Antibacterial peptide is a small molecule protein with antibacterial activity and is an important component of the host's innate non-specific defense system. To date, more than 100 endogenous antimicrobial peptides have been found in various organisms such as insects, tunicates, birds, mammals, and plants. Antibacterial peptides have the advantages of small molecular weight, stable physical and chemical properties, broad antibacterial spectrum and abundant material sources. Antibacterial peptides can not only act on bacteria, fungi, viruses and other prokaryotes, but also have a certain effect on tumor treatment. Antibacterial peptides play a key role in the natural immunity of living things. In the early 1980s, Steiner [1] and Salsted et al [2] first isolated insect antibacterial peptides cecropin and defensin. Since then, people have begun to pay attention to such antibacterial peptides and studied their genetic immunity. , host defense systems, membrane protein interactions, protein modification and secretion, etc., to develop these antimicrobial peptides into application food additives and drugs.
1 Types of insect antibacterial peptides
The antibacterial polypeptide produced by insects is an effector of insect immunity. Insect antibacterial peptides are cationic basic peptides, which can be roughly classified into the following four categories: the first type is cecropin, which contains 31 to 39 amino acid residues, generally does not contain cysteine, and is composed of Lepidoptera and Diptera. Insects are produced. The second category is the insect defensin, which contains 38 to 43 amino acid residues and is similar in structure to the defensins of animals and certain plants. Defensins are abundantly present in the hemolymph of insects. More than 30 kinds of defensins have been found in Diptera, Coleoptera, Hymenoptera, Hemiptera and Acarina, mainly killing Gram-positive bacteria. Recently, the antibacterial polypeptide drosomycin was isolated from Drosophila, and 38% of the amino acid sequence was similar to plant defensin, indicating that it has a certain homology with the evolution of plant polypeptides. The third type is an antibacterial peptide rich in proline and arginine with a molecular weight of 2 to 4 kD, such as the bee peptide apidaecin and abeecin (molecular weight of 2.0 kD and 4.0 kD, respectively) isolated from Italian honeybees. The peptide mainly inhibits Gram-negative bacteria. The fourth type is a glycine-rich protein with a molecular weight of 8 to 30 kD, such as sarcotoxin II (molecular weight of about 27 kD) isolated from the larvae of the larvae, isolated from the larvae of the green fly larvae and the larvae of the larvae The dipterin diptericin (molecular weight is about 9kD) and the coleopteran coleopterinin, etc., these large peptides are far apart, and belong to the superfamily members of attacin.
2 Antibacterial mechanism of insect antibacterial peptide
Insects can induce the rapid production of blood cells and fat bodies and release a large number of different components of antimicrobial peptides into the bloodstream, killing foreign bacteria. These antimicrobial peptides are synthesized similarly to immune response and mammalian acute phase reactions. The expression of these genes is regulated by the κB-associated pattern regulatory element, which is regulated by mammalian similarity to the NF κB-associated transactivator encoding the immune and contingency protein genes [3].
When the antimicrobial peptide gene is induced in vivo by bacterial lipopolysaccharides (LPS) or dead cells, the foreign stimuli signal activates the transcriptional activator NF κB, and the activated NF κB recognizes and binds to the antimicrobial peptide gene 5' A specific region upstream of B, thereby activating the expression of the antimicrobial peptide gene [4, 5]. The antibacterial peptide produced after expression has no stable conformation in aqueous solution, but forms a high-order structure only when it is bound or close to the cell membrane. Some scholars have put forward their own views: Merrifield et al [6] believe that when the antimicrobial peptide is in contact with the hydrophobic region of the cell membrane, the entire molecule (from the N-terminus to the C-terminus) induces an amphipathic helix, and the bacteriostatic process may first be The charged amino acid at the N-terminus of the antimicrobial peptide is electrostatically attracted to the polar group of the cell membrane, and forms an α-helix, which is inverted along the helical axis, embedded in the cell membrane, and disrupts the membrane structure. Fink et al [7] showed that the N-terminal (residues 1 to 11) of the antimicrobial peptide forms an amphipathic helix, the middle part (residues 12 to 24) forms an elastic corner, and the C-terminus forms a hydrophobic helix. When the antimicrobial peptide acts on the cell membrane, the C-terminal hydrophobic helix is ​​inserted into the membrane, and the N-terminus is bound to the membrane surface, and the middle is connected by elastic corners. Christensen et al. [8] proposed that antibacterial peptides are attracted to the surface of the membrane by electrostatic interaction, and then the hydrophobic tail is inserted into the hydrophobic region of the cell membrane. By changing the membrane conformation, multiple antimicrobial peptides polymerize to form ion channels on the membrane. Among the above four types of antibacterial polypeptides, the antibacterial mechanism of cecropin and defensin is studied in the most detailed manner.
