CN115948377A - Vibrio parahaemolyticus phage lyase peptidase M15, gene thereof, preparation method and application - Google Patents
Vibrio parahaemolyticus phage lyase peptidase M15, gene thereof, preparation method and application Download PDFInfo
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Abstract
The invention provides a vibrio parahaemolyticus phage lyase peptidaseM15, the amino acid sequence of which is shown in SEQ ID No.3, and the nucleotide sequence of which is shown in SEQ ID No. 2. The invention also discloses a preparation method of the lyase peptidase M15, and the lyase peptidaseM15 expressed and purified by the escherichia coli prokaryotic expression system has the advantages of wide cracking host spectrum and high-efficiency antibacterial action, and has important application value in the aspect of antibiotic-resistant vibrio parahaemolyticus infection in the fields of aquatic products and public health.
Description
Technical Field
The invention belongs to the technical field of recombinant DNA biology, and particularly relates to vibrio parahaemolyticus phage lyase peptase M15, a gene thereof, a preparation method and application thereof.
Background
Vibrio parahaemolyticus is a gram-negative halophilic bacterium, is widely distributed in seawater, sludge, fish, shrimp, shellfish and other aquatic animals, can cause shrimp erythrozoosis, fish skin ulceration and other aquatic animal diseases, and is one of the main pathogenic bacteria in the aquaculture industry. Meanwhile, vibrio parahaemolyticus can also cause various human diseases including wound infection, septicemia or more commonly acute gastroenteritis. Because the problem of antibiotic resistance becomes more serious, most antibiotics gradually lose their effects, and at the same time, the development speed of novel antibiotics is relatively slow, and the development of an effective antibacterial drug from the viewpoint of biological safety is urgently needed.
Bacteriophages are a class of viruses that specifically infect bacteria, known as bacteriophages, discovered by Tport and Felix d' Herelle in 1917. The phage is hosted by bacteria and replicates and proliferates in the body of the bacteria. At the end of the infection of the bacteria, lytic bacteriophages release cell wall hydrolases, thereby releasing progeny phage particles, and causing lysis and death of the bacteria.
At present, the bacteriophage is applied to a plurality of fields of food, aquaculture, livestock and poultry, disease prevention and control and the like to control the infection of drug-resistant pathogenic bacteria. However, the application of the phage has a plurality of limitations, such as narrow phage lysis spectrum, and most of the phage can only specifically lyse one host bacterium; resistance is easy to generate, and the like, so overcoming the limitations becomes a problem to be solved for phage application. The cell wall hydrolase expressed by the phage is also called lyase, has the characteristics of high specificity, quick action, high efficiency, low drug resistance risk and the like, can hydrolyze bacterial pathogen peptidoglycan to kill bacteria, and is considered as a substitute therapeutic agent of antibiotics in the post-antibiotic age. However, some currently reported phage lytic enzymes such as Lysin1902 need to exert antibacterial action under the action of exogenous additives (Wang Yuxin, etc., phage lytic enzyme Lysin1902 prokaryotic expression and its effect evaluation in combination with epsilon-polylysine [ J/OL ]. Microbiological report), so, it is one of the important concerns in the field of antibiotic substitution to develop lytic enzymes with independent antibacterial activity without the assistance of exogenous additives.
Disclosure of Invention
The invention aims to provide a lyase gene derived from vibrio parahaemolyticus phage coding and an in vitro preparation method.
The technical method of the invention is as follows:
a vibrio parahaemolyticus phage lyase peptidase M15, wherein the amino acid sequence of the vibrio parahaemolyticus phage lyase M15 is shown as SEQ ID No. 3.
The gene for coding the vibrio parahaemolyticus phage lyase M15 has an original nucleotide sequence shown as SEQ ID No.1, is optimized through escherichia coli genetic code preference, and has an optimized nucleotide sequence shown as SEQ ID No.2, namely the coding gene of the vibrio parahaemolyticus lyase.
