CN115057945A

CN115057945A - Hybrid antibacterial peptide NK-LPd, gene, vector, preparation method and application thereof

Info

Publication number: CN115057945A
Application number: CN202210751172.7A
Authority: CN
Inventors: 姚波; 刘子琦; 刘正宇; 朱蠡庆; 柴水琴; 郭银虹
Original assignee: Chongqing University of Science and Technology
Current assignee: Chongqing University of Science and Technology
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2022-09-16
Anticipated expiration: 2042-06-29
Also published as: CN115057945B

Abstract

The invention discloses a hybrid antibacterial peptide NK-LPd, a gene, a vector, a preparation method and application thereof.A glycine joint GG is adopted to be connected in series with two Piscidin antibacterial peptide repetitive sequences shown in SEQ ID No.4 and an NK-lysin antibacterial peptide active fragment shown in SEQ ID No.3 to obtain an amino acid sequence of the hybrid peptide NK-LPd, and the amino acid sequence of the hybrid peptide NK-LPd is shown in SEQ ID No. 5. The optimized codon of Pichia pastoris is selected to design and synthesize the heterozygous peptide NK-LPd gene, so that the expression efficiency in Pichia pastoris is improved, the heterozygous peptide NK-LPd obtained by yeast expression and purification has strong antibacterial activity and high selectivity, and can be massively prepared and used for preparing antibacterial drugs, feed additives or immunopotentiators.

Description

Hybrid antibacterial peptide NK-LPd, gene, vector, preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a hybrid antibacterial peptide NK-LPd, a gene and a vector thereof, and a preparation method and application of the hybrid antibacterial peptide NK-LPd.

Background

The widespread and indiscriminate use of conventional antibiotics has led to the emergence and spread of multiple drug-resistant bacteria, including pathogens resistant to antibiotics. Antimicrobial peptides (AMPs) target multiple pathogens at low concentrations as one of the drug candidates, with little evidence of resistance to date. However, the natural antibacterial peptide has the defects of cytotoxicity, immunogenicity, loss of in vivo activity and the like, and the defects are overcome, so that the development and utilization of the antibacterial peptide are facilitated.

NK-lysin is an antibacterial peptide produced from cytotoxic T cells and NK cells, contains 120-168 amino acid residues, and is present in intracellular toxic granules. NK-lysin belongs to the saporin-like protein (SAPLIP) family of lipid binding proteins, members of which all have six conserved cysteines and a Saposin B domain. Many studies have now confirmed that short peptides of NK-lysin have antibacterial effects. For example, NKLP27 is a 27-residue truncated peptide of cynoglossus semilaevis, having lytic activity against gram-negative and gram-positive bacteria, binds to Lipopolysaccharide (LPS) of several bacteria by reacting with the lipid moiety of LPS, enters the cytoplasm of target cells, and positively charged residues electrostatically interact with negatively charged DNA, resulting in DNA degradation and modulation of the innate immune response of the host fish. However, NK-lysin has a long amino acid sequence compared with most of antibacterial peptides, and the chemical synthesis method has certain difficulty.

Piscidin is a linear cationic alpha-helical peptide with a broad spectrum of activity. The first cationic AMP from Piscidin was isolated from hybrid striped bass and had antibacterial, antifungal and antiviral properties. Studies have shown that Piscidin is sensitive to fish and human pathogens as well as to multi-drug resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, etc.). Piscidin disrupts the bacterial cell membrane by a loop pore mechanism. The alpha helix of the peptide is inserted between the lipids of the membrane. Piscidin forms a ring-shaped pore on the membrane when it interacts with acidic phospholipids, permeabilizing the plasma membrane of the pathogen. Most of the antibiotic peptides of the Piscidin family are small peptides consisting of 20-26 amino acids, the molecular weight is small, the degradation is easy, the expression yield is low, and the major bottleneck of the production and application of the antibiotic peptides is formed.