2.1 Antibacterial mechanism of cecropin
Cecropin is a cationic molecule containing two alpha helix structures, which have dual affinity. The N-terminus is hydrophilic and contains a basic amino acid. The basic part containing the basic amino acid is affinity with the phospholipid of the bacterial cell membrane, and the hydrophobic part penetrates the bacterial cell membrane, so that the electromotive force of the bacterial cell membrane disappears, and the ATP content rapidly decreases, so that the bacterial cell membrane function is lost and the bacteria die. The amidation of the C-terminal amino acid is extremely important for the antibacterial activity of the antibacterial protein [9].
2.2 Antibacterial mechanism of defensins
Insect defensins have a killing effect on Gram-positive and negative bacteria. Relatively speaking, Gram-positive bacteria are more lethal, which may be due to the cell wall of Gram-negative bacteria that prevents defensin from contacting the cell membrane. The defensin molecule contains six cysteines, forming three disulfide bonds and having a stable molecular structure. This structure is identical to the structure of the defensin molecule isolated from the mammalian body. The bactericidal mechanism of defensin antibacterial peptides is also that defensin molecules act on bacterial cell membranes. The disulfide bond in the molecule has a strong affinity with the phospholipid of the bacterial cell membrane. After binding, an ion channel is formed, thereby changing the permeability of the cell membrane and killing the bacteria. The bactericidal active center of the defensin is α-helical structure [10]. The amidation of the C-terminal amino acid is also extremely important for maintaining its antibacterial activity. The study found that after the defensin adheres to the surface of the bacteria, in addition to its own damage to the bacteria, it can also cause the bacteria to be phagocytized and digested by the phagocytic cells, acting as an opsonin. Other studies have shown that hydrogen peroxide (H2O2) has a synergistic effect with defensins, and this synergistic effect increases with the increase of H2O2 concentration, making it easy for defensins to enter its target cell membrane or intracellular environment. In addition, studies have shown that the antibacterial mechanism of cecropin antibacterial peptides hinders the synthesis of bacterial outer membrane proteins, and the antibacterial mechanism of glycine-rich antimicrobial peptides hinders the synthesis of bacterial peptidoglycans.
3 insect antibacterial peptide application
3.1 transgenic peptide gene plant
Insect antibacterial peptides also exhibit extremely strong antibacterial activity against plant pathogenic bacteria. It is a hotspot of research at home and abroad to use genetic engineering methods to introduce the gene encoding the antibacterial peptide of eukaryote into the crop and express it to achieve the purpose of antibacterial. Jaymes [11] introduced the cecropin B gene into tobacco plants to produce tobacco plants resistant to tobacco bacterial wilt (Pseudomonassolanacearum). Beltan et al [12] transferred the gene of cecropin B analog SB 37 into tobacco and found that it can delay the onset of tobacco and reduce morbidity and mortality. It has also been reported that the transfer of the attacinE gene into apple trees can increase the resistance of apple trees to Erwinia amylovora [13]. Recent studies have shown that a gene encoding a stable cecropin analog MB39 is expressed in transgenic tobacco against tobacco wildfire (Pseudomonassyringaepv. tabaci) [14].
Transgenic tobacco against Tobaccomosaic virus (TMV) has also been developed in China. Jian Yuyu et al [15] transferred the cecropin B gene into rice and cultivated plants resistant to rice bacterial blight (Xanthomonasoryzaepv. oryzae). Jia Shirong et al [16] began to select the antibacterial peptide cecropin produced by insect cells in 1986 to carry out research on potato resistance to bacterial wilt genetic engineering, and reconstructed and constructed antibiotics such as cecropinB, shivaA and WHD which are more resistant to cecropin. The peptide gene was introduced into 7 main potato varieties in China, and 1050 transgenic lines were transformed.
