Clostridium perfringens bacteriophage lyase produced by utilizing pichia pastoris as well as preparation method and application of clostridium perfringens bacteriophage lyase
By using the Pichia cerevisia expression system to optimize the coding nucleic acid sequence of C. perfringens phage lyase, it can achieve efficient expression and secretion, and solve the problems of complex preparation process and low expression in the prior art. High purity and high expression volume of C. perfringens phage lyase is obtained, which has broad-spectrum and efficient inhibitory effect.
Patent Information
- Application Number
- CN202510242611.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-09-27
- Filing Date
- 2025-03-03
- Publication Date
- 2025-05-30
AI Technical Summary
In the prior art, the preparation process of Clostridium perfringens phage lyase is complex and requires cell lysis and purification steps, resulting in a large number of proteins and low expression, making it difficult to meet the needs of large-scale production.
Using the Pichia cerevisia expression system, the codon optimization and the construction of the recombinant expression plasmid pPIC9K-PMQ0416 was used to achieve efficient expression and secretion of Clostridium perfringens, and the cell lysis and purification steps were omitted.
The purity and expression of C. perfringens phage lyase was improved, the preparation process was simplified, and it was suitable for large-scale production, and it showed a broad-spectrum and efficient inhibitory effect on C. perfringens.
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Abstract
Description
Technical Field
[0001] This application belongs to the technical field of molecular biology, and specifically relates to a Clostridium perfringens phage lyase produced by Pichia pastoris, its preparation method, and application. Background Art
[0002] Clostridium perfringens, also known as Clostridium welchii, is an important zoonotic pathogen that can cause severe infectious diseases such as food poisoning, necrotic enteritis, and gas gangrene. Clostridium perfringens not only causes harm to animals during livestock and poultry breeding, but also contaminates animal-derived products during slaughter. Bacteriophages are able to infect bacteria and are also known as "bacterial viruses". Bacteriophage lyase is a protein expressed in the late stage of Clostridium perfringens phage proliferation. It can accurately recognize and degrade the peptidoglycan cell wall of Clostridium perfringens, and has synergy with antibacterial drugs and can effectively inhibit biofilm growth. It is a novel antibacterial molecule. Bacteriophage lyase can act directly on bacteria or can be developed as a chassis material for precise detection of pathogenic bacteria. While killing pathogenic bacteria, bacteriophage lyase does not interfere with normal flora and is more efficient than bacteriophages, showing the potential to become a novel antibacterial agent.
[0003] The most representative expression system in eukaryotic expression systems is Pichia pastoris, which has the advantages of high-density fermentation, high expression level, few heterologous proteins secreted by itself, and can simplify subsequent purification work. The promoter of its alcohol oxidase AOX1 can effectively control the high-level expression of foreign genes after induction by methanol and secrete them into the culture medium supernatant. There are already many precedents of a large number of enzyme proteins being expressed by Pichia pastoris.
[0004] Currently, most Clostridium perfringens phage lyases are prepared by means of prokaryotic expression systems. Prokaryotic expression is mainly intracellular expression. After lysing the host cells, further purification is required to obtain the lyase for further research. Therefore, how to simplify the preparation process and obtain Clostridium perfringens phage lyase with few heterologous proteins and high expression level has become an urgent problem to be solved. Summary of the Invention
[0005] Aiming at the deficiencies of the prior art, this application provides a Clostridium perfringens phage lyase produced by Pichia pastoris, its preparation method, and application. This application uses the Pichia pastoris system, omitting the steps of lysing cells and purifying and extracting; Pichia pastoris is suitable for high-density fermentation. Compared with the shake-flask fermentation level of Pichia pastoris, the expression level in the fermenter can be increased by more than 10 times, which better meets the requirements of large-scale production.
[0006] In the first aspect, this application provides a Clostridium perfringens phage lyase, adopting the following technical solution: A Clostridium perfringens phage lyase optimized according to the codon preference of the Pichia pastoris expression system. The optimized coding nucleic acid sequence of the Clostridium perfringens phage lyase is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. The applicant names it Clostridium perfringens phage lyase PMQ0416.
