Preparation of fusion antibacterial peptide for resisting main pathogenic bacteria of dairy cow mastitisTechnical Field
The invention relates to a fusion antibacterial peptide and a preparation method of yeast for expressing the fusion antibacterial peptide.
Background
Cow mastitis (bovine mastitis) is an inflammatory disease caused by a plurality of pathogenic microorganisms, and has the characteristics of invasion of mammary tissue and long-term infection. Is divided into serous mastitis, cellulose catarrh mastitis, suppurative mastitis, hemorrhagic mastitis, gangrene mastitis and recessive mastitis. In recent years, the dairy industry in China rapidly develops, the dairy stock, the raw milk yield and the dairy product yield are multiplied, the dairy product consumption is steadily improved, and the total amount of the imported and exported trade of the dairy products is continuously increased. The industrial chain length and the industrial association degree of the dairy industry are extremely high, and the healthy development of the dairy industry plays an important role in optimizing rural industrial structures, increasing peasant income, improving resident dietary structures and driving the development of national economy related industries. As the first of three major diseases in dairy cow industry, dairy cow mastitis affects the global dairy industry, causing great economic loss and animal health decline. The main harm is that the milk yield is obviously reduced, the quality of milk is seriously reduced, the health care effect is greatly reduced, the reproductive capacity of cows is reduced, in addition, the treatment of the disease also generates high treatment cost, the service life of the cows is greatly shortened, and the like, the existence of the disease severely restricts the rapid and healthy development of the dairy cow breeding industry, and becomes the bottleneck of the development of the dairy cow breeding industry in China (YangNa,Zhang Qingjuan,Mao Ruoyu,Hao Ya,Ma Xuanxuan,Teng Da,Fan Huan,Wang Jianhua.Effect ofNZ2114 against Streptococcus dysgalactiae biofilms and its application in murine mastitis model[J].Frontiers in Microbiology,2022,13.).
Antibacterial peptides (AMPs) are a class of short peptides with antibacterial ability that are widely present in organisms, and generally consist of 15 to 30 amino acids. Is an important component of the immune system of the organism, in the 80 s of the (Na Dong,Chensi Wang,Tingting Zhang,Lei Zhang,Chenyu Xue,Xinjun Feng,Chongpeng Bi,Anshan Shan.Bioactivity and Bactericidal Mechanism of Histidine-Richβ-Hairpin PeptideAgainst Gram-Negative Bacteria[J].International Journal of Molecular Sciences,2019,20(16).).20 th century, the first antibacterial peptide namely 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 antibacterial peptide has the characteristics of resisting gram-positive bacteria and gram-negative bacteria. 2. Has small molecular weight, rapid action and degradation, and no residue. 3. Has unique action mechanism, no damage to eukaryotic cells, safety and reliability (Exley Sarah E,Paslay Lea C,Sahukhal Gyan S,Abel BrooksA,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).).
Thus, the antibacterial peptide is expected to provide a powerful help for solving the increasingly serious cow mastitis. At present, the antibacterial peptide is more and more paid attention to, and the application of the antibacterial peptide preparation to the treatment of cow mastitis has become a hot spot for domestic and foreign research (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).).
Disclosure of Invention
The invention provides a transgenic yeast, and the transgenic yeast provided by the invention can efficiently express a fusion antibacterial peptide capable of inhibiting main pathogenic bacteria of cow mastitis, and the provided fusion antibacterial peptide is named as AP-14829.
The fusion antibacterial peptide with antibacterial activity provided by the invention can resist main pathogenic bacteria of cow mastitis, is named as AP-14829, and is characterized in that the fusion antibacterial peptide with antibacterial activity is obtained by connecting two antibacterial peptides 'RGLRRLGRKIAHGVKKYGPTVLRIIRIAG' and 'LKRVWKRVFKLLKRYWRQLKKPVR' through a Flexible Linker 'GGGGS', and the sequence of the obtained fusion antibacterial peptide AP-14829 is RGLRRLGRKIAHGVKKYGPTVLRIIRIAGGGGGSLKRVWKRVFKLLKRYWRQLKKPVR.
The invention also provides a preparation method of the transgenic yeast capable of efficiently expressing the fusion antibacterial peptide, which comprises the steps of selecting an amino acid sequence of the fusion antibacterial peptide to design a nucleotide sequence, selecting pichia pastoris preferential codons for optimization, adding an initiation codon ATG, inserting the target sequence between EcoRI and NotI of a pPICZ alpha A vector multiple cloning site, and transferring into yeast cells.
The transgenic yeast of the invention is applied to preventing and treating cow mastitis.
The preparation method of the fusion antibacterial peptide with antibacterial activity is to use transgenic yeast to express and separate from yeast cell lysate.
The fusion antibacterial peptide with antibacterial activity is applied to the prevention and treatment of cow mastitis.