3.2 transgenic peptide gene animals
The main means of controlling mosquito-borne diseases such as malaria and yellow fever are the development of highly effective drugs and vaccines, as well as the elimination of mediators (mosquitoes, etc.) and the cessation of transmission routes. In recent years, the antibacterial peptide gene that inhibits pathogenic organisms has been introduced into the mediator (mosquito) by genetic engineering methods, so that it is efficiently and stably expressed in insects, thereby cutting off the path of pathogens [17-19] .
The hybrid peptide shiva 3 constructed on the basis of cecropin B has a toxic effect on the development of Plasmodium perghei spores. A glutathione (GST) transferase gene is ligated in front of the shiva 3 gene to form a fusion gene Gst shiva 3 as a reporter gene, thereby obtaining a transgenic mosquito which can synthesize and secrete shiva 3 in the intestine, so it can be used for Control the spread of human malaria [18]. Recently, Vladimir et al. [19] used the Aedesaegypti vitel logeningene (vg) promoter to link a defensin A (DefA) gene and integrated vg DefA into the genome of Aedes aegypti. The GM mosquitoes, after taking blood, activate the biosynthesis of defensins and accumulate in the hemolymph, and their activity can be maintained for up to 22 days.
3.3 Anticancer effect of antimicrobial peptides
Antibacterial peptides can not only act on bacteria, viruses and other prokaryotes, but also play a role in tumor treatment. In recent years, domestic and foreign competitions have reported the anticancer effect of antimicrobial peptides. After the antibacterial peptide acts on the cancer cells, the cell membrane can be damaged, the internal structure of the cells changes significantly, the mitochondria become vacuolized, the sputum falls off, the boundary of the nuclear membrane is blurred, and even the nuclear contents are leaked. Antimicrobial peptides can also rupture the nuclear DNA of cancer cells and inhibit the re-synthesis of DNA, thereby killing cancer cells [20]. On the other hand, the antibacterial peptide regulates the immune function of the motility body and acts against cancer cells from the aspect of humoral immunity.
4 Problems and prospects of insect antibacterial peptide research
The antibacterial peptide has the advantages of small molecular weight, stable physical and chemical properties, good water solubility and wide spectrum of sterilization. The introduction of insect antibacterial peptide gene into animals and plants and the cultivation of new disease-resistant varieties have become a very attractive research field, and have been successfully reported at home and abroad. It can be predicted that as new antibiotics and new drugs, antimicrobial peptides will play an important role in agriculture, food industry, industry, sanitary products and clinical medicine. However, insect antibacterial peptide molecules are small and easily degraded by proteases, affecting the high level expression of genes. In addition, the separation and purification of small molecular substances are difficult, and these are all problems that need to be solved in antimicrobial peptide genetic engineering. The relationship between the primary structure of the antimicrobial peptide and the spatial molecular structure and biological activity of the antibacterial peptide needs further study.
1 Types of insect antibacterial peptides
The antibacterial polypeptide produced by insects is an effector of insect immunity. Insect antibacterial peptides are cationic basic peptides, which can be roughly classified into the following four categories: the first type is cecropin, which contains 31 to 39 amino acid residues, generally does not contain cysteine, and is composed of Lepidoptera and Diptera. Insects are produced. The second category is the insect defensin, which contains 38 to 43 amino acid residues and is similar in structure to the defensins of animals and certain plants. Defensins are abundantly present in the hemolymph of insects. More than 30 kinds of defensins have been found in Diptera, Coleoptera, Hymenoptera, Hemiptera and Acarina, mainly killing Gram-positive bacteria. Recently, the antibacterial polypeptide drosomycin was isolated from Drosophila, and 38% of the amino acid sequence was similar to plant defensin, indicating that it has a certain homology with the evolution of plant polypeptides. The third type is an antibacterial peptide rich in proline and arginine with a molecular weight of 2 to 4 kD, such as the bee peptide apidaecin and abeecin (molecular weight of 2.0 kD and 4.0 kD, respectively) isolated from Italian honeybees. The peptide mainly inhibits Gram-negative bacteria. The fourth type is a glycine-rich protein with a molecular weight of 8 to 30 kD, such as sarcotoxin II (molecular weight of about 27 kD) isolated from the larvae of the larvae, isolated from the larvae of the green fly larvae and the larvae of the larvae The dipterin diptericin (molecular weight is about 9kD) and the coleopteran coleopterinin, etc., these large peptides are far apart, and belong to the superfamily members of attacin.
