Clostridium perfringens bacteriophage lyase produced by utilizing pichia pastoris as well as preparation method and application of clostridium perfringens bacteriophage lyase

Abstract

The invention belongs to the technical field of molecular biology, and relates to clostridium perfringens bacteriophage lyase produced by utilizing pichia pastoris as well as a preparation method and application of the clostridium perfringens bacteriophage lyase, and the amino acid sequence of the clostridium perfringens bacteriophage lyase is shown as SEQ ID NO.2; a preparation method for producing clostridium perfringens bacteriophage lyase by using pichia pastoris comprises the following steps: linearizing a recombinant expression plasmid pPIC9K-PMQ0416 containing an optimized gene, then electrically transferring the recombinant expression plasmid pPIC9K-PMQ0416 into a yeast expression host GS115 to obtain a recombinant engineering bacterium, screening thousands of positive transformants by using geneticin, carrying out methanol induced expression on the transformants, and carrying out freeze-drying on the transformants to obtain the clostridium perfringens bacteriophage lyase. A highly expressed clostridium perfringens bacteriophage lyase yeast engineering strain is obtained; the clostridium perfringens bacteriophage lyase prepared by the invention is few in impure protein and high in purity, and has a broad-spectrum and efficient inhibition effect on clostridium perfringens.

Description

Technical Field

[0001] The present application belongs to the technical field of molecular biology, and specifically relates to a Clostridium perfringens phage lytic enzyme produced by Pichia pastoris, and a preparation method and application thereof. Background Art

[0002] Clostridium perfringens, also known as Clostridium weigeni, is an important zoonotic pathogen that can cause serious infectious diseases such as food poisoning, necrotic enteritis, and gas gangrene. Clostridium perfringens not only harms animals during livestock and poultry breeding, but also contaminates animal products during slaughter. Bacteriophages are capable of infecting bacteria and are also called "bacterial viruses." Phage lytic enzymes are proteins expressed in the late stage of Clostridium perfringens phage proliferation. They can accurately identify and degrade the peptidoglycan cell wall of Clostridium perfringens, and are synergistic with antimicrobial drugs and can effectively inhibit the growth of biofilms. They are a new type of antibacterial molecule. Phage lytic enzymes can act directly on bacteria, and can also be developed as chassis materials for accurate detection of pathogens. While killing pathogens, phage lytic enzymes do not interfere with normal flora, and are more efficient than phages, and have the potential to become new antibacterial agents.

[0003] The most representative expression system among eukaryotic expression systems is Pichia pastoris, which has the advantages of high-density fermentation, high expression, less impurity proteins secreted by itself, and can simplify subsequent purification work. Its alcohol oxidase AOX1 promoter can effectively control the efficient expression of exogenous genes after methanol induction and secrete them into the culture medium supernatant. There are many precedents for the large-scale expression of enzyme proteins by Pichia pastoris.

[0004] At present, Clostridium perfringens phage lytic enzymes are mostly prepared by prokaryotic expression systems, and prokaryotic expression is mainly intracellular expression. After lysing the host cells, further purification is required to obtain the lytic enzyme for further research. Therefore, how to simplify the preparation process and prepare Clostridium perfringens phage lytic enzymes with less impurities and high expression levels has become an urgent problem to be solved. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present application provides a Clostridium perfringens phage lytic enzyme produced by Pichia pastoris and its preparation method and application. The present application adopts the Pichia pastoris system, omitting the steps of cell lysis and purification and extraction; Pichia pastoris is suitable for high-density fermentation, and the expression level of Pichia pastoris fermentation level can be increased by more than 10 times compared with that of Pichia pastoris shake flask fermentation level, which is more in line with the needs of large-scale production.

[0006] In a first aspect, the present application provides a Clostridium perfringens phage lytic enzyme, which adopts the following technical solution: A Clostridium perfringens phage lytic enzyme optimized according to the codon preference of a Pichia pastoris expression system. The encoding nucleic acid sequence of the optimized Clostridium perfringens phage lytic enzyme is shown in SEQ ID NO.1, and the amino acid sequence thereof is shown in SEQ ID NO.2. The applicant named it Clostridium perfringens phage lytic enzyme PMQ0416.