[0007] In a second aspect, the present application provides a recombinant expression plasmid, a recombinant engineering bacterium, and a high-copy recombinant bacterium containing the coding nucleic acid of Clostridium perfringens phage lyase, and the following technical solutions are adopted: A recombinant expression plasmid, which contains the coding nucleic acid of Clostridium perfringens phage lyase PMQ0416.
[0008] Preferably, the recombinant expression plasmid uses pPIC9K as the expression plasmid.
[0009] A recombinant engineering bacterium, which contains the above recombinant expression plasmid.
[0010] Preferably, the expression host is Pichia pastoris GS115.
[0011] A high-copy recombinant bacterium, which is obtained by screening the above recombinant engineering bacterium.
[0012] In a third aspect, the present application provides the application of the coding nucleic acid of Clostridium perfringens phage lyase PMQ0416, its recombinant expression plasmid, recombinant engineering bacterium, and high-copy recombinant bacterium in the preparation of Clostridium perfringens phage lyase, and the following technical solutions are adopted: The application of the coding nucleic acid of Clostridium perfringens phage lyase PMQ0416 in the preparation of Clostridium perfringens phage lyase.
[0013] The application of a recombinant expression plasmid containing Clostridium perfringens phage lyase PMQ0416 in the preparation of Clostridium perfringens phage lyase.
[0014] The application of a recombinant engineering bacterium containing the above recombinant expression plasmid in the preparation of Clostridium perfringens phage lyase.
[0015] The application of the above high-copy recombinant bacterium in the preparation of Clostridium perfringens phage lyase.
[0016] In a fourth aspect, the present application provides a preparation method of Clostridium perfringens phage lyase, and the following technical solutions are adopted: A preparation method of Clostridium perfringens phage lyase PMQ0416, which includes the following steps: (1) Optimize the nucleic acid sequence encoding the Clostridium perfringens phage lyase according to the codon preference of Pichia pastoris. The optimized nucleic acid sequence encoding the Clostridium perfringens phage lyase is shown as SEQ ID NO.1; (2) Synthesize a recombinant expression plasmid with the optimized nucleic acid encoding the Clostridium perfringens phage lyase. The specific steps are as follows: Insert the optimized nucleic acid sequence encoding the Clostridium perfringens phage lyase between the EcoRI and NotI restriction enzyme sites of the pPIC9K expression plasmid to construct the recombinant expression plasmid pPIC9K-PMQ0416 for the Clostridium perfringens phage lyase gene. The optimized sequence is synthesized by BGI.
[0017] (3) After linearizing the synthesized recombinant expression plasmid, electrotransform it into the expression host to obtain a recombinant engineering bacterium, and obtain a high-copy recombinant bacterium after screening; (4) Inoculate the recombinant bacterium monoclonal into a medium for culture, centrifuge to discard the supernatant, resuspend and culture with the medium, centrifuge after induction of expression, and collect the supernatant to obtain the crude enzyme solution of the Clostridium perfringens phage lyase.
[0018] Preferably, in step (2), the recombinant expression plasmid uses pPIC9K as the expression plasmid.
[0019] Preferably, in step (3), the expression host is Pichia pastoris GS115.
[0020] Preferably, in step (4), the medium inoculated with the recombinant bacterium monoclonal is BMGY liquid medium.
[0021] Preferably, the steps of resuspending and culturing with the medium in step (4) are as follows: Resuspend with an equal volume of BMMY liquid medium and continue culturing, and supplement methanol every 24 h.
[0022] In this application, the codons of the nucleic acid encoding the Clostridium perfringens phage lyase are optimized, and the Clostridium perfringens phage lyase is expressed in Pichia pastoris GS115 using the pPIC9K secretion expression plasmid. The fermentation supernatant is the sample of the Clostridium perfringens phage lyase.
[0023] Since the yeast cells secrete few proteins by themselves, the Clostridium perfringens phage lyase obtained from the fermentation supernatant has a high purity, which simplifies the subsequent purification process.
[0024] It has been verified that the Clostridium perfringens phage lyase prepared by the method of this application has an obvious inhibitory effect on Clostridium perfringens.
