huge economic losses and declining animal health. The main hazards include a significant decrease in milk production, a serious decline in milk quality, which greatly reduces its health benefits, and a decrease in the reproductive capacity of cows. In addition, the treatment of the disease will incur high treatment costs and greatly shorten the life of cows. The existence of this disease seriously restricts the rapid and healthy development of the dairy cattle farming industry and has become a bottleneck in the development of my country's dairy cattle farming industry (Yang Na, Zhang Qingjuan, Mao Ruoyu, Hao Ya, Ma Xuanxuan, Teng Da, Fan Huan, Wang Jianhua. Effect of NZ2114 against Streptococcusdysgalactiae biofilms and its application in murine mastitis model[J]. Frontiers in Microbiology, 2022, 13.).

[0003] Antimicrobial peptides (AMPs) are a class of short peptides with antimicrobial ability that are widely present in organisms. They are usually composed of 15 to 30 amino acids. They are an important part of the body's immune system (Na Dong, Chensi Wang, Tingting Zhang, Lei Zhang, Chenyu Xue, Xinjun Feng, Chongpeng Bi, Anshan Shan. Bioactivity and Bactericidal Mechanism of Histidine-Rich β-Hairpin Peptide Against Gram-Negative Bacteria [J]. International Journal of Molecular Sciences, 2019, 20 (16).). In the 1980s, Swedish scientists used Bacillus cereus to induce Cecropin and discovered the first antimicrobial peptide, Cecropin (Steiner H, Hultmark D, A, Bennich H, Boman H G. Sequence and specificity of two antibacterial proteins involved in insect immunity. [J]. Nature, 1981, 292 (5820).). The characteristics of antimicrobial peptides are: 1. Ability to fight against Gram-positive and Gram-negative bacteria. 2. Small molecular weight, rapid onset and degradation, no residue. 3. Unique mechanism of action, no harm to eukaryotic cells, safe and reliable (Exley Sarah E, Paslay Lea C, Sahukhal Gyan S, Abel Brooks A, Brown Tyler D, McCormick Charles L, Heinhorst Sabine, Koul Veena, Choudhary Veena, Elasri Mohamed O, Morgan Sarah E. Antimicrobial Peptide Mimicking Primary Amine and Guanidine Containing Methacrylamide Copolymers Prepared by Raft Polymerization. [J]. Biomacromolecules, 2015, 16 (12).).

[0004] Therefore, antimicrobial peptides are expected to provide effective help in solving the increasingly serious problem of bovine mastitis. At present, antimicrobial peptides are attracting more and more attention, and the application of antimicrobial peptide preparations to treat bovine mastitis has become a hot topic of research at home and abroad (Pen Guihong, Yang Na, Teng Da, Mao Ruoyu, Hao Ya, Wang Jianhua. A Review on the Use of Antimicrobial Peptides to Combat Porcine Viruses. [J]. Antibiotics (Basel, Switzerland), 2020, 9 (11).). Summary of the invention

[0005] The invention provides a transgenic yeast. The transgenic yeast provided by the invention can efficiently express a fusion antimicrobial peptide capable of inhibiting major pathogenic bacteria of cow mastitis. The provided fusion antimicrobial peptide is named AP-14829.

[0006] The present invention provides a fusion antimicrobial peptide with antibacterial activity, which can resist the main pathogenic bacteria of bovine mastitis and is named AP-14829. The peptide is characterized by being obtained by connecting two antimicrobial peptides "RGLRRLGRKIAHGVKKYGPTVLRIIRIAG" and "LKRVWKRVFKLLKRYWRQLKKPVR" through FlexibleLinker "GGGGS", and the sequence of the obtained fusion antimicrobial peptide AP-14829 is as follows: RGLRRLGRKIAHGVKKYGPTVLRIIRIAGGGGGSLKRVWKRVFKLLKRYWRQLKKPVR.

[0007] The present invention also provides a method for preparing transgenic yeast capable of efficiently expressing the above-mentioned fusion antimicrobial peptide: the amino acid sequence of the above-mentioned fusion antimicrobial peptide is selected to design its nucleotide sequence, the preferred codon of Pichia pastoris is selected for optimization, the start codon ATG is added, the target sequence is inserted between EcoRI and NotI of the multiple cloning site of the pPICZαA vector, and then transferred into yeast cells.

[0008] The invention discloses an application of the transgenic yeast in preventing and treating mastitis in dairy cows.

[0009] The preparation method of the fusion antimicrobial peptide with antimicrobial activity of the present invention is to separate and obtain it from yeast cell lysate after expressing it with transgenic yeast.

[0010] The invention discloses an application of the fusion antimicrobial peptide with antibacterial activity in the prevention and treatment of mastitis in dairy cows.

[0011] The present invention provides a fusion antimicrobial peptide that has a significant inhibitory effect on the main pathogenic bacteria of bovine mastitis. Further, the fusion antimicrobial peptide is designed by designing its nucleotide sequence through the amino acid sequence of the fusion antimicrobial peptide, after optimizing the preferred codon of Pichia pastoris, adding the start codon ATG, inserting the target sequence between EcoRI and NotI of the multi-cloning site of the pPICZαA vector, transferring into yeast cells, and is isolated and obtained in yeast cell lysate after yeast expression.

[0012] As a fusion antimicrobial peptide, AP-14829 can resist both Gram-positive and Gram-negative bacteria. It has a small molecular weight and can be used and degraded quickly. At the same time, the various modifications made by cells to proteins during yeast expression are not available in chemical synthesis and prokaryotic expression. The peptides obtained through eukaryotic expression are closer to the natural state, so they are more active, thus ensuring the normal function of the peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a tricine SD-PAGE identification diagram of the fusion antimicrobial peptide.