[0007] In a second aspect, the present application provides a recombinant expression plasmid, a recombinant engineered bacterium, and a high-copy recombinant bacterium containing a nucleic acid encoding a Clostridium perfringens phage lytic enzyme, using the following technical solution: A recombinant expression plasmid comprising a nucleic acid encoding a Clostridium perfringens phage lytic enzyme PMQ0416.

[0008] Preferably, the recombinant expression plasmid is pPIC9K.

[0009] A recombinant engineered bacterium, comprising the above-mentioned recombinant expression plasmid.

[0010] Preferably, the expression host is Pichia pastoris GS115.

[0011] A high-copy recombinant bacterium is obtained by screening the above-mentioned recombinant engineering bacterium.

[0012] In the third aspect, the present application provides a nucleic acid encoding a phage lytic enzyme of Clostridium perfringens PMQ0416, and its recombinant expression plasmid, recombinant engineered bacteria, and high-copy recombinant bacteria for the preparation of a phage lytic enzyme of Clostridium perfringens, using the following technical solution: The invention discloses an application of a nucleic acid encoding a Clostridium perfringens phage lytic enzyme PMQ0416 in preparing the Clostridium perfringens phage lytic enzyme.

[0013] The invention discloses an application of a recombinant expression plasmid containing a Clostridium perfringens phage lytic enzyme PMQ0416 in preparing the Clostridium perfringens phage lytic enzyme.

[0014] The invention discloses an application of a recombinant engineering bacterium containing the recombinant expression plasmid in preparing Clostridium perfringens phage lytic enzyme.

[0015] Application of the high-copy recombinant bacteria in the preparation of Clostridium perfringens phage lytic enzyme.

[0016] In a fourth aspect, the present application provides a method for preparing a Clostridium perfringens phage lytic enzyme, using the following technical solution: A method for preparing Clostridium perfringens phage lytic enzyme PMQ0416 comprises the following steps: (1) Optimizing the nucleic acid sequence encoding the lytic enzyme of Clostridium perfringens phage according to the codon preference of Pichia pastoris. The optimized nucleic acid sequence encoding the lytic enzyme of Clostridium perfringens phage is shown in SEQ ID NO.1; (2) The optimized nucleic acid encoding the lytic enzyme of Clostridium perfringens phage was synthesized into a recombinant expression plasmid. The specific steps are as follows: the optimized nucleic acid sequence encoding the lytic enzyme of Clostridium perfringens phage was inserted between the EcoRI and NotI restriction sites of the pPIC9K expression plasmid to construct the recombinant expression plasmid pPIC9K-PMQ0416 of the lytic enzyme gene of Clostridium perfringens phage. The optimized sequence was synthesized by BGI.

[0017] (3) The synthesized recombinant expression plasmid is linearized and then electroporated into the expression host to obtain recombinant engineered bacteria, and high-copy recombinant bacteria are obtained after screening; (4) The recombinant bacterial monoclone is inoculated into the culture medium for cultivation, the supernatant is discarded after centrifugation, the culture medium is used for resuspending and cultivation, the expression is induced and the supernatant is collected to obtain the crude enzyme solution of Clostridium perfringens phage lytic enzyme.

[0018] Preferably, the recombinant expression plasmid in step (2) is pPIC9K.

[0019] Preferably, the expression host in step (3) is Pichia pastoris GS115.

[0020] Preferably, the culture medium for inoculating the recombinant bacterial monoclonal in step (4) is BMGY liquid culture medium.

[0021] Preferably, the step of resuspending and culturing with culture medium in step (4) is as follows: resuspending with an equal volume of BMMY liquid culture medium and continuing the culture, and supplementing methanol every 24 hours.

[0022] In the present application, the codons of the nucleic acid encoding the Clostridium perfringens phage lytic enzyme are optimized, and the Clostridium perfringens phage lytic enzyme is expressed in Pichia pastoris GS115 using the pPIC9K secretion expression plasmid, and the fermentation supernatant is the Clostridium perfringens phage lytic enzyme sample.

[0023] Since yeast cells themselves secrete and express little protein, the Clostridium perfringens phage lytic enzyme obtained from the fermentation supernatant has high purity, which simplifies the subsequent purification process.