A preparation method of a vibrio parahaemolyticus phage lyase peptidase M15 is realized by the following scheme:
s1, carrying out escherichia coli genetic codon optimization on a nucleotide sequence shown as SEQ ID No.1 of a lyase coded by a parahaemolytic phage vB _ VpaM _ VPs20, wherein the optimized nucleotide sequence of the lyase is shown as SEQ ID No.2, and the optimized nucleotide sequence is a coding gene of the vibrio parahaemolyticus lyase;
s2, constructing a recombinant expression vector pCzn1-M15 by artificially synthesizing a target gene, and screening and identifying recombinant Escherichia coli Arctic Express-M15;
s3, optimizing the induced expression condition of recombinant Escherichia coli Arctic Express-M15, renaturing the generated inclusion body, and purifying by a Ni column to obtain a large amount of correctly folded recombinant protein, wherein the amino acid sequence of the correctly folded recombinant protein is shown as SEQ ID No. 3.
The NdeI and XbaI enzyme cutting sites of the recombinant expression vector are inserted with a nucleotide sequence shown in SEQ ID No. 2. The recombinant expression vector is a prokaryotic low-temperature (11-37 ℃) induced expression vector, and is specifically named as pCzn1-M15 plasmid.
Escherichia coli Arctic Express is used as a host bacterium, and the recombinant vector contains the nucleotide sequence shown in SEQ ID No. 2.
The preparation method of the vibrio parahaemolyticus phage lyase specifically comprises the following steps:
s1, constructing a recombinant expression vector pCzn 1-M15:
the original nucleotide sequence of the phage lyase is shown as SEQ ID No.1, the nucleotide sequence is optimized by referring to an escherichia coli genetic codon preference table, the optimized lyase has the nucleotide sequence shown as SEQ ID No.2, so that the lyase is more beneficial to expression in host bacteria, but the coded amino acid sequence is unchanged, then the lyase is cloned to NdeI and XbaI enzyme cutting sites of pCzn1 plasmid through artificial synthesis genes, and the constructed recombinant expression vector pCzn1-M15 plasmid is introduced into an escherichia coli T1 strain to serve as a clone strain to store the plasmid;
s2, constructing a recombinant escherichia coli Arctic Express-M15 expression strain:
extracting plasmids from an escherichia coli T1 strain with pCzn1-M15 plasmids by small extraction of the plasmids, converting the extracted plasmids into escherichia coli Arctic Express competent cells by a chemical conversion method according to 80-100ng/mL, screening positive strains by using an Amp resistant culture medium, and subculturing for two generations until the strains grow stably to obtain recombinant escherichia coli Arctic Express-M15;
s3, induced expression of recombinant M15 protein:
carrying out fermentation liquid culture on the recombinant Escherichia coli Arctic Express-M15 strain, adding IPTG (isopropyl-beta-D-thiogalactoside) into the strain with the OD600 of 0.6-0.8 until the final concentration is 0.2mM, shaking at 15 ℃ at 220r/min overnight, and inducing the expression of the fusion protein; the pellet was resuspended in 20mL of lysate (20 mM Tris-HCl containing 1mM PMSF and bacterial protease inhibitor cocktail, pH 8.0), sonicated and then treated with inclusion body wash to obtain renatured soluble protein, which was then affinity purified by Ni column.
The invention also provides application of the vibrio parahaemolyticus phage lyase peptidase M15 in vitro bacteriostasis.
The invention has the advantages that:
1. the phage lyase provided by the invention can perform a lytic action on gram-negative bacteria without being treated by an outer membrane permeabilizing agent, and has high application potential.
2. The phage lyase has strong lytic effect on bacteria, and can reduce 6-7 orders of magnitude of bacteria within 1h. Compared with the bacteriophage, the antibacterial agent has quick action effect and no generation of resistant bacteria.
3. The phage lyase has wider host spectrum than phage encoding genes, and can cleave multiple gram-negative bacteria including vibrio parahaemolyticus, vibrio anguillarum and the like.