A number of different mechanisms of action of antimicrobial peptides have been disclosed, such as membrane pore formation, inhibition of DNA replication, or modulation of other immune responses. When different AMPs are present together, they may exhibit additive, potentiating or synergistic effects depending on the mechanism of action involved, and AMPs may also exhibit synergistic effects with conventional antibiotics. Because different drug targets require multiple adaptations at the same time and the effective dose of each compound is low, the probability of the pathogen developing drug resistance is reduced. However, the co-administration of multiple AMPs requires the synthesis and formulation of different products, which increases the cost involved and the likelihood of treatment failure. Hybrid antimicrobial peptides can effectively overcome this disadvantage. Therefore, the development of NK-lysin and Piscidin hybrid antibacterial peptides with enhanced antibacterial activity and high selectivity is urgently needed.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a hybrid antimicrobial peptide NK-LPd; the second object of the present invention is to provide a gene encoding the hybrid antimicrobial peptide NK-LPd; the third purpose of the invention is to provide a recombinant expression vector containing the hybrid antibacterial peptide NK-LPd gene; the fourth object of the present invention is to provide a host cell containing the NK-LPd gene or the recombinant expression vector; the fifth object of the present invention is to provide a method for preparing the hybrid antimicrobial peptide NK-LPd; the sixth purpose of the invention is to provide the application of the hybrid antibacterial peptide NK-LPd in the preparation of antibacterial drugs, feed additives or immunopotentiators.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a hybrid antibacterial peptide NK-LPd is characterized in that a glycine linker GG is adopted to be connected in series with two Piscidin antibacterial peptide repetitive sequences shown in SEQ ID No.4 and an NK-lysin antibacterial peptide active fragment shown in SEQ ID No.3 to obtain a hybrid peptide NK-LPd sequence, and the amino acid sequence of the hybrid peptide NK-LPd is shown in SEQ ID No. 5.

2. A gene encoding said hybrid antimicrobial peptide NK-LPd.

Preferably, the gene of the hybrid antibacterial peptide NK-LPd is an optimized gene suitable for yeast expression, and the nucleotide sequence of the gene is shown in SEQ ID NO. 6.

3. A recombinant expression vector containing the gene of the hybrid antibacterial peptide NK-LPd.

Preferably, the recombinant expression vector is obtained by connecting a sequence shown in SEQ ID NO.6 to EcoR I and Not I enzyme cutting sites of a plasmid pPIC 9K.

4. A host cell containing said gene or said recombinant expression vector.

Preferably, the host cell is a pichia pastoris cell.

5. A method for preparing the hybrid antibacterial peptide NK-LPd, which comprises transforming a yeast host cell with the recombinant expression vector of claim 5, screening positive yeast transformants, inducing the expression of the target peptide, and isolating and purifying the expression product.

6. The application of the hybrid antibacterial peptide NK-LPd in preparing antibacterial drugs, feed additives or immunopotentiators.

Preferably, the antibacterial drug has good antibacterial effect on staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, salmonella and aeromonas hydrophila.

The invention has the beneficial effects that:

1. the Pichia pastoris preferential codon is selected, the hybrid peptide NK-LPd gene is designed and synthesized, and the expression efficiency in the Pichia pastoris is improved, so that the use cost of the hybrid peptide is reduced, and the application in actual production is expanded.

2. By adopting a tandem repeat expression mode, the copy number of the Piscidin is increased, the structure of the hybrid antibacterial peptide is more balanced, and the length of an effective antibacterial fragment is increased.