[0024] It has been verified that the Clostridium perfringens phage lytic enzyme prepared by the method of the present application has a significant inhibitory effect on Clostridium perfringens.

[0025] By adopting the above technical scheme, the present application discloses a preparation method for producing Clostridium perfringens phage lytic enzyme using Pichia pastoris, comprising the following steps: linearizing the recombinant expression plasmid pPIC9K-PMQ0416 containing the optimized gene and then electrotransforming it into the yeast expression host GS115 to obtain a recombinant engineered bacterium, and screening more than a thousand positive transformants with geneticin and methanol-induced expression to obtain a highly efficient expression of Clostridium perfringens phage lytic enzyme PMQ0416 yeast engineered strain. The engineered strain has an α-factor signal peptide, which can secrete Clostridium perfringens phage lytic enzyme to express outside the cell, so after centrifuging the fermentation broth, the Clostridium perfringens phage lytic enzyme PMQ0416 crude enzyme solution can be obtained.

[0026] The crude enzyme sample of the Clostridium perfringens phage lytic enzyme PMQ0416 prepared in the present application has less impurities and high purity, and has a broad-spectrum and highly effective inhibitory effect on Clostridium perfringens. The lytic enzyme PMQ0416 has the effect of inhibiting and killing Clostridium perfringens at a certain concentration, and can be used to treat diseases and pollution caused by Clostridium perfringens infection, providing a new idea for clinical treatment of diseases caused by Clostridium perfringens infection, and can also be used as a food additive to kill Clostridium perfringens contaminated in food.

[0027] In a specific embodiment, a method for preparing a Clostridium perfringens phage lytic enzyme comprises the following steps: The nucleic acid sequence encoding the Clostridium perfringens phage lytic enzyme is optimized according to the codon preference of Pichia pastoris. The optimized nucleic acid sequence encoding the Clostridium perfringens phage lytic enzyme is shown in SEQ ID NO.1; The optimized coding nucleic acid of Clostridium perfringens phage lytic enzyme was synthesized into a recombinant expression plasmid pPIC9K-PMQ0416. The specific steps are as follows: the optimized coding nucleic acid sequence of Clostridium perfringens phage lytic enzyme was inserted between the EcoRI and NotI restriction sites of the pPIC9K expression plasmid to construct the Clostridium perfringens phage lytic enzyme gene recombinant expression plasmid pPIC9K-PMQ0416. The optimized sequence was synthesized by BGI Corporation.

[0028] The synthesized recombinant expression plasmid pPIC9K-PMQ0416 was linearized and electroporated into the yeast expression host GS115 to obtain the recombinant engineered bacteria, and the high-copy recombinant bacteria were obtained after screening on G418-YPD plates of different concentrations. The high-copy recombinant bacteria were inoculated into BMGY liquid culture medium, centrifuged and discarded the supernatant after 24 hours, and resuspended with an equal volume of BMMY (containing a final concentration of 0.5% methanol) liquid culture medium to continue culturing, supplemented with methanol every 24 hours, induced expression for 48 hours, and centrifuged. The supernatant was the crude enzyme solution of Clostridium perfringens phage lytic enzyme PMQ0416.

[0029] In summary, this application has at least the following beneficial effects.

[0030] (1) The present application discloses a Clostridium perfringens phage lytic enzyme produced by Pichia pastoris and its preparation method and application. The Clostridium perfringens phage is obtained by expressing exogenous proteins in Pichia pastoris. The lytic enzyme in the obtained Clostridium perfringens phage crude enzyme liquid has high purity, which reduces the subsequent purification work; (2) The Clostridium perfringens phage lytic enzyme prepared by the method of the present application has a certain inhibitory and killing effect on Clostridium perfringens.

[0031] Instruction Manual Figure 1 It is a PCR verification picture, where 1 is a water control; 2 and 3 are single colonies of pPIC9K-PMQ0416; M is DL2000.