[0014] Figure 2 This is the inhibition zone diagram of the fusion antimicrobial peptide.

[0015] Figure 3 It is the hemolytic activity of the fusion antimicrobial peptide. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0017] The preparation steps of fusion antimicrobial peptide are as follows:

[0018] Screening and fusion of antimicrobial peptides:

[0019] 1. Screening of antimicrobial peptides

[0020] Twelve antimicrobial peptides were screened from the antimicrobial peptide database, and their activity was verified by inhibition zone experiment after chemical synthesis. The two antimicrobial peptides with the best antibacterial effect, "RGLRRLGRKIAHGVKKYGPTVLRIIRIAG" and "LKRVWKRVFKLLKRYWRQLKKPVR", were selected and connected by Flexible Linker "GGGGS" to obtain a fusion antimicrobial peptide. The obtained fusion antimicrobial peptide was named AP-14829.

[0021] Construction of recombinant yeast expression vector:

[0022] 1. Construction of pPICZαA-AP-14829 plasmid vector

[0023] The nucleotide sequence of the hybrid antimicrobial peptide AP-14829 was designed according to its amino acid sequence, and the start codon ATG was added after the Pichia pastoris preferred codon optimization. The target sequence was synthesized and connected between EcoRI and NotI of the multi-cloning site of the pPICZαA vector.

[0024] 2. Plasmid transfer into yeast x33

[0025] The plasmid pPICZαA-AP-14829 was linearized and then electroporated into competent yeast X33. Yeast X33 with the plasmid was cultured on a Zeocin-resistant plate, and after positive transformants grew out, a single colony was picked for colony PCR, and yeast with the plasmid successfully transferred was selected for expansion culture.

[0026] Expression and detection of fusion antimicrobial peptides:

[0027] The yeast obtained above was placed in a liquid culture medium at 28°C and 220rpm for 96h. 1ml of bacterial solution was taken out at intervals and stored at -80°C, and methanol was added at a final concentration of 0.8%. The sampling time points were: 24h, 48h, 72h, and 96h. The bacterial solution was centrifuged at 8000rpm for 1min at each time point. The precipitate was mixed with an equal volume of glass beads and 100μL of lysis buffer was added. The mixture was shaken vigorously for 100s and quickly frozen with liquid nitrogen. This was repeated 8 times. The mixture was centrifuged at 8000rpm for 10min. 20μL of the supernatant was mixed with an equal volume of loading buffer and boiled for 10min. Tricine-SDS-PAGE electrophoresis was performed.

[0028] Activity detection of fusion antimicrobial peptides:

[0029] 1. Antibacterial activity detection

[0030] The recombinant yeast cells were lysed and purified to obtain the fusion antimicrobial peptide AP-14829, and the purified antimicrobial peptide was used to perform an inhibition zone experiment on several harmful bacteria to verify its antimicrobial activity. Using the punching method (the diameter of each hole is 7.8 mm), 150 μL of purified protein was added to each well (the elution buffer of the purified protein was used as a negative control, and Zeocin was used as a positive control). The size of each inhibition zone was measured, and each bacterium was repeated three times.

[0031] 2. Hemolytic activity detection

[0032] Collect 1mL of bovine blood in an anticoagulant tube, centrifuge at 2000rpm for 5min, discard the supernatant, and collect red blood cells; wash the red blood cells with phosphate buffer (pH=7.4) PBS, repeat 3 times, and finally resuspend the cells with 10mL PBS to make a red blood cell suspension. Take 100μL of purified polypeptide and add it to the No. 1 well of each row of the 96-well plate, and add 50μL PBS buffer to each other well. Pipette out 50μL of polypeptide solution in well No. 1, add it to the adjacent wells in turn, and dilute it to well No. 12. Take 50μL from the last well and discard it. Add 50μL 0.2% Tritonx-100 and 50μL red blood cell suspension to each well of the positive control group, and add 50μL PBS and 50μL red blood cell suspension to the negative control group. Incubate at 37℃ for 1h, measure the absorbance at a wavelength of 570nm with an enzyme marker, repeat the experiment 3 times, and take the average value of the results. Substitute the formula to calculate the hemolysis rate: Hemolysis rate (%) = (A sample - A negative) / (A positive - A negative) × 100%.

Claims

1. A fusion antimicrobial peptide AP-14829 for resisting the main pathogenic bacteria of bovine mastitis, characterized by: The fusion antimicrobial peptide AP-14829 is obtained by connecting two antimicrobial peptides "RGLRRLGRKIAHGVKKYGPTVLRIIRIAG" and "LKRVWKRVFKLLKRYWRQLKKPVR" through FlexibleLinker "GGGGS". The sequence of the fusion antimicrobial peptide AP-14829 is as follows: RGLRRLGRKIAHGVKKYGPTVLRIIRIAGGGGGSLKRVWKRVFKLLKRYWRQLKKPVR.

2. A transgenic yeast capable of efficiently expressing the fusion antimicrobial peptide according to claim 1, characterized in that: The fusion antimicrobial peptide AP-14829 for resisting the main pathogenic bacteria of bovine mastitis according to claim 1 is selected, and its nucleotide sequence is designed according to the amino acid sequence of the antimicrobial peptide. After optimizing the codons preferred by Pichia pastoris, the start codon ATG is added, and the above-mentioned target sequence is inserted between EcoRI and NotI of the multiple cloning site of the pPICZαA vector, and the recombinant plasmid is transferred into yeast cells.

3. The method for preparing the fusion antimicrobial peptide AP-14829 for resisting the main pathogenic bacteria of bovine mastitis according to claim 1, characterized in that: Separated from the yeast cell lysate obtained in claim 2.

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