2 Antibacterial mechanism of insect antibacterial peptide
Insects can induce the rapid production of blood cells and fat bodies and release a large number of different components of antimicrobial peptides into the bloodstream, killing foreign bacteria. These antimicrobial peptides are synthesized similarly to immune response and mammalian acute phase reactions. The expression of these genes is regulated by the κB-associated pattern regulatory element, which is regulated by mammalian similarity to the NF κB-associated transactivator encoding the immune and contingency protein genes [3].
When the antimicrobial peptide gene is induced in vivo by bacterial lipopolysaccharides (LPS) or dead cells, the foreign stimuli signal activates the transcriptional activator NF κB, and the activated NF κB recognizes and binds to the antimicrobial peptide gene 5' A specific region upstream of B, thereby activating the expression of the antimicrobial peptide gene [4, 5]. The antibacterial peptide produced after expression has no stable conformation in aqueous solution, but forms a high-order structure only when it is bound or close to the cell membrane. Some scholars have put forward their own views: Merrifield et al [6] believe that when the antimicrobial peptide is in contact with the hydrophobic region of the cell membrane, the entire molecule (from the N-terminus to the C-terminus) induces an amphipathic helix, and the bacteriostatic process may first be The charged amino acid at the N-terminus of the antimicrobial peptide is electrostatically attracted to the polar group of the cell membrane, and forms an α-helix, which is inverted along the helical axis, embedded in the cell membrane, and disrupts the membrane structure. Fink et al [7] showed that the N-terminal (residues 1 to 11) of the antimicrobial peptide forms an amphipathic helix, the middle part (residues 12 to 24) forms an elastic corner, and the C-terminus forms a hydrophobic helix. When the antimicrobial peptide acts on the cell membrane, the C-terminal hydrophobic helix is ​​inserted into the membrane, and the N-terminus is bound to the membrane surface, and the middle is connected by elastic corners. Christensen et al. [8] proposed that antibacterial peptides are attracted to the surface of the membrane by electrostatic interaction, and then the hydrophobic tail is inserted into the hydrophobic region of the cell membrane. By changing the membrane conformation, multiple antimicrobial peptides polymerize to form ion channels on the membrane. Among the above four types of antibacterial polypeptides, the antibacterial mechanism of cecropin and defensin is studied in the most detailed manner.
2.1 Antibacterial mechanism of cecropin
Cecropin is a cationic molecule containing two alpha helix structures, which have dual affinity. The N-terminus is hydrophilic and contains a basic amino acid. The basic part containing the basic amino acid is affinity with the phospholipid of the bacterial cell membrane, and the hydrophobic part penetrates the bacterial cell membrane, so that the electromotive force of the bacterial cell membrane disappears, and the ATP content rapidly decreases, so that the bacterial cell membrane function is lost and the bacteria die. The amidation of the C-terminal amino acid is extremely important for the antibacterial activity of the antibacterial protein [9].
2.2 Antibacterial mechanism of defensins
Insect defensins have a killing effect on Gram-positive and negative bacteria. Relatively speaking, Gram-positive bacteria are more lethal, which may be due to the cell wall of Gram-negative bacteria that prevents defensin from contacting the cell membrane. The defensin molecule contains six cysteines, forming three disulfide bonds and having a stable molecular structure. This structure is identical to the structure of the defensin molecule isolated from the mammalian body. The bactericidal mechanism of defensin antibacterial peptides is also that defensin molecules act on bacterial cell membranes. The disulfide bond in the molecule has a strong affinity with the phospholipid of the bacterial cell membrane. After binding, an ion channel is formed, thereby changing the permeability of the cell membrane and killing the bacteria. The bactericidal active center of the defensin is α-helical structure [10]. The amidation of the C-terminal amino acid is also extremely important for maintaining its antibacterial activity. The study found that after the defensin adheres to the surface of the bacteria, in addition to its own damage to the bacteria, it can also cause the bacteria to be phagocytized and digested by the phagocytic cells, acting as an opsonin. Other studies have shown that hydrogen peroxide (H2O2) has a synergistic effect with defensins, and this synergistic effect increases with the increase of H2O2 concentration, making it easy for defensins to enter its target cell membrane or intracellular environment. In addition, studies have shown that the antibacterial mechanism of cecropin antibacterial peptides hinders the synthesis of bacterial outer membrane proteins, and the antibacterial mechanism of glycine-rich antimicrobial peptides hinders the synthesis of bacterial peptidoglycans.