[0025] By adopting the above technical solution, the present application discloses a preparation method of using Pichia pastoris to produce Clostridium perfringens phage lyase, which includes the following steps: linearize the recombinant expression plasmid pPIC9K-PMQ0416 containing the optimized gene and electrotransform it into the yeast expression host GS115 to obtain a recombinant engineering bacterium. After screening thousands of positive transformants by geneticin and inducing expression with methanol, a yeast engineering strain of Clostridium perfringens phage lyase PMQ0416 with high expression efficiency is obtained. This engineering strain has an α-factor signal peptide, which can secrete Clostridium perfringens phage lyase to be expressed extracellularly. Therefore, after centrifuging the fermentation broth, the crude enzyme solution of Clostridium perfringens phage lyase PMQ0416 can be obtained.
[0026] The crude enzyme sample of Clostridium perfringens phage lyase PMQ0416 prepared in the present application has few impurities and high purity, and has a broad-spectrum and high-efficiency inhibitory effect on Clostridium perfringens. Lyase PMQ0416 has the effect of inhibiting and killing Clostridium perfringens at a certain concentration, and can be applied to the treatment of diseases and contaminations caused by Clostridium perfringens infection, providing new ideas for the 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 feasible embodiment, a preparation method of Clostridium perfringens phage lyase includes the following steps: Optimize the nucleic acid sequence encoding Clostridium perfringens phage lyase according to the codon preference of Pichia pastoris. The optimized nucleic acid sequence encoding Clostridium perfringens phage lyase is shown in SEQ ID NO.1; Synthesize the recombinant expression plasmid pPIC9K-PMQ0416 with the optimized nucleic acid sequence encoding Clostridium perfringens phage lyase. The specific steps are as follows: insert the optimized nucleic acid sequence encoding Clostridium perfringens phage lyase between the EcoRI and NotI restriction enzyme sites of the pPIC9K expression plasmid to construct the recombinant expression plasmid pPIC9K-PMQ0416 of Clostridium perfringens phage lyase gene. The optimized sequence is synthesized by BGI.
[0028] Linearize the synthesized recombinant expression plasmid pPIC9K-PMQ0416 and electrotransform it into the yeast expression host GS115 to obtain a recombinant engineering bacterium. After screening with G418-YPD plates with different concentrations, high-copy recombinant bacteria are obtained. Inoculate the high-copy recombinant bacteria into BMGY liquid medium, centrifuge and discard the supernatant after 24 h, resuspend with an equal volume of BMMY (containing 0.5% methanol in the final concentration) liquid medium and continue to culture. Supplement methanol every 24 h. After inducing expression for 48 h, centrifuge, and the supernatant is the crude enzyme solution of Clostridium perfringens phage lyase PMQ0416.
[0029] In summary, the present application has at least the following beneficial effects.
[0030] (1) The present application discloses a Clostridium perfringens phage lyase produced by Pichia pastoris, its preparation method and application. The Clostridium perfringens phage is obtained through the Pichia pastoris expression of foreign proteins. The lyase in the obtained crude enzyme solution of Clostridium perfringens phage has high purity, reducing the later purification work; (2) The Clostridium perfringens phage lyase prepared by the method of the present application has a certain inhibitory and killing effect on Clostridium perfringens.
[0031] Description of the Drawings Figure 1 It is a PCR verification diagram. Among them, 1 is the water control; 2 and 3 are both single colonies of pPIC9K-PMQ0416; M is DL2000.
[0032] Figure 2 It is an SDS-PAGE electrophoresis diagram. Among them, 1 and 2 are both the supernatants of the recombinant fermentation broth, and M is the protein marker; Figure 3 It is a Western-blot diagram. Among them, 1 and 2 are both the supernatants of the recombinant fermentation broth, and M is the protein marker; Figure 4 It is the activity detection by the plate spotting method. Among them, 1, 2, 3, 4, 5 are the supernatants of the recombinant transformant fermentation broth, and 6 is the 10-fold concentrated solution; Figure 5 It is the turbidity method for detecting the enzyme activity of PMQ0416; Figure 6 It is the effect of pH on the enzyme activity of the lyase PMQ0416; Figure 7 It is the effect of temperature on the enzyme activity of the lyase PMQ0416. Detailed Embodiments
[0033] The technical solutions of the present invention are further described below through specific embodiments. The specific embodiments do not represent a limitation on 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] All raw materials involved in the present application are commercially available products. Among them, Pichia pastoris GS115 is purchased from Invitrogen.