Drawings
FIG. 1 is a protein electrophoresis diagram showing the expression of peptase M15, wherein lane M is protein marker, lane 1 is pCzn1 (empty vector) induced, lane 2 is not induced, lane 3 is induced whole mycoprotein, 4 is induced and crushed supernatant, and lane 5 is induced and crushed precipitate;
FIG. 2 is an electrophoretogram of the purified protein, lane M is a protein marker, lane 1 is a treated sample after disruption, lane 2 is an effluent, and lanes 3-4 are eluents;
FIG. 3 shows the results of western blot assay of example 2;
FIG. 4 shows the lytic activity of the lyase on Vibrio parahaemolyticus.
FIG. 5 is a diagram of the bacteriostatic effect of lyase.
FIG. 6 results of bacterial counts after lyase treatment.
FIG. 7 cleavage activity of lyase on Vibrio anguillarum.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1
A method for expressing a phage lyase peptidase M15 (preparation method), comprising the steps of:
obtaining of S1 phage lyase peptidase M15 coding gene:
the phage lyase M15 gene is from a lytic phage vB _ VpaM _ VPs20 aiming at drug-resistant vibrio parahaemolyticus, the whole genome of the phage vB _ VpaM _ VPs is sequenced, the result is uploaded to an ncbi database with the accession number of OP056089, the original coding nucleotide sequence of the phage lyase M15 is obtained by blast comparison, as shown in SEQ ID No.1, the optimization is carried out through the genetic codon preference of escherichia coli, the nucleotide sequence of the optimized lyase is shown in SEQ ID No.2, so that the phage lyase M15 is more beneficial to the expression in host bacteria, but the coding amino acid sequence is not changed, then the phage lyase M15 gene is added to NdeI and XbaI enzyme cutting sites of a pCzn1 plasmid according to an artificial gene synthesis mode to obtain a recombinant pCzn1-M15 plasmid, and the recombinant pCzn1-M15 plasmid is introduced into escherichia coli T1 competent cells, and the specific method is as follows:
(1) Plasmid 1. Mu.L was added to 100. Mu.L of competent bacteria and placed on ice for 20min.
(2) The mixture was heat-shocked at 42 ℃ for 90sec, rapidly placed in ice for 5min, and 600. Mu.LLB medium was added.
(3) Shaking at 37 deg.C for 1h at 220r/min, centrifuging, spreading on LB plate containing 50 μ g/ml of pump, and culturing at 37 deg.C for overnight.
Selecting successfully transformed monoclonal strains (the colonies growing on the plate are the required successfully transformed monoclonal strains), extracting plasmids after amplification culture according to the instructions on the plasmid extraction kit, and transforming the plasmids to escherichia coli Arctic Express, wherein the specific method comprises the following steps:
(1) 1. Mu.L of pCzn1-M15 plasmid was added to 100. Mu.L of competent bacteria and placed on ice for 20min.
(2) The mixture was heat-shocked at 42 ℃ for 90sec, rapidly placed in ice for 5min, and 600. Mu.LLB medium was added.
(3) Shaking at 37 deg.C for 1h at 220r/min, centrifuging, coating on LB plate containing 50 μ g/mLAmp, and culturing at 37 deg.C for overnight to obtain recombinant Escherichia coli Arctic Express-M15.
S2 IPTG induced expression of pCzn1-M15 carrier fusion protein
(1) The monoclonal strains on the transformation plates were picked and inoculated into tubes containing 50. Mu.g/mLAmp 3mLLB medium and shaken overnight at 220r/min at 37 ℃.
(2) The following day, the cells were inoculated into 30mL of LB medium at 50. Mu.g/mLAmp in an amount of 1.
(3) 1mL of the culture was removed, centrifuged at 10000r/min at room temperature for 2min, the supernatant was discarded, and the pellet was resuspended in 100. Mu.L of 1 Xloading buffer.
(4) IPTG was added to the remaining culture to a final concentration of 0.2mM, and shaking was carried out overnight at 15 ℃ at 220r/min to induce expression of the fusion protein.