3 insect antibacterial peptide application
3.1 transgenic peptide gene plant
Insect antibacterial peptides also exhibit extremely strong antibacterial activity against plant pathogenic bacteria. It is a hotspot of research at home and abroad to use genetic engineering methods to introduce the gene encoding the antibacterial peptide of eukaryote into the crop and express it to achieve the purpose of antibacterial. Jaymes [11] introduced the cecropin B gene into tobacco plants to produce tobacco plants resistant to tobacco bacterial wilt (Pseudomonassolanacearum). Beltan et al [12] transferred the gene of cecropin B analog SB 37 into tobacco and found that it can delay the onset of tobacco and reduce morbidity and mortality. It has also been reported that the transfer of the attacinE gene into apple trees can increase the resistance of apple trees to Erwinia amylovora [13]. Recent studies have shown that a gene encoding a stable cecropin analog MB39 is expressed in transgenic tobacco against tobacco wildfire (Pseudomonassyringaepv. tabaci) [14].
Transgenic tobacco against Tobaccomosaic virus (TMV) has also been developed in China. Jian Yuyu et al [15] transferred the cecropin B gene into rice and cultivated plants resistant to rice bacterial blight (Xanthomonasoryzaepv. oryzae). Jia Shirong et al [16] began to select the antibacterial peptide cecropin produced by insect cells in 1986 to carry out research on potato resistance to bacterial wilt genetic engineering, and reconstructed and constructed antibiotics such as cecropinB, shivaA and WHD which are more resistant to cecropin. The peptide gene was introduced into 7 main potato varieties in China, and 1050 transgenic lines were transformed.
3.2 transgenic peptide gene animals
The main means of controlling mosquito-borne diseases such as malaria and yellow fever are the development of highly effective drugs and vaccines, as well as the elimination of mediators (mosquitoes, etc.) and the cessation of transmission routes. In recent years, the antibacterial peptide gene that inhibits pathogenic organisms has been introduced into the mediator (mosquito) by genetic engineering methods, so that it is efficiently and stably expressed in insects, thereby cutting off the path of pathogens [17-19] .
The hybrid peptide shiva 3 constructed on the basis of cecropin B has a toxic effect on the development of Plasmodium perghei spores. A glutathione (GST) transferase gene is ligated in front of the shiva 3 gene to form a fusion gene Gst shiva 3 as a reporter gene, thereby obtaining a transgenic mosquito which can synthesize and secrete shiva 3 in the intestine, so it can be used for Control the spread of human malaria [18]. Recently, Vladimir et al. [19] used the Aedesaegypti vitel logeningene (vg) promoter to link a defensin A (DefA) gene and integrated vg DefA into the genome of Aedes aegypti. The GM mosquitoes, after taking blood, activate the biosynthesis of defensins and accumulate in the hemolymph, and their activity can be maintained for up to 22 days.
3.3 Anticancer effect of antimicrobial peptides
Antibacterial peptides can not only act on bacteria, viruses and other prokaryotes, but also play a role in tumor treatment. In recent years, domestic and foreign competitions have reported the anticancer effect of antimicrobial peptides. After the antibacterial peptide acts on the cancer cells, the cell membrane can be damaged, the internal structure of the cells changes significantly, the mitochondria become vacuolized, the sputum falls off, the boundary of the nuclear membrane is blurred, and even the nuclear contents are leaked. Antimicrobial peptides can also rupture the nuclear DNA of cancer cells and inhibit the re-synthesis of DNA, thereby killing cancer cells [20]. On the other hand, the antibacterial peptide regulates the immune function of the motility body and acts against cancer cells from the aspect of humoral immunity.
4 Problems and prospects of insect antibacterial peptide research
The antibacterial peptide has the advantages of small molecular weight, stable physical and chemical properties, good water solubility and wide spectrum of sterilization. The introduction of insect antibacterial peptide gene into animals and plants and the cultivation of new disease-resistant varieties have become a very attractive research field, and have been successfully reported at home and abroad. It can be predicted that as new antibiotics and new drugs, antimicrobial peptides will play an important role in agriculture, food industry, industry, sanitary products and clinical medicine. However, insect antibacterial peptide molecules are small and easily degraded by proteases, affecting the high level expression of genes. In addition, the separation and purification of small molecular substances are difficult, and these are all problems that need to be solved in antimicrobial peptide genetic engineering. The relationship between the primary structure of the antimicrobial peptide and the spatial molecular structure and biological activity of the antibacterial peptide needs further study.
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