[0035] The pPIC9K expression plasmid is purchased from Invitrogen.
[0036] The Clostridium perfringens strain ATCC13124 is a gift from Qingdao Agricultural University.
[0037] The MD, YPD, BMGY, and BMMY media were prepared according to the Pichia pastoris expression instruction manual of Invitrogen; G418 was purchased from Sangon Biotech (Shanghai) Co., Ltd.; restriction endonucleases were purchased from TaKaRa Bio Inc. (Dalian); plasmid extraction kits and DNA recovery kits were purchased from Omega; other reagents and equipment, unless otherwise specified, were commercially available.
[0038] MD solid plate (1 L): Weigh 15 g of agar powder into 800 ml of water, heat to boiling until completely dissolved, autoclave at 121 °C for 15 min, and add 100 ml of 10×YNB, 2 ml of 500×B, and 100 ml of 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 into 1 L of water, heat to boiling until completely dissolved, autoclave at 115 °C for 30 min, and cool for standby (if preparing solid medium, add 20 g of agar before autoclaving).
[0040] BMGY liquid medium (1 L): Weigh 10 g of yeast extract, 20 g of peptone, KH 2 PO 4 11.8 g, K 2 HPO 4 3 g, and 10 g of glycerol into 900 ml of deionized water, autoclave at 121 °C for 20 min, cool to 60 °C, and add 100 ml of 10×YNB (final concentration 13.4 g / L) and 2 ml of 500×B (final concentration 4×10 -4 g / L) in a laminar flow hood, and mix well before use.
[0041] BMMY liquid medium (1 L): Weigh 10 g of yeast extract, 20 g of peptone, KH 2 PO 4 11.8 g, K 2 HPO 4 3 g, dissolve in 895 ml of deionized water, autoclave at 121 °C for 20 min, cool to 60 °C, and add 100 ml of 10×YNB (final concentration 13.4 g / L), 5 ml of anhydrous methanol, and 2 ml of 500×B (final concentration 4×10 -4 g / L) in a laminar flow hood, and mix well before use.
[0042] 100 mg / ml G418 solution: Prepare a 100 mg / ml G418 stock solution with sterile water, filter sterilize it, and store it at -20°C. Used to prepare G418-YPD plates with different final concentrations: 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml, etc. After the YPD solid medium is sterilized and cooled to 60°C, prepare plates with different final concentrations.
[0043] YPD solid medium (1 L): Glucose 20 g, peptone 20 g, yeast extract 10 g, agar 20 g, sterilize at 115°C for 30 min.
[0044] 10×YNB (13.4% yeast nitrogen base (YNB) containing ammonium sulfate without amino acids): Dissolve 134 g of YNB in 1000 ml of water, heat until YNB is completely dissolved, filter sterilize it, and store it at 4°C.
[0045] 500×B (0.02% biotin): Dissolve 20 mg of biotin in 100 ml of water, filter sterilize it.
[0046] 10×D (20% glucose): Dissolve 200 g of D-glucose in 1000 ml of water, autoclave at 115°C for 30 min or filter sterilize it.
[0047] LB liquid medium containing 100 μg / mL ampicillin resistance: Weigh 2.5 g of LB broth powder, dissolve it in 100 mL of water, sterilize at 121°C for 20 min. When cooled to 55°C, add ampicillin to a final concentration of 100 μg / mL and store at 4°C.
[0048] 0.75% TSA upper layer medium: Weigh 3 g of TSB powder and 0.75 g of agar powder, dissolve them in 100 mL of water, heat and dissolve, then dispense into test tubes, 5 mL per tube, sterilize at 121°C for 20 min. When cooled to 55°C, pour it onto a 1.5% NA lower layer plate.
[0049] 1.5% NA lower layer plate: Weigh 1.8 g of nutrient agar powder and 1.5 g of agar powder, dissolve them in 100 mL of water, sterilize at 121°C for 20 min. When cooled to 60°C, prepare plates and use after solidification.