[0032] Figure 2 It is the SDS-PAGE electrophoresis diagram, where 1 and 2 are the supernatants of the recombinant fermentation broth, and M is a protein marker; Figure 3 It is a Western-blot image, where 1 and 2 are the supernatants of the recombinant fermentation broth, and M is a protein marker; Figure 4 This is a plate spot method activity test, where 1, 2, 3, 4, and 5 are the supernatants of the fermentation broth of the recombinant transformant, and 6 is a 10-fold concentrate; Figure 5 The turbidimetric method was used to detect the enzyme activity of PMQ0416; Figure 6 The effect of pH on the activity of lyase PMQ0416; Figure 7 The effect of temperature on the activity of lytic enzyme PMQ0416. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further illustrated by specific embodiments below. The specific embodiments do not limit the protection scope of the present invention; some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0034] The raw materials involved in this application are all commercially available products, among which: Pichia pastoris GS115 was purchased from Invitrogen.

[0035] The pPIC9K expression plasmid was purchased from Invitrogen.

[0036] Clostridium perfringens strain ATCC13124 was a gift from Qingdao Agricultural University.

[0037] MD, YPD, BMGY, and BMMY culture media were prepared according to the Pichia pastoris expression instructions of Invitrogen; G418 was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; restriction endonucleases were purchased from Dalian Bao Biotechnology Co., Ltd.; plasmid extraction kits and DNA recovery kits were purchased from Omega. Other reagents and equipment, unless otherwise specified, can be obtained from commercial channels.

[0038] MD solid plate (1 L): weigh 15 g agar powder into 800 ml water, heat and boil until completely dissolved, autoclave at 121°C for 15 min, add 100 ml 10×YNB, 2 ml 500×B, and 100 ml 10×D at 60°C to prepare the plate.

[0039] YPD liquid medium (1 L): weigh 20 g of glucose, 20 g of peptone, and 10 g of yeast extract in 1 L of water, heat and boil until completely dissolved, autoclave at 115°C for 30 min, and cool for use (if preparing solid culture medium, add 20 g of agar before autoclaving).

[0040] BMGY liquid medium (1 L): weigh 10 g of yeast extract, 20 g of peptone, 11.8 g of KH2PO4, 3 g of K2HPO4, and 10 g of glycerol and dissolve them in 900 ml of deionized water. Sterilize at 121 °C for 20 min, cool to 60 °C, and add 100 ml of 10×YNB (final concentration 13.4 g / L), 2 ml of 500×B (final concentration 4×10 -4 g / L), mix well before use.

[0041] BMMY liquid medium (1 L): weigh 10 g yeast extract, 20 g peptone, 11.8 g KH2PO4, and 3 g K2HPO4, dissolve in 895 ml deionized water, sterilize at 121 °C for 20 min, cool to 60 °C, add 100 ml 10×YNB (final concentration 13.4 g / L), 5 ml anhydrous methanol, and 2 ml 500×B (final concentration 4×10 -4 g / L), mix well before use.

[0042] 100mg / ml G418 solution: Prepare 100mg / ml G418 stock solution with sterile water, filter sterilize, and store at -20℃. Use it to prepare G418-YPD plates with different final concentrations: 0.5mg / ml, 1.0mg / ml, 2.0mg / ml, 3.0mg / ml, 4.0mg / ml, etc. After the YPD solid medium is sterilized and cooled to 60℃, prepare plates with different final concentrations.

[0043] YPD solid medium (1 L): 20 g glucose, 20 g peptone, 10 g yeast extract, 20 g agar, sterilized at 115 °C for 30 min.

[0044] 10×YNB (13.4% Yeast Nitrogen Base (YNB) containing ammonium sulfate but not amino acids): Dissolve 134 g YNB in ​​1000 ml water, heat until YNB is completely dissolved, filter and sterilize, and store at 4 degrees.

[0045] 500×B (0.02% biotin): Dissolve 20 mg of biotin in 100 ml of water and filter sterilize.

[0046] 10×D (20% glucose): Dissolve 200 g D-glucose in 1000 ml water and sterilize by autoclaving at 115°C for 30 min or by filtration.

[0047] LB liquid culture medium containing 100ug / mL ampicillin resistance: weigh 2.5g of LB broth powder, dissolve in 100mL of water, sterilize at 121℃ for 20min, cool to 55℃, add ampicillin to a final concentration of 100ug / mL, and store at 4℃.