3. The active fragments of the NK-lysin and the Piscidin are connected in series by adopting the joint GG of glycine, so that steric hindrance is avoided, and the hybrid antibacterial peptide can form a stable secondary structure more easily and is beneficial to exerting the function.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows the result of comparison of NK-lysin sequences;

FIG. 2 shows the alignment results of Piscidin sequences;

FIG. 3 shows the electrophoresis results of the first PCR product of SOE-PCR (1: extension solution 1; 2: extension solution 2; 3: DL500 DNA Marker);

FIG. 4 shows the electrophoresis results of the second PCR products of SOE-PCR (1: DL500 DNA Marker; 2: NK-LPd);

FIG. 5 is a PCR analysis of the recombinant strain KM71 (1: DL 2000 DNA Marker; 2: KM71/pPIC 9K-NK-LPd);

FIG. 6 shows the results of electrophoresis analysis of Tricine-SDS-PAGE of induced expression supernatant (M: protein molecular weight standard; 1: KM71 strain; 2: KM71-pPIC 9K; 3: 1.0mg/mL G418 screening; 4: 2.0mg/mL G418 screening; 5-7: 3.0mg/mL G418 screening for 4, 5, 6 days);

FIG. 7 shows the experimental results of the inhibition zone of hybrid peptide NK-LPd.

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Example 1 hybrid peptide design

(1) NK-lysin design

Extracting total RNA in the liver tissue of the prussian carp, carrying out reverse transcription to obtain a cDNA sequence as a template for carrying out gene cloning to obtain a prussian carp NK-lysin gene sequence:

the amino acid sequence is obtained by translation:

the sequence is aligned with the amino acid sequence of NKLP27, the result of the alignment of the NK-lysin sequence is shown in figure 1, and homologous parts are obtained: IKIKLGMICDEIGFLKSMCRNLVN (SEQ ID NO. 3).

(2) Piscidin design

Amino acid sequences of the Piscidin family are collected from the NCBI database for sequence alignment, and the result of the sequence alignment of the Piscidin is shown in FIG. 2, so that conserved sequences are obtained: HHIFRGIVH (SEQ ID NO. 4).

(3) Hybrid peptide NK-LPd sequence design

And (3) connecting two Piscidin repeated sequences and an NK-lysin active fragment in series by using a glycine linker (GG) to obtain a hybrid peptide NK-LPd sequence: (HHIFRGIVH)2GGIKIKLGMICDEIGFLKSMCRNLVN (SEQ ID NO. 5).

Example 2 Synthesis of hybrid peptide NK-LPd Gene

(1) Hybrid peptide NK-LPd Gene sequence design

In order to make the expressed antibacterial peptide have a natural N terminal, a KEX2 protease cleavage site is added at the N terminal of the hybrid peptide sequence, and a 6 XHis tag is added at the C terminal for subsequent purification. The 5 'end of the hybrid antibacterial peptide nucleotide sequence is added with an EcoRI enzyme cutting site GAATTC, and the 3' end is added with a stop codon (TAA) and a Not I enzyme cutting site GCGGCCGC. Obtaining an optimized nucleotide sequence by referring to pichia pastoris preference codon translation:

(2) SOE-PCR Synthesis

According to the nucleotide sequence of the hybrid antibacterial peptide, four primers are designed:

overlap extension polymerase chain reaction (SOE-PCR) was performed using primers NL 1, NL 2 and NL 3, NL 4 as templates, respectively. And (3) PCR reaction conditions: denaturation at 94 deg.C for 5min, annealing at 55 deg.C for 5min, extension at 72 deg.C for 5min, circulation for 1 time, and storage at 4 deg.C. Product extension 1 (a fragment synthesized with NL 1 and NL 2 as primers) and extension 2 (a fragment synthesized with NL 3 and NL 4 as primers) were collected as templates for the second round of PCR. The electrophoresis result of the first round of PCR product of SOE-PCR is shown in FIG. 3, the size of the product fragment in the extension solution 1 is 95bp, and the size of the product fragment in the extension solution 2 is 102 bp. ddH was added first during the second PCR ₂ O, 10 XPCR Buffer, 2.5mM dNTPs, 3 cycles of reaction, Pfu enzyme and primers NL 1 and NL 4. Reaction conditions are as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, and amplification cycle for 25 times; final extension at 72 deg.C for 5 min; storing at 4 ℃. The electrophoresis result of the second PCR product of SOE-PCR is shown in FIG. 4, and the size of the target fragment is 179 bp.

