Mutant of Clostridium perfringens phage CPS2 lytic enzyme and its application

Abstract

The present invention belongs to the field of protein engineering and biotechnology, and specifically relates to a mutant of the CPS2 lyase of the phage of Clostridium perfringens and its application. The present invention improves the anti-trypsin enzymatic performance of the CPS2 lyase of the phage of Clostridium perfringens in the intestinal environment by a directed modification method, and the obtained mutant enzyme has higher antibacterial activity than the wild-type enzyme under the action of trypsin in the intestinal environment. The lyase mutant of the present invention can be used as an alternative antibiotic product to exert an antibacterial effect in the intestine through oral administration, prevent and treat related diseases caused by Clostridium perfringens, and improve intestinal health.

Description

Technical Field

[0001] The invention belongs to the fields of protein engineering and biotechnology, and in particular relates to a Clostridium perfringens bacteriophage CPS2 lyase mutant and application thereof. Background Art

[0002] Clostridium perfringens is an important zoonotic pathogen, and is widely found in water sources, soil, and the intestines of humans and animals. Type A Clostridium perfringens is the main pathogen that causes food poisoning in humans, and can also cause necrotic enteritis and enterotoxemia in animals; Type C Clostridium perfringens can cause necrotic enteritis in humans and enterotoxemia in various animals; Type B and Type D Clostridium perfringens are mainly found in the intestines of animals, and as a conditional pathogen, they can cause enterotoxemia in animals. The increased mortality, economic losses, and contamination of livestock and poultry products caused by Clostridium perfringens are important issues of concern to people.

[0003] Bacteriophages are a type of virus that has a very strong specific killing effect on pathogenic bacteria. Bacteriophages targeting pathogenic bacteria are naturally alternatives to antibiotics. The ability of bacteriophages themselves also shows potential in this regard. For example, it is very specific and only targets a certain type of bacteria, so it can avoid accidentally injuring beneficial bacteria and the body's own cells, and it is extremely difficult to develop drug resistance. Phage lytic enzymes are peptidoglycan hydrolases encoded by phages. They are produced in the late biosynthesis of the lytic cycle. Phages rely on lytic enzymes to hydrolyze the peptidoglycan in the cell wall to release progeny phages. As early as 1959, humans had discovered the characteristics of phage lytic enzymes in lysing bacteria and named them endolysin. However, due to the significant efficacy of antibiotics at the time, the exploration of bacteriophage antibacterial was once shelved. Today, bacterial resistance to antibiotics has increased dramatically, and people have turned their attention back to bacteriophages and their lytic enzymes. Phages themselves are viruses, contain genetic material, and have toxic risks to the body. Product development investment is large and screening is difficult. The development of bacteriophage lytic enzymes can effectively solve the safety issues of bacteriophages, and is more conducive to industrialization and market promotion.

[0004] In 2018, a Clostridium perfringens phage CPS2 was isolated from chicken manure. At the same time, through bioinformatics analysis of the CPS2 genome, its lytic enzyme was discovered. The Clostridium perfringens phage CPS2 lytic enzyme has strong lytic activity against Clostridium perfringens and has high thermal stability, and has great application prospects as an alternative antibiotic product.

[0005] Trypsin is a proteolytic enzyme present in the intestine that can selectively hydrolyze peptide chains composed of lysine or arginine carboxyl groups in proteins. The protein tertiary structure modeling and analysis of the Clostridium perfringens phage CPS2 lyase revealed that its Loop region contained multiple trypsin cleavage sites, which were easily hydrolyzed by trypsin and lost their antibacterial activity. Therefore, based on this technical problem, the present invention improves the anti-trypsin enzymatic performance of the Clostridium perfringens phage CPS2 lyase in the intestinal environment through a directional transformation method. Solving this short board problem will be more conducive to the lyase as an alternative antibiotic product to play an antibacterial effect in the intestine through oral administration, prevent and treat Clostridium perfringens related diseases, and improve intestinal health. Summary of the invention

[0006] The present invention performs mutation screening on the Clostridium perfringens phage CPS2 lyase (wild-type CPS2-WT) disclosed in the prior art. The nucleic acid coding sequence of the Clostridium perfringens phage CPS2 lyase is shown by SEQ ID NO.1, and the amino acid sequence is shown by SEQ ID NO.2. The Clostridium perfringens phage CPS2 lyase has a strong lysis activity against Clostridium perfringens and has a high thermal stability. It has great application prospects as an alternative antibiotic product, but it is very easy to be hydrolyzed by trypsin and lose its antibacterial activity. Therefore, by performing site-directed mutation on the wild-type enzyme, it is expected to obtain a Clostridium perfringens phage CPS2 lyase mutant with enhanced anti-trypsin enzymolysis performance in the intestinal environment.