[0048] 0.75% TSA upper medium: Weigh 3g TSB powder and 0.75g agar powder and dissolve in 100mL water. Heat and dissolve, dispense into test tubes, 5mL per tube, sterilize at 121℃ for 20min. Pour onto 1.5% NA lower plate when cooled to 55℃.

[0049] 1.5% NA lower plate: weigh 1.8 g of nutrient agar powder and 1.5 g of agar powder, dissolve in 100 mL of water, sterilize at 121°C for 20 min, cool to 60°C to prepare the plate, and use after solidification.

[0050] Anaerobic liver soup liquid culture medium: Weigh 3.2 g of anaerobic liver soup powder, dissolve in 100 mL of water, and dispense 5 mL into each test tube. Add 1 beef cube to each test tube, cover the liquid in each test tube with 4 mm of liquid paraffin, and sterilize at 116°C for 30 min.

[0051] Example 1. Construction of yeast expression strain of Clostridium perfringens phage lytic enzyme and high copy screening. With reference to the GeneBank Clostridium perfringens phage PMQ04 sequence (accession number: MZ995505.1), an amino acid fragment (hereinafter identified as PMQ0416) predicted to have lytic enzyme properties in the Clostridium perfringens phage (PMQ04) sequence was used, as shown in SEQ ID NO.2, and the nucleic acid sequence was optimized according to the codon preference of Pichia pastoris. The optimized nucleic acid sequence is shown in SEQ ID NO. As shown in NO.1, a 6×his tag was added to the C-terminus of the PMQ0416 sequence to facilitate subsequent purification; the sequence was inserted between the EcoRI and NotI restriction sites of the pPIC9K expression plasmid to construct the recombinant expression plasmid pPIC9K-PMQ0416 of the Clostridium perfringens phage lytic enzyme gene, and the optimized sequence was synthesized by BGI to obtain the expression plasmid pPIC9K-PMQ0416 and the DH5a strain containing the recombinant expression plasmid pPIC9K-PMQ0416.

[0052] Preparation of Pichia competent cells: Resuscitate Pichia GS115 cells, streak on YPD plates, and culture at 30°C for 2-3 days. Randomly pick a single clone and inoculate it into 5 mL YPD liquid medium, and shake it overnight at 30°C and 220 rpm. Inoculate the overnight culture into 50 mL YPD liquid medium at a 1% (V / V) inoculum, and grow the bacteria at 30°C and 220 rpm to an OD of 600 The cell density is about 1.0. Centrifuge at 4000rpm for 2min at 4℃, resuspend twice with 20mL ice-cold sterile water, centrifuge at 4000rpm for 2min at 4℃, resuspend once with 20mL ice-cold 1mol / L sorbitol, centrifuge at 4000rpm for 2min at 4℃, resuspend with 1mL 1mol / L sorbitol after centrifugation, and obtain Pichia competent cells. Dispense 80uL in 1.5mL centrifuge tubes and place on ice for later use.

[0053] The DH5a strain containing the recombinant expression plasmid pPIC9K-PMQ0416 was amplified and identified by PCR using the universal primers (sequence SEQ ID NO.3, sequence SEQ ID NO.4) on the pPIC9K vector.

[0054] Primer 1: 5'AOX1-GACTGGTTCCAATTGACAAGC, as shown in SEQ ID NO.3.

[0055] Primer 2: 3'AOX1-GGCAAATGGCATTCTGACAT, as shown in SEQ ID NO.4.

[0056] PCR reaction system: 2×Hieff PCR Mix 10uL, Primer 1 1uL, Primer 2 1uL, Water 8uL, PCR template is a single colony grown on the plate.

[0057] PCR reaction conditions: 94°C for 5 min; 94°C for 30 s, 50°C for 30 s, 72°C for 60 s, for a total of 35 cycles; extension at 72°C for 10 min.

[0058] The PCR products were subjected to 1% agarose gel electrophoresis. Figure 1 (where 1 is a water control; 2 and 3 are single colonies of pPIC9K-PMQ0416; M is DL2000) as shown, the amplified product of pPIC9K-PMQ0416 is approximately 1.1 kb, which is consistent with the expected size.