(5) 1mL of the culture was removed, centrifuged at 10000r/min at room temperature for 2min, the supernatant was discarded, and the pellet was resuspended in 100. Mu.L of 1 Xloading buffer. Centrifuging the rest culture at 4000r/min for 10min, discarding the supernatant, and resuspending the thallus precipitate with PBS; after the resuspension liquid is subjected to ultrasonic crushing, supernatant and precipitation liquid are respectively taken and added into a sample loading buffer solution for resuspension.
(6) 12% SDS-PAGE detection analysis was performed and the bands were visualized by Coomassie brilliant blue staining, see FIG. 1.
S3 renaturation of inclusion body protein, the concrete operation steps are as follows:
(1) The pellet was resuspended in 20mL of lysate (20 mM Tris-HCl containment 1mM PMSFNDBACTERIAPROTEASE inhibitor cocktail, pH 8.0) and sonicated (power 400W, work 4sec, pause 8sec, total 20 min).
(2) Centrifuging the cell lysate subjected to ultrasonic disruption at 4 ℃ and 10000r/min for 20min, and collecting the precipitate.
(3) The inclusion bodies were washed 3 times with an inclusion body wash (20mM Tris,1mM EDTA,2M urea, 1M NaCl,1% Triton X-100, pH 8.0).
(4) Dissolving inclusion body with dissolving buffer solution (20mM Tris,5mM DTT,8M urea, pH 8.0) according to a certain proportion, and standing overnight at 4 ℃; centrifuging at room temperature at 10000r/min for 15min.
(5) The solution was added dropwise to 20mM Tris-HCl,0.15M NaCl, pH 8.0 buffer solution, gradually diluted in a stepwise gradient and slowly stirred, and the protein solution was placed in a dialysis bag and dialyzed overnight in 20mM Tris-HCl,0.15M NaCl, pH 8.0 solution.
Ni column affinity purification of S4 fusion protein
(1) The supernatant solution was loaded onto a Ni-IDA Binding-Buffer pre-equilibrated Ni-IDA-Sepharose Cl-6B affinity column using a low pressure chromatography system at a flow rate of 0.5 mL/min.
(2) Washed with Ni-IDABinding-Buffer at a flow rate of 0.5mL/min until the effluent OD280 reached baseline.
(3) The column was flushed with Ni-IDAWashing-Buffer (20 mM Tris-HCl,20mM imidazole, 0.15M NaCl, pH 8.0) at a flow rate of 1mL/min until the effluent OD280 reached baseline.
(4) The target protein was eluted with Ni-IDAElution-Buffer (20 mM Tris-HCl,250mM imidazole, 0.15M NaCl, pH 8.0) at a flow rate of 1mL/min, and the effluent was collected.
(5) The protein solution collected above was added to a dialysis bag and dialyzed overnight using PBS. The amino acid sequence is shown in SEQ ID No. 3.
(6) 12% SDS-PAGE analysis was performed, see FIG. 2.
Example 2Western Blot to identify the immunogenicity of recombinant proteins, based on the 6His tag carried by the N-terminus of the recombinant protein.
Dilution ratio of primary antibody and secondary antibody
The specific operation steps are as follows:
(1) Samples were loaded at 3 μ L.
(2) After the sample loading is finished, the polyacrylamide gel runs out of laminated gel at 90V, and then the voltage is increased to 200V until the electrophoresis is finished.
(3) After electrophoresis is finished, the gel is taken down and membrane conversion is carried out, the membrane is converted at constant voltage of 100V for about 1.5h, and the constant current is 250mA.
(4) After the electrotransfer was complete, the membrane was removed and washed with PBST 4 times for 5min each.
(5) The membrane was placed in a 5% nonfat dry milk blocking solution and blocked at 37 ℃ for 1h.
(6) Primary antibody was diluted with blocking solution and membranes were incubated overnight at 4 ℃ in primary antibody dilution.
(7) The following day the membranes were removed and washed with PBST for 5min 4 times.