[0007] The purpose of the present invention is to provide a mutant of Clostridium perfringens phage CPS2 lyase and its application. The present invention finally obtains a mutant of Clostridium perfringens phage CPS2 lyase with enhanced anti-trypsin enzymolysis performance in a simulated intestinal environment through a large number of site-directed mutagenesis screenings, and constructs a genetically engineered strain that recombinantly expresses Clostridium perfringens phage CPS2 lyase, laying a foundation for realizing its application.

[0008] First, the present invention provides a Clostridium perfringens phage CPS2 lyase mutant having higher antibacterial activity than the wild-type enzyme under the action of trypsin in the intestinal environment, wherein the amino acid sequence is based on the amino acid sequence shown in SEQ ID NO:2, and the amino acid residues at at least one of K58, L91, K139, L140, R141, K147, K212, L213, and K214 are mutated; or the amino acid sequence of the Clostridium perfringens phage CPS2 lyase mutant has the mutation site in the mutated amino acid sequence.

[0009] More preferably, the Clostridium perfringens phage CPS2 lytic enzyme mutant includes mutations corresponding to SEQ ID NO: 2, and the following sites are present: K58P, L91F, K139G, L140S, R141P, K147P, K212G, L213P, K214P, one or a combination of two or three or more of them.

[0010] More specifically, corresponding to SEQ ID NO: 2, there are mutations at the following sites:

[0011] (1) Lysine 58 was mutated to proline;

[0012] (2) leucine at position 91 mutated to phenylalanine;

[0013] (3) Lysine 214 was mutated to proline;

[0014] (4) Lysine at position 212 mutated to glycine, and Leucine at position 213 mutated to proline;

[0015] (5) Lysine at position 139 mutated to glycine, leucine at position 140 mutated to serine, arginine at position 141 mutated to proline, and lysine at position 147 mutated to proline.

[0016] The present invention also provides a gene encoding the above-mentioned Clostridium perfringens phage CPS2 lytic enzyme.

[0017] The present invention further provides a recombinant vector and a recombinant cell containing the gene encoding the Clostridium perfringens bacteriophage CPS2 lytic enzyme.

[0018] The present invention also provides a method for recombinant expression of the coding gene of the Clostridium perfringens phage CPS2 lyase, which is obtained by expression in a recombinant bacterium containing a recombinant expression vector. More specifically, pET-30a(+) is used as an expression vector, Escherichia coli BL21(DE3) is used as an expression host, and the obtained recombinant strain is inoculated in an LB expression medium containing 50 μg / mL kanamycin resistance, and cultured at 37°C and 220 rpm until the OD 600 =0.6-0.8, add inducer IPTG to a final concentration of 0.5 mM, transfer to 16°C, 150 rpm and culture for 16-18 h to induce the expression of Clostridium perfringens phage CPS2 lytic enzyme protein. The LB medium contains 10 g / L peptone, 5 g / L yeast extract and 10 g / L NaCl.

[0019] Furthermore, the method further includes crushing the recombinant bacteria, collecting the supernatant, purifying the target protein with a Ni column, and eluting with a high concentration of imidazole buffer to obtain the target protein.

[0020] The present invention also provides the use of the Clostridium perfringens phage CPS2 lytic enzyme mutant in inhibiting Clostridium perfringens under the action of trypsin in the intestinal environment.

[0021] The present invention further provides the use of the Clostridium perfringens phage CPS2 lyase mutant in the preparation of a drug for preventing and / or treating related diseases caused by Clostridium perfringens. Preferably, the drug is suitable for oral administration; the disease is food poisoning, necrotic enteritis or enterotoxemia caused by Clostridium perfringens. The Clostridium perfringens phage CPS2 lyase mutant can be used as an alternative antibiotic product to exert an antibacterial effect in the intestine through oral administration, so as to prevent and treat related diseases caused by Clostridium perfringens.

[0022] The present invention has the beneficial effect of constructing a large number of mutants by means of genetic engineering, through the method of protein structural analysis and directional transformation, and then screening and obtaining the CPS2 lyase mutant of Clostridium perfringens bacteriophage with enhanced anti-trypsin enzymolysis performance in the intestinal environment. The obtained mutant enzyme has higher antibacterial activity than the wild-type enzyme under the action of trypsin in the intestinal environment. Among them, the antibacterial activity of some mutants was increased by 2.08 times or even 2.47 times, indicating that its anti-trypsin enzymolysis performance was significantly enhanced. Therefore, the mutant is more conducive to the lyase as an alternative antibiotic product to play an antibacterial effect in the intestine through oral administration, prevent and treat related diseases caused by Clostridium perfringens, and improve intestinal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : SDS-PAGE of the purified wild-type and mutant proteins of CPS2 lytic enzyme from Clostridium perfringens phage.