[0050] Anaerobic liver broth liquid medium: Weigh 3.2 g of anaerobic liver broth powder, dissolve it in 100 mL of water, dispense 5 mL into each test tube, add 1 beef granule 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 and high-copy screening of a yeast expression strain of Clostridium perfringens phage lyase Refer to the GeneBank sequence of Clostridium perfringens phage PMQ04 (accession number: MZ995505.1). An amino acid fragment predicted to have lyase properties in the Clostridium perfringens phage (PMQ04) sequence (hereinafter designated as PMQ0416), as shown in SEQ ID NO.2, was optimized for nucleic acid sequence according to the codon preference of Pichia pastoris. The optimized nucleic acid sequence is shown in SEQ ID NO.1. A 6×his tag was added to the C-terminus of the PMQ0416 sequence to facilitate later 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 lyase gene. The optimized sequence was synthesized by BGI, and the expression plasmid pPIC9K-PMQ0416 and the DH5a strain containing the recombinant expression plasmid pPIC9K-PMQ0416 were obtained.
[0052] Preparation of Pichia pastoris competent cells: Revive Pichia pastoris GS115 cells and streak them on a YPD plate, and culture at 30 °C for 2 - 3 days. Randomly pick a single colony and inoculate it into 5 mL of YPD liquid medium, and shake the culture overnight at 30 °C and 220 rpm. Inoculate the overnight culture into 50 mL of YPD liquid medium at an inoculation amount of 1% (V / V), and grow the bacteria at 30 °C and 220 rpm until the OD 600 is about 1.0. Centrifuge at 4000 rpm for 2 min at 4 °C, resuspend twice with 20 mL of ice-cold sterile water, centrifuge at 4000 rpm for 2 min at 4 °C, then resuspend once with 20 mL of ice-cold 1 mol / L sorbitol, centrifuge at 4000 rpm for 2 min at 4 °C, and after centrifugation, resuspend with 1 mL of 1 mol / L sorbitol. This is the Pichia pastoris competent cell, which is aliquoted in 1.5 mL centrifuge tubes at 80 uL per unit and placed on ice for standby.
[0053] The DH5a strain containing the recombinant expression plasmid pPIC9K-PMQ0416 was identified by PCR amplification 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: 10 μL of 2×Hieff PCR Mix, 1 μL of primer 1, 1 μL of primer 2, 8 μL of water, and the PCR template is the single colony grown on the plate.
[0057] PCR reaction conditions: 5 min at 94 °C; 30 s at 94 °C, 30 s at 50 °C, 60 s at 72 °C, for a total of 35 cycles; 10 min of extension at 72 °C.
[0058] The PCR products were subjected to 1% agarose gel electrophoresis, and the results were as Figure 1 (where 1 is the water control; 2 and 3 are both single colonies of pPIC9K-PMQ0416; M is DL2000). The amplified product of pPIC9K-PMQ0416 is about 1.1 kb, which is consistent with the expected size.
[0059] Pick a single colony (i.e., the DH5α strain containing the recombinant expression plasmid pPIC9K-PMQ0416) and culture it overnight in LB liquid medium containing 100 μg / mL ampicillin resistance. Extract the plasmid using the Omega plasmid extraction kit, digest it with SalI restriction endonuclease for 2 h, and recover the linearized recombinant expression plasmid using a DNA recovery kit. Electroporate the recovered product (voltage 2 kV, resistance 200 Ω, capacitance 25 μF) into 80 μL of freshly prepared Pichia pastoris competent cells GS115 using a gene electroporator (Ningbo Xinzhi Biotechnology Co., Ltd.). Immediately after electroporation, add 1 mL of pre-cooled sorbitol (1 mol / L) and mix well quickly. Incubate in a 30 °C water bath for 1 h. Spread an appropriate amount of the bacterial solution on an MD solid plate and incubate it upside down at 30 °C for 3 - 5 days until single colonies grow. Use the sterile toothpick spotting method to spot all the transformants on G418-YPD plates with different concentrations (0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL) in sequence and incubate at 30 °C for 2 - 5 days; after screening with the 4.0 mg / mL G418-YPD plate, high-copy recombinant strains can be obtained.