(8) The secondary antibody was diluted with blocking solution containing 5% milk and the membrane was reacted in the secondary antibody at 37 ℃ for 1h.
(9) After the reaction, the membrane was taken out and placed in a clean box to wash the membrane for 4 times, 5min each time.
(10) And ECL developing and exposing.
The Western Blot results are shown in FIG. 3. The results show that the expressed recombinant protein size of about 15.63kDa corresponds to the calculated theoretical value.
Example 3 validation of lyase Activity by the Spot method and measurement of bacteriostatic Activity
The specific operation steps are as follows: 1mL of Vibrio parahaemolyticus cultured to logarithmic phase is uniformly coated on a 2216E solid plate, the Vibrio parahaemolyticus is naturally dried at room temperature, then 5 mu LPBS and 5 mu Lpeptidase M15 lyase are dropwise added on the dried plate, the plate is placed in a constant temperature incubator at 28 ℃ for 10 minutes until liquid drops are dried, the plate is placed in the incubator upside down for 10 hours for culture, and the result is observed, wherein the result is shown in figure 4, no plaque appears in a PBS control group, and clear and transparent plaque appears in a peptidase M15 lyase group.
In order to further explore the bacteriostatic effect of the lyase, the bacteriostatic activity of the lyase is determined, and the specific operation is as follows: the host bacteria vibrio parahaemolyticus is divided into two groups, one group is an EDTA untreated group, the other group is an EDTA pretreated group, the EDTA untreated group is a bacterial liquid cultured to a logarithmic phase, 2mL of the bacterial liquid 6000x g is taken for centrifugation for 5min, the supernatant is discarded, washed twice by PBS and resuspended in 2mL of LPSB.
The EDTA pretreatment group was performed by taking 2mL of the cell suspension 6000x g, centrifuging for 5min, discarding the supernatant, washing twice with PBS, resuspending in PBS containing 0.5mM EDTA, washing twice with PBS after 15min in 37 ℃ water bath and centrifuging 6000x g for 5min, and resuspending in 2mL PBS.
Taking two parts of the 450 mu LEDTA untreated bacteria liquid and the EDTA pretreated bacteria liquid, respectively adding 50 mu LPBS and 50 mu LPeptidase M15 lyase solutions, carrying out viable bacteria counting on the mixed bacteria liquid after 60min of water bath at 28 ℃, and repeating the experiment for 3 times. The results are shown in FIG. 5, which shows that the PBS control bacteria had no significant change, while the peptdase M15 lyase solution treatment bacteria became clear. The results of counting viable bacteria on the bacterial liquid by a flat plate are shown in FIG. 6, and the viable bacteria number of the lyase treatment group is reduced by 6-7 orders of magnitude after 1h of action.
Example 4 lyase host Spectroscopy
The cleavage activity of the cleavage enzyme with respect to Vibrio anguillarum was determined by the dot-drop method, which was carried out as described in example 3. As shown in FIG. 7, the peptidase M15 lyase also exhibited a cleavage effect on Vibrio anguillarum.
The experimental results show that the lyase recombinant protein peptidase M15 expressed by the invention has stronger lytic capacity to host bacteria, can have better inhibition effect on vibrio parahaemolyticus without the treatment of EDTA outer membrane permeabilizing agent, has rapid action effect, and has greatly improved lytic time and capacity compared with bacteriophage. The phage lyase has wider host spectrum than phage encoding the gene thereof, and can cleave various gram-negative bacteria including Vibrio parahaemolyticus, vibrio anguillarum and the like.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (7)
1. A Vibrio parahaemolyticus phage lyase peptidaseM15, wherein the amino acid sequence of the Vibrio parahaemolyticus phage lyase peptidaseM15 is shown in SEQ ID No. 3.
2. A gene encoding the phage lytic enzyme peptidaseM15 of vibrio parahaemolyticus of claim 1, wherein: the original nucleotide sequence is shown as SEQ ID No.1, the escherichia coli genetic code preference is optimized, and the optimized nucleotide sequence for coding the lyase is shown as SEQ ID No.2, namely the coding gene of the vibrio parahaemolyticus lyase.