[0059] A single colony (i.e., DH5a strain containing the recombinant expression plasmid pPIC9K-PMQ0416) was picked and cultured overnight in LB liquid medium containing 100ug / mL ampicillin resistance. The plasmid was extracted using an Omega plasmid extraction kit, digested with SalI restriction endonuclease for 2h, and the linearized recombinant expression plasmid was recovered using a DNA recovery kit. The recovered material was electroporated using a gene electroporator (Ningbo Xinzhi Biotechnology Co., Ltd.) (voltage 2kv, resistance 200Ω, capacitance 25μF) into 80uL of freshly prepared Pichia pastoris competent cells GS115. After electroporation, 1mL of pre-cooled sorbitol (1mol / L) was immediately added and mixed quickly, and cultured in a 30°C water bath for 1h. Spread an appropriate amount of bacterial liquid on MD solid plates, invert and culture at 30°C for 3-5 days until a single colony grows, and use a sterile toothpick spotting method to spot all transformants on G418-YPD plates of different concentrations (0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL) in turn, and culture at 30°C for 2-5 days; high-copy recombinant strains can be obtained after screening on 4.0 mg / mL G418-YPD plates.

[0060] Example 2. Expression verification of recombinant Clostridium perfringens phage lytic enzyme shake flask culture: The high-copy recombinant bacterial single colony in Example 1 was fermented in a 48-well plate using a high-throughput screening method, and the single colony (high-copy recombinant bacterial strain) was placed in 1 mL of BMGY liquid culture medium. After culturing at 30°C and 220 rpm for 20 hours, the supernatant was discarded by centrifugation at 4000 rpm for 10 minutes, and the precipitate was resuspended in 1 mL of BMMY (containing a final concentration of 0.5% (v / v) methanol) liquid culture medium for methanol induction culture. After the start of induction, methanol was supplemented every 24 hours to make the final concentration of methanol 0.5% (v / v). After 72 hours of induction, centrifugation was performed at 10000 rpm for 10 minutes. The supernatant was the crude enzyme solution of Clostridium perfringens phage lytic enzyme. The supernatant of the 72-hour fermentation broth was taken for SDS-PAGE electrophoresis detection (12% separation gel, 5% concentration gel) and Western-blot detection. The results of SDS-PAGE electrophoresis are shown as follows. Figure 2 (In which, 1 and 2 are both supernatants of recombinant pPIC9K-PMQ0416 / GS115 yeast fermentation broth, M is a protein marker) As shown, there is a protein band of target size between the standard molecular weight of 25-35kDa, and the band size is similar to the molecular weight of Clostridium perfringens phage lytic enzyme, which is about 26kDa. The content of impurities is small, and the purification process is simple and safe. The Western-blot results are shown in Figure 3 As shown in the figure (where 1 and 2 are both supernatants of recombinant fermentation broth, and M is a protein marker), the recombinant protein has a 6×his tag at the C-terminus. After specific binding and staining with the antibody, the protein with the his-tag can be stained blue. In the figure, there is a blue-stained protein near 25KDa, which is consistent with the molecular weight of the recombinant protein and the position of the protein band in the SDS-PAGE figure, indicating that the recombinant protein PMQ0416 is effectively expressed in the fermentation broth supernatant.

[0061] Example 3. Activity detection of recombinant Clostridium perfringens phage lytic enzyme. Activity detection by plate spot method: Clostridium perfringens strain ATCC13124 was activated and enriched in anaerobic meat liver soup liquid culture medium in a test tube. 100uL of the culture was mixed with 0.75% TSA upper culture medium and evenly spread on a 1.5% NA lower plate. After anaerobic culture overnight, a uniform biofilm was visible.

[0062] Take 10uLPMQ0416 fermentation liquid and spot it on the appropriate position of the plate with biofilm. After the liquid is absorbed, place it in a 37℃ incubator for further culture (no anaerobic conditions are required) and observe the results after about 10 hours. Observe whether an inhibition zone is formed. For specific results, see Figure 4 ; The results showed that the fermentation broth of Clostridium perfringens phage lytic enzyme ( Figure 41, 2, 4, and 5) can produce obvious inhibition zones, indicating that the Clostridium perfringens phage lytic enzyme PMQ0416 expressed by Pichia pastoris has a good bactericidal effect on Clostridium perfringens.