[0060] Example 2. Expression verification of recombinant Clostridium perfringens phage lyase by shake flask culture: The single colony of the high-copy recombinant bacterium in Example 1 was fermented in a 48-well plate by high-throughput screening. The single colony (high-copy recombinant bacterium strain) was placed into 1 mL of BMGY liquid medium and cultured at 30 °C and 220 rpm for 20 h. Then, the supernatant was discarded by centrifugation at 4000 rpm for 10 min, and the precipitate was resuspended with 1 mL of BMMY (containing methanol at a final concentration of 0.5% (v / v)) liquid medium for methanol induction culture. Methanol was supplemented every 24 h after the induction started to make the final concentration of methanol 0.5% (v / v). After 72 h of induction, centrifugation was performed at 10000 rpm for 10 min, and the supernatant was the crude enzyme solution of Clostridium perfringens phage lyase. Take the supernatant of the 72-h fermentation broth for SDS-PAGE electrophoresis detection (12% separating gel, 5% stacking gel) and Western-blot detection. The results of SDS-PAGE electrophoresis are as Figure 2 (where 1 and 2 are both the supernatants of the fermentation broth of recombinant pPIC9K-PMQ0416 / GS115 yeast, and M is the protein Marker). There is a protein band of the target size between the standard molecular weights of 25 - 35 kDa. The band size is similar to the molecular weight of Clostridium perfringens phage lyase, about 26 kDa. The content of miscellaneous proteins is small, and the purification process is simple and safe. The results of Western-blot are as Figure 3 (where 1 and 2 are both the supernatants of the fermentation broth of the recombinant, and M is the protein marker). The C-terminus of the recombinant protein has a 6×his tag. After specific binding and staining with the antibody, the protein with his-tag can be stained blue. There is a blue-stained protein near 25 KDa in the figure, 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 lyase by plate spotting method: Activate the Clostridium perfringens strain ATCC13124. After enrichment in anaerobic liver broth liquid medium in a test tube, take 100 μL of the culture and mix it with 0.75% TSA upper-layer medium, and then evenly spread it on a 1.5% NA lower-layer plate. After anaerobic culture overnight, a uniform bacterial film can be seen.
[0062] Take 10 μL of the PMQ0416 fermentation broth and spot it at an appropriate position on the plate with the grown bacterial film. After the liquid is absorbed, place it in an incubator at 37 °C for continued culture (anaerobic condition is not required), and observe the results after about 10 h. Observe whether an inhibition zone is formed. The specific results are shown in Figure 4 ; The results show that the fermentation broth of Clostridium perfringens phage lyase ( Figure 4Among 1, 2, 4, and 5), obvious inhibition zones can be produced, indicating that the Clostridium perfringens phage lyase PMQ0416 expressed by Pichia pastoris has good bactericidal effects on Clostridium perfringens.
[0063] Enzyme activity detection by turbidimetry: To further evaluate the enzyme activity of Clostridium perfringens phage lyase, the bactericidal activity of Clostridium perfringens phage lyase against Clostridium perfringens was detected by turbidimetry. The specific steps are as follows: Wash Clostridium perfringens ATCC13124 twice with PBS, and then resuspend the bacterial solution with PBS to an OD600 of about 1.0 to obtain the Clostridium perfringens ATCC13124 bacterial solution; Take 500 μl of the prepared bacterial solution and add it to a 48-well plate. Add 500 μl of the PMQ0416 crude enzyme solution to the experimental group and 500 μl of PBS solution to the control group. Place it in a microbial growth analyzer, set the incubation temperature at 37 °C, continuously shake, and read the OD 600 value every 1 hour, and record the change in OD 600 value within 10 h. As can be seen from Figure 5 , the OD 600 value of the experimental group decreased from 6.4 to 1.0, showing an obvious contrast with the decrease in the OD 600 value of the control group. The decrease in turbidity indicates that the bacteria in the reaction system were lysed by the lyase PMQ0416. Therefore, it shows that this lyase has in vitro lysis activity.