Example 3 construction of Pichia expression vector

The obtained PCR product and the plasmid pPIC9K are subjected to double enzyme digestion by restriction enzymes EcoR I and Not I respectively, then T4 DNA ligase is used for splicing to obtain a recombinant plasmid pPIC9K-NK-LPd, an escherichia coli competent cell DH5 alpha is transformed by the recombinant plasmid, and a positive clone is obtained by screening an LB plate (containing Amp). 10 colonies are picked for colony PCR verification, and positive clones are sent to a sequencing company for recombinant plasmid sequencing identification. And (3) overnight culturing by using an ampicillin-containing LB culture solution, extracting a plasmid, carrying out enzyme digestion linearization by Sac I, and then transforming to pichia pastoris KM 71. After screening with an MD plate not containing histidine, 1mL of sterile water was added to resuspend the transformants, and the bacterial solution was spread on YPD plates with G418 concentrations of 1.0, 2.0 and 3.0mg/mL in this order to screen high copy yeast transformants. The positive recombinant strain KM71 was obtained by PCR detection using 5' -AOX of the vector and the gene specific primer NL-4, and the detection results are shown in FIG. 5. The transformants were resuspended, and the bacterial suspension was plated in sequence on YPD plates with G418 concentrations of 1.0, 2.0, and 3.0mg/mL to screen for high copy yeast transformants.

Example 4 Induction of expression by recombinant Yeast

The selected high copy yeast transformants were inoculated into YPD medium and cultured overnight. Inoculating 1mL of the bacterial solution into 100mL of BMGY medium, and performing shaking culture until OD ₆₀₀ Centrifuging the strain 2-6, collecting the strain, discarding the supernatant, washing the strain precipitate with sterile water, centrifuging the precipitate, and discarding the supernatant. Resuspending the thallus precipitate with 20mL BMMY culture solution, transferring into a conical flask for shaking culture, performing induced expression with 100% methanol, adding methanol every 24h until the final concentration is still 0.5%, and sampling 1mL respectively when performing induced expression for 0h, 24h, 48h, 72h, 96h and 120 h.

Sampling 1mL, centrifuging to obtain 900 μ L of supernatant, adding 140 μ L of 100% TCA solution, and standing overnight at 4 ℃; centrifuging, removing supernatant, washing with 1mL of precooled acetone, centrifuging again, drying the precipitate, and adding 40 μ L of 50mM NaOH solution to dissolve. Add 40. mu.L of 2 XSDS Loading Buffer and cook at 100 ℃ for 10 minutes. The empty vector without the target gene is used as a control, the same treatment is carried out according to the preparation method of the sample, and Tricine-SDS-PAGE is carried out by 15 percent of separation gel and 4 percent of concentrated gel to detect the expression condition of the target protein.

The electrophoresis process is as follows: adding 10 μ L of the treated sample into each gel hole, setting the voltage of the concentrated gel at 80V, stopping electrophoresis after 20min to the upper layer of the separation gel and 150V, stopping electrophoresis when the bromophenol blue reaches the bottom layer of the separation gel, taking down the gel, and staining with Coomassie brilliant blue-R250, wherein the detection result is shown in FIG. 6.

Example 5 purification of recombinant polypeptide and detection of bacteriostatic Activity

(I) Ni ²⁺ Affinity chromatography purification

Mixing the supernatant with Binding Buffer (20mM imidazole) with the same volume, loading the mixture on a column, washing the column by using Binding Buffer with 15 times of column volume, washing off the hybrid protein, washing the column by using Eluent Buffer (25mM imidazole) until the absorbance value of the collected Eluent at 280nm is close to the baseline level, collecting the Eluent, and desalting the purified protein by dialysis. The concentration of purified recombinant peptide was determined to be 98.5. mu.g/mL by BCA method.