[0063] Turbidimetric enzyme activity detection: In order to further evaluate the enzymatic activity of Clostridium perfringens phage lytic enzyme, the bactericidal activity of Clostridium perfringens phage lytic enzyme against Clostridium perfringens was detected by turbidimetric method. The specific steps are as follows: Wash Clostridium perfringens ATCC13124 with PBS twice, and then resuspend the bacterial solution with PBS to an OD600 of about 1.0 to obtain Clostridium perfringens ATCC13124 bacterial solution; Take 500ul of the prepared bacterial solution and add it to a 48-well plate, add 500ul PMQ0416 crude enzyme solution to the experimental group, and add 500ul PBS solution to the control group, put it in a microbial growth analyzer, set the incubation temperature to 37°C, keep shaking, and read the OD every 1 hour. 600 Record the OD value within 10 hours. 600 The value changes from Figure 5 It can be seen that the experimental group OD 600 The value decreased from 6.4 to 1.0, which was comparable to the control group OD 600 The decrease in turbidity indicates that the bacteria in the reaction system are lysed by the lytic enzyme PMQ0416, thus indicating that the lytic enzyme has in vitro lytic activity.

[0064] Example 4: Identification of the cleavage spectrum of the recombinant Clostridium perfringens phage lytic enzyme Twenty strains of Clostridium perfringens were revived and cultured, and the recombinant protein PMQ0416 and phage PMQ04 were detected by plate spot method for lysis spectrum identification. The specific steps are as follows: After 20 strains of Clostridium perfringens proliferated overnight (i.e., Clostridium perfringens ATCC13124 proliferated overnight in Example 3) were mixed with 5 mL of 0.75% TSA upper layer culture medium (temperature 60°C), they were spread on 1.5% NA lower layer plates, and after the culture medium solidified, they were cultured anaerobically for 12-16 hours, and 5 uL of the recombinant protein PMQ0416 crude enzyme solution and phage PMQ04 were spotted on each Clostridium perfringens double-layer plate, placed at 4°C for 20 minutes, and placed at 37°C after the enzyme solution was absorbed, and the results were observed for 10 hours. The results are shown in Table 1, + represents cleavable, and - represents non-cleavable. Among them, bacteriophage PMQ04 can lyse 7 out of 20 bacterial strains, with a lysis rate of 35%; PMQ0416 crude enzyme solution can lyse 13 out of 20 strains of Clostridium perfringens, with a lysis rate of 65%. Compared with the bacteriophage from which it is derived, the lytic enzyme has a wider lysis spectrum, can act on more pathogens, and has a broader application space.

[0065] Table 1 Lysis rate of recombinant protein PMQ0416 and its bacteriophage PMQ04 on Clostridium perfringens Among them, + represents cleavable, and - represents non-cleavable.

[0066] Example 5. Effect of pH on the activity of lyase PMQ0416 The overnight-grown Clostridium perfringens ATCC13124 bacterial solution (i.e., the overnight-grown Clostridium perfringens ATCC13124 bacterial solution in Example 3) was centrifuged to collect the bacteria, washed twice with PBS (pH 7.5), and then resuspended with PBS (pH values ​​of 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, and 9.0, respectively) to an OD600 of about 1.3 (100 uL of the bacterial solution was detected by an ELISA instrument to obtain OD 600 500ul of the prepared bacterial solution was added to a 48-well plate. The experimental group was added with 500ul of PMQ0416 crude enzyme solution (the pH of the crude enzyme solution had been adjusted to the pH corresponding to that of PBS in advance), and the control group was added with 500ul of PBS solution (the pH was the same as that of the PBS used to dilute the bacterial solution). After mixing, the 48-well plate was placed in a microbial growth analyzer, the incubation temperature was set to 37°C, and the incubation was continuously shaken. The OD was read every hour. 600 Record the OD value within 10 hours. 600 The result is as follows: Figure 6 As shown, when the pH value of the reaction system is 5-9, OD 600 The values ​​decreased, especially at pH 7 and pH 8. 600 The value decreased the most, which shows that the activity of the lyase PMQ0416 is highest when the pH value is 7 to 8.