The invention provides a fusion antibacterial peptide with obvious inhibition effect on main pathogenic bacteria of cow mastitis. Further, the amino acid sequence of the fusion antibacterial peptide is used for designing the nucleotide sequence of the fusion antibacterial peptide, after optimization of the preference codon of pichia pastoris is selected, an initiation codon ATG is added, a target sequence is inserted between EcoRI and NotI of a pPICZ alpha A vector multiple cloning site, the fusion antibacterial peptide is transferred into a yeast cell, and the fusion antibacterial peptide is obtained by separating in a yeast cell lysate after yeast expression.
As a fusion antimicrobial peptide, AP-14829 is resistant to both gram-positive and gram-negative bacteria. Small molecular weight, rapid action and degradation. Meanwhile, various modifications of proteins by cells during yeast expression are not available for chemical synthesis and prokaryotic expression. The polypeptide obtained through eukaryotic expression is more similar to a natural state, so that the activity is higher, and the normal running of the polypeptide function is ensured.
Drawings
FIG. 1 is a TRICINE SD-PAGE identification of fusion antimicrobial peptides.
FIG. 2 is a diagram of the zone of inhibition of the fusion of antimicrobial peptides.
FIG. 3 is the hemolytic activity of the fusion antimicrobial peptide.
Detailed Description
The following detailed description of the invention is given in connection with specific embodiments thereof, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The preparation method of the fusion antibacterial peptide comprises the following steps:
screening and fusion of antibacterial peptides:
1. Screening for antimicrobial peptides
Twelve antibacterial peptides are screened from an antibacterial peptide database, the activity of the antibacterial peptide is verified through a bacteriostasis circle experiment after chemical synthesis, two antibacterial peptides 'RGLRRLGRKIAHGVKKYGPTVLRIIRIAG' and 'LKRVWKRVFKLLKRYWRQLKKPVR' with the best bacteriostasis effect are selected and connected through a Flexible Linker 'GGGGS', and the obtained fusion antibacterial peptide is named as AP-14829.
Construction of recombinant yeast expression vectors:
1. construction of pPICZαA-AP-14829 plasmid vector
The nucleotide sequence of the hybrid antibacterial peptide AP-14829 is designed according to the amino acid sequence, and after the optimization of the preference codon of pichia pastoris is selected, the initiation codon ATG is added. The sequence of interest was synthesized and ligated between EcoRI and NotI at the multicloning site of the pPICZ alpha A vector.
2. Plasmid transfer into Yeast x33
Plasmid pPICZαA-AP-14829 was linearized and then electroporated into competent yeast X33. The plasmid-transferred yeast X33 is spread and cultured on a Zeocin-resistant plate, and after the positive transformant grows out, a single colony is picked for colony PCR, and yeast which is successfully transferred into the plasmid is selected for expansion culture.
Expression and detection of fusion antibacterial peptide:
The yeast obtained above was placed in a liquid medium at 28℃and cultured at 220rpm for 96 hours, 1ml of the bacterial liquid was taken out at intervals and stored at-80℃and supplemented with 0.8% final concentration of methanol, and sampling time points were 24 hours, 48 hours, 72 hours, 96 hours. Bacterial solutions were taken at each time point and centrifuged at 8000rpm for 1min. Mixing the precipitate with equal volume of glass beads, adding 100 μl of lysis buffer, shaking vigorously for 100s, quick freezing with liquid nitrogen, repeating for 8 times, centrifuging at 8000rpm for 10min, mixing 20 μl of supernatant with equal volume of loading buffer, boiling for 10min, and performing Tricine-SDS-PAGE electrophoresis.
Activity detection of fusion antibacterial peptide:
1. Antibacterial activity detection
The recombinant yeast cells are subjected to pyrolysis, purification and separation to obtain fusion antibacterial peptide AP-14829, and antibacterial activity of the fusion antibacterial peptide AP-14829 is verified by performing a bacteriostasis zone experiment on a plurality of harmful bacteria by using the purified antibacterial peptide. Using a well-punching method (diameter of each well is 7.8 mm), 150. Mu.L of purified protein was added to each well (using an elution buffer for purified protein as a negative control and Zeocin as a positive control). The size of each zone of inhibition was measured and each bacteria was repeated three times.
2. Hemolytic activity assay
1ML of bovine blood was collected in an anticoagulant tube, centrifuged at 2000rpm for 5min, the supernatant was discarded, red blood cells were collected, washed with phosphate buffer (pH=7.4) PBS, repeated 3 times, and finally resuspended with 10mL PBS to prepare a red blood cell suspension. 100. Mu.L of purified polypeptide was added to each row 1 of 96-well plates and 50. Mu. LPBS buffer was added to each of the other wells. The polypeptide solution in well 1 was aspirated to 50 μl, sequentially added to adjacent wells, diluted to well 12, and finally 50 μl was discarded. The positive control group was added with 50. Mu.L of 0.2% Tritonx-100 and 50. Mu.L of red blood cell suspension per well, and the negative control group was added with 50. Mu. LPBS and 50. Mu.L of red blood cell suspension. Incubation was carried out at 37℃for 1h, absorbance at 570nm was measured with an enzyme-labeled instrument, the test was repeated 3 times, and the results were averaged. The ratio of hemolysis was calculated by substituting the formula = (sample a-negative)/(sample a-positive-sample a-negative) ×100%.