3. A process for preparing the bacteriophage lytic enzyme peptidaseM15 of claim 1, wherein: the method comprises the following steps:
s1, carrying out Escherichia coli genetic codon preference optimization on a lyase coded by a parahemolytic phage vB _ VpaM _ VPs20 and shown in SEQ ID No.1, wherein the nucleotide sequence of the optimized lyase is shown in SEQ ID No.2, and the optimized lyase is a coding gene of the vibrio parahemolyticus lyase;
s2, constructing a recombinant expression vector pCzn1-M15 by artificially synthesizing a target gene, and screening and identifying recombinant Escherichia coli Arcticexpress-M15;
s3, optimizing the induced expression condition of the recombinant Escherichia coli ArcticExpress-m15, renaturing the generated inclusion body, and purifying by a Ni column to obtain a large amount of correctly folded recombinant protein, wherein the amino acid sequence of the correctly folded recombinant protein is shown in SEQ ID No. 3.
4. The method for producing phage lyase peptidaseM15 for Vibrio parahaemolyticus according to claim 3, wherein the method comprises the steps of: the NdeI and XbaI enzyme cutting sites of the recombinant expression vector are inserted with a nucleotide sequence shown in SEQ ID No. 2; the recombinant expression vector is a prokaryotic low-temperature induction expression vector and is specifically named as pCzn1-M15 plasmid.
5. The method for producing phage lyase peptidaseM15 for Vibrio parahaemolyticus according to claim 3, wherein the method comprises the steps of: the Escherichia coli ArcticExpress is used as a host bacterium and contains a recombinant vector of the nucleotide sequence shown in SEQ ID No. 2.
6. The method for producing phage lyase peptidaseM15 for Vibrio parahaemolyticus according to claim 3, wherein the method comprises the steps of: the method specifically comprises the following steps:
s1, constructing a recombinant expression vector pCzn 1-M15:
the original nucleotide sequence of the phage lyase is shown as SEQ ID No.1, the nucleotide sequence is optimized by referring to an escherichia coli genetic codon preference table, the optimized lyase has the nucleotide sequence shown as SEQ ID No.2, so that the lyase is more beneficial to expression in host bacteria, but the coded amino acid sequence is unchanged, then the lyase is cloned to NdeI and XbaI enzyme cutting sites of pCzn1 plasmid through artificial synthesis genes, and the constructed recombinant expression vector pCzn1-M15 plasmid is introduced into an escherichia coli T1 strain to serve as a clone strain to store the plasmid;
s2, constructing a recombinant escherichia coli Arcticexpress-M15 expression strain:
extracting plasmids from an escherichia coli T1 strain with pCzn1-M15 plasmids, converting 1 mu L of plasmids with the concentration of 80-100ng/mL into an escherichia coli ArcticExpress competent cell by a chemical conversion method, screening positive strains by using an Amp resistance-containing culture medium, and subculturing for two generations until the strains grow stably to obtain recombinant escherichia coli ArcticExpress-M15;
s3, induced expression of recombinant M15 protein:
performing fermentation liquid culture on recombinant Escherichia coli Arcticexpress-M15 strain, and culturing in thallus OD 600 When the concentration is 0.6-0.8, IPTG is added to the final concentration of 0.2mM, and the mixture is shaken overnight at the temperature of 15 ℃ and the speed of 220r/min to induce the expression of the fusion protein; and (3) resuspending the thallus precipitate in 20mL of lysate, carrying out ultrasonic crushing, treating with an inclusion body washing solution to obtain renatured soluble protein, and then carrying out affinity purification on the renatured soluble protein through a Ni column.
7. Use of the bacteriophage lytic enzyme peptidaseM15 of claim 1 for lysing Vibrio parahaemolyticus.
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| CN116732006A (en) * | 2023-08-09 | 2023-09-12 | 山东省农业科学院畜牧兽医研究所 | Vibrio parahaemolyticus phage depolymerase and application thereof |
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