[0067] Example 6. Effect of temperature on the activity of lyase PMQ0416 The overnight-grown Clostridium perfringens ATCC13124 bacterial solution (i.e., the overnight-grown Clostridium perfringens ATCC13124 bacterial solution in Example 3) was centrifuged to collect the bacteria, washed twice with PBS, and then resuspended with PBS (pH 7.5) to an OD600 of about 1.3 (100 uL of the bacterial solution was detected by an ELISA instrument to obtain OD 600 500ul of the prepared bacterial solution was added to a 48-well plate. 500ul of PMQ0416 crude enzyme solution was added to the experimental group, and 500ul of PBS solution was added to the control group. After mixing, the plate was placed in a microbial growth analyzer. The incubation temperatures were set to 20°C, 25°C, 30°C, 35°C, 40°C, and 50°C, respectively. The plate was shaken continuously, and the OD was read every hour. 600 Record the OD value within 10 hours. 600 The result is as follows: Figure 7 As shown, when the reaction temperature is 20℃-50℃, OD 600The values ​​decreased, especially in the 35℃ and 40℃ groups. 600 The value decreased the most, which shows that the optimal reaction temperature of the lytic enzyme PMQ0416 is 35℃-40℃.

[0068] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, all equivalent changes made according to the structure, structure, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A Clostridium perfringens phage lytic enzyme, characterized in that: The amino acid sequence of the Clostridium perfringens phage lytic enzyme is shown in SEQ ID NO.

2.

2. The nucleic acid sequence encoding the Clostridium perfringens phage lytic enzyme according to claim 1, characterized in that: The nucleic acid sequence encoding the Clostridium perfringens phage lytic enzyme is shown in SEQ ID NO.

1.

3. A recombinant expression plasmid, characterized in that: The recombinant expression plasmid comprises the encoding nucleic acid according to claim 2; preferably, the recombinant expression plasmid uses pPIC9K as the expression plasmid.

4. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria comprises the recombinant expression plasmid according to claim 3.

5. The recombinant engineered bacterium according to claim 4, characterized in that: The expression host was Pichia pastoris GS115.

6. A high-copy recombinant bacterium, characterized in that: The high-copy recombinant bacteria are obtained by screening the recombinant engineered bacteria described in claim 4 or 5.

7. Use of the encoding nucleic acid according to claim 2, the recombinant expression plasmid according to claim 3, the recombinant engineered bacteria according to claim 4 or 5, or the high-copy recombinant bacteria according to claim 6 in the preparation of Clostridium perfringens phage lytic enzyme.

8. A method for preparing Clostridium perfringens phage lytic enzyme, characterized in that: The following steps are involved: (1) Optimizing the nucleic acid sequence encoding the lytic enzyme of Clostridium perfringens phage according to the codon preference of Pichia pastoris. The optimized nucleic acid sequence encoding the lytic enzyme of Clostridium perfringens phage is shown in SEQ ID NO.1; (2) synthesizing the optimized Clostridium perfringens phage lytic enzyme encoding nucleic acid into a recombinant expression plasmid; (3) The synthesized recombinant expression plasmid is linearized and then electroporated into the expression host to obtain recombinant engineered bacteria, and high-copy recombinant bacteria are obtained after screening; (4) The high-copy recombinant bacterial monoclone is inoculated into the culture medium for cultivation, the supernatant is discarded by centrifugation, the culture is resuspended in the culture medium, the expression is induced and the supernatant is collected by centrifugation to obtain the crude enzyme solution of Clostridium perfringens phage lytic enzyme.

9. The method for preparing a Clostridium perfringens phage lytic enzyme according to claim 8, characterized in that: In the step (2), the recombinant expression plasmid is pPIC9K; The expression host in step (3) is Pichia pastoris GS115; The culture medium for inoculating the recombinant bacterial monoclonal in step (4) is BMGY liquid culture medium.

10. Use of the encoding nucleic acid according to claim 2, the recombinant expression plasmid according to claim 3, the recombinant engineered bacteria according to claim 4 or 5, or the high expression strain according to claim 6 in efficiently lysing Clostridium perfringens.

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