(II) detection of antibacterial Activity

And (2) streaking and inoculating the frozen and preserved escherichia coli, staphylococcus aureus, salmonella, pseudomonas aeruginosa and aeromonas hydrophila separated and identified by the laboratory into a plate culture medium, culturing for 12 hours at 37 ℃, selecting a single colony, inoculating into 10mL of LB liquid culture medium, performing shake culture at 37 ℃ and 200r/min overnight, and keeping the bacterial liquid for later use. Diluting to 1X 10 with liquid culture medium ⁶ And (3) CFU/mL for standby, injecting a certain amount of bacterial liquid into LB solid culture medium cooled to about 50 ℃, uniformly mixing, pouring a flat plate (about 30 mL/flat plate), horizontally standing and solidifying for standby. A6 mm hole was punched with a punch and a mark was made behind the plate next to the corresponding hole. Mu.l of recombinant hybrid peptide NK-LPd (98.5. mu.g/ml), NK-lysin (100. mu.g/ml), Pisicidn (100. mu.g/ml) and ampicillin (10mg/ml) were added to the wells as positive controls. Placing the flat plate in a refrigerator at 4 ℃ for pre-diffusion for 2h, then placing the flat plate upside down in a constant temperature incubator at 37 ℃ for culture until a bacteriostatic circle (12-18 h) is generated, taking out the cultured test flat plate after the culture is finished, measuring the diameter of the bacteriostatic circle by using a vernier caliper, and expressing the size of the bacteriostatic circle by using the diameter, wherein the detection result is shown in figure 7. The results show that the hybrid peptide NK-LPd shows good bacteriostatic activity on Escherichia coli (2.402cm), Staphylococcus aureus (2.58cm), Salmonella (1.992cm), Pseudomonas aeruginosa (2.14cm) and Aeromonas hydrophila (2.648 cm). Compared with the parent peptide, the antibacterial activity of the hybrid peptide NK-LPd on pseudomonas aeruginosa is between NK-lysin (2.202cm) and Piscidin (1.814cm), but the antibacterial activity on escherichia coli, staphylococcus aureus, salmonella and aeromonas hydrophila is enhanced, and particularly the antibacterial activity of the hybrid peptide on the aeromonas hydrophila is twice that of the Piscidin.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or the change made by the technical personnel in the technical field on the basis of the invention are all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

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Claims

1. A hybrid antimicrobial peptide NK-LPd, characterized in that: glycine linker GG is adopted to be connected in series with two Piscidin antibacterial peptide repetitive sequences shown in SEQ ID No.4 and an NK-lysin antibacterial peptide active fragment shown in SEQ ID No.3 to obtain a hybrid peptide NK-LPd sequence, and the amino acid sequence of the hybrid peptide NK-LPd is shown in SEQ ID No. 5.

2. A gene encoding the hybrid antimicrobial peptide NK-LPd of claim 1.

3. The hybrid antibacterial peptide NK-LPd gene according to claim 2, wherein the hybrid antibacterial peptide NK-LPd gene is an optimized gene suitable for yeast expression, and the nucleotide sequence of the gene is shown in SEQ ID No. 6.

4. A recombinant expression vector comprising the gene of the hybrid antimicrobial peptide NK-LPd according to claim 2 or 3.

5. The recombinant expression vector according to claim 4, wherein the vector is obtained by connecting the sequence shown in SEQ ID No.6 to EcoR I and Not I cleavage sites of plasmid pPIC 9K.

6. A host cell comprising the gene of claim 2 or 3 or the recombinant expression vector of claim 4 or 5.

7. The host cell of claim 6, wherein the host cell is a Pichia pastoris cell.

8. A method for preparing the hybrid antimicrobial peptide NK-LPd according to claim 1, which comprises transforming a yeast host cell with the recombinant expression vector of claim 5, selecting a positive yeast transformant, inducing the expression of the target peptide, and isolating and purifying the expression product.

9. Use of the hybrid antimicrobial peptide NK-LPd of claim 1 for the preparation of an antimicrobial drug, a feed additive or an immunopotentiator.

10. The use according to claim 9, wherein said hybrid antimicrobial peptide has a good antimicrobial effect against staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, salmonella, aeromonas hydrophila.