[0064] Example 4. Identification of the lysis spectrum of recombinant Clostridium perfringens phage lyase Resuscitate and culture 20 strains of Clostridium perfringens, and use the plate spotting method to detect the lysis spectra of the recombinant protein PMQ0416 and the phage PMQ04. The specific steps are as follows: Mix 20 strains of Clostridium perfringens (i.e., Clostridium perfringens ATCC13124 proliferated overnight in Example 3) that proliferated overnight with 5 mL of 0.75% TSA upper-layer medium (temperature 60 °C) respectively, and spread them on a 1.5% NA lower-layer plate. After the medium solidifies, anaerobically culture for 12 - 16 h. Spot 5 μL of the PMQ0416 crude enzyme solution of the recombinant protein and the phage PMQ04 respectively on each double-layer plate of Clostridium perfringens, place it at 4 °C for 20 min, and then place it at 37 °C for 10 h to observe the results. The results are shown in Table 1, where + represents lysable and - represents non-lysable. Among them, the phage PMQ04 can lyse 7 out of 20 strains of bacteria, and the lysis rate is 35%; the PMQ0416 crude enzyme solution can lyse 13 out of 20 strains of Clostridium perfringens, and the lysis rate is 65%. Compared with its source phage, the lyase has a wider lysis spectrum, can act on more pathogenic bacteria, and has a broader application space.
[0065] Table 1 Lysis rates of the recombinant protein PMQ0416 and its phage PMQ04 against Clostridium perfringens Among them, '+' represents cleavable, and '-' represents non-cleavable.
[0066] Example 5. Effect of pH on the enzyme activity of lyase PMQ0416 The Clostridium perfringens ATCC13124 bacterial solution that had proliferated overnight (i.e., the Clostridium perfringens ATCC13124 bacterial solution that had proliferated overnight in Example 3) was centrifuged to collect the bacterial cells, washed twice with PBS (pH 7.5), and then resuspended in PBS (with pH values of 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, and 9.0 respectively) until the OD600 was about 1.3 (the OD 600 value was detected by a microplate reader for 100 uL of the bacterial solution). 500 μL of the prepared bacterial solution was added to a 48-well plate. In the experimental group, 500 μL of the crude PMQ0416 enzyme solution (the pH of the crude enzyme solution had been adjusted in advance to the corresponding pH value of PBS) was added, and in the control group, 500 μL of PBS solution (with the same pH as the PBS used to dilute the bacterial solution) was added. After mixing, the 48-well plate was placed in a microbial growth analyzer, the incubation temperature was set at 37 °C, and continuous oscillation was carried out. The OD 600 value was read every 1 hour, and the change in the OD 600 value within 10 h was recorded. The results are as Figure 6 shown. When the pH value of the reaction system was 5 - 9, the OD 600 value decreased in all cases. The OD 600 value decreased the most at pH 7 and pH 8. It can be seen from this that when the pH value was 7 to 8, the activity of lyase PMQ0416 was the highest.
[0067] Example 6. Effect of temperature on the enzyme activity of lyase PMQ0416 The Clostridium perfringens ATCC13124 bacterial solution that had proliferated overnight (i.e., the Clostridium perfringens ATCC13124 bacterial solution that had proliferated overnight in Example 3) was centrifuged to collect the bacterial cells, washed twice with PBS, and then resuspended in PBS (pH 7.5) until the OD600 was about 1.3 (the OD 600 value was detected by a microplate reader for 100 uL of the bacterial solution). 500 μL of the prepared bacterial solution was added to a 48-well plate. In the experimental group, 500 μL of the crude PMQ0416 enzyme solution was added, and in the control group, 500 μL of PBS solution was added. After mixing, it was placed in a microbial growth analyzer, and the incubation temperatures were set at 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, and 50 °C respectively. Continuous oscillation was carried out, the OD 600 value was read every 1 hour, and the change in the OD 600 value within 10 h was recorded. The results are as Figure 7 shown. When the reaction temperature was 20 °C - 50 °C, the OD 600The values all decreased, and the OD value in the 35°C and 40°C groups decreased the most. Thus, it can be seen that the optimal reaction temperature of the lyase PMQ0416 is 35°C - 40°C. 600 The value decreased the most. Thus, it can be seen that the optimal reaction temperature of the lyase PMQ0416 is 35°C - 40°C.
[0068] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereby. Therefore, all equivalent changes made according to the structure and principle of the present application shall be covered within 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.