[0024] Figure 2 :The antibacterial effect of wild-type and mutant CPS2 lytic enzyme from Clostridium perfringens phage under the action of trypsin in the intestinal environment. DETAILED DESCRIPTION

[0025] The method of the present invention is further described below in conjunction with the examples. The experimental methods for which specific conditions are not specified in the examples can usually be operated according to the conditions in conventional experiments in the field of molecular biology, or according to the instructions of commercial manufacturers such as plasmids and strains. Those skilled in the art can better understand and grasp the present invention with the help of the examples. Mutation PCR is a site-directed mutagenesis PCR familiar to those skilled in the art. Unless otherwise specified, the reagents and instruments used in the following examples are all commercially available products. The specific implementation cases are as follows:

[0026] Example 1: Gene cloning of wild-type CPS2-WT of Clostridium perfringens bacteriophage CPS2 lytic enzyme

[0027] The present invention uses the Clostridium perfringens phage CPS2 lyase (wild-type CPS2-WT) disclosed in the prior art for mutation screening. The nucleic acid coding sequence of Clostridium perfringens phage CPS2 lyase is shown in SEQ ID NO.1 and the amino acid sequence is expressed in Escherichia coli. GenScript Biotech Co., Ltd. is commissioned to synthesize the gene artificially after codon optimization, and then connected between the NdeI and XhoI restriction sites of the pET-30a (+) vector, transformed into the Escherichia coli cloning host Top10, and screened on an LB plate containing 50 μg / mL of kanamycin. The expression vector contained in the positive bacteria is named pET30a-CPS2, and the plasmid is extracted and sequenced to verify that it is correct.

[0028] Among them, SEQ ID NO.1:

[0029]

[0030] SEQ ID NO.2:

[0031]

[0032] Example 2: Mutation Screening

[0033] 1. Construction of mutants

[0034] In order to further improve the anti-trypsin enzymatic performance of the Clostridium perfringens phage CPS2 lyase (amino acid sequence is SEQID NO.2) in the intestinal environment in Example 1, firstly, according to the literature (Ha, E.; Son, B.; Ryu, S. Clostridium perfringens virulent bacteriophage CPS2 and its thermostableendolysinLysCPS2. Viruses 2018, 10, 251.), it is determined that the amino acids 16 to 134 of the CPS2 lyase are the catalytic active region, the amino acids 166 to 224 are the cell wall binding region, and the 134 to 166 positions are Linker; then the tertiary structure of the CPS2 lyase is modeled to find a total of 13 trypsin cleavage sites in the Loop region of the catalytic domain and the binding domain, and 19 mutants (Table 1, Mutant 1 to 19) are designed for these 13 mutation sites. At the same time, a combined mutant (Table 1, Mutant 20) is designed for the 4 trypsin cleavage sites on Linker. Using the plasmid pET30a-CPS2 constructed in Example 1 as a template, site-directed mutagenesis primers were designed for mutants Mutant1 to 20, and reverse PCR was performed. The PCR purified products were transformed into E. coli cloning host Top10, respectively, and screened on LB plates containing 50 μg / mL of kanamycin. The expression vectors contained in the positively screened bacteria were named pET30a-Mutant1 to 20, respectively, and the plasmids were extracted and sequenced to verify correctness.

[0035] 2. Protein expression and purification methods

[0036] The plasmid was transformed into competent cells of Escherichia coli BL21 (DE3) by a method known in the art. The competent cells of Escherichia coli BL21 (DE3) were purchased from Beijing Qingke Biotechnology Co., Ltd. The wild-type expression vector pET30a-CPS2 of Example 1 and 20 successfully constructed mutant vectors pET30a-Mutant1 to 20 were transformed into competent cells by heat shock method. Positive clones were screened for PCR verification and sequencing verification and then used for later use.

[0037] The wild-type and mutant positive monoclonal strains were picked up with an inoculation needle and inoculated into 5 mL LB medium, cultured at 37°C, 220 rpm overnight, and then inoculated into 250 mL LB medium at a 2% (V / V) inoculation volume, cultured at 37°C, 220 rpm for 2-3 h. 600 When the p-value reached 0.8, IPTG was added at a final concentration of 0.5 mM to induce expression. To prevent the formation of inclusion bodies, the expression conditions were 150 rpm and 16°C overnight to induce protein expression.

[0038] The cells were collected by centrifugation at 7000 rpm, 4°C, for 10 min, and resuspended in Tris-HCl / NaCl (pH 7.5) buffer and broken by ultrasonication in an ice bath; the supernatant was collected by centrifugation at 12000 rpm for 30 min, which was the crude enzyme extract expressed in the cells and filtered through a 0.22 μm water system membrane. 2+ The crude enzyme extract expressed above was purified by affinity chromatography using a chromatography column, eluted with imidazole and then desalted by a desalting column. The protein was stored in a Tris-HCl / NaCl (pH 7.5) buffer and the protein concentration was determined. The wild-type protein was named CPS2-WT, and the mutant proteins were named Mutant plus numbers. The purified proteins were tested by SDS-PAGE. The SDS-PAGE results of the purified CPS2 wild-type and some mutant proteins are shown in Figure 1 .

[0039] 3. Determination of the anti-trypsin enzymatic performance of Clostridium perfringens phage CPS2 lyase and its mutant enzymes

[0040] Take a 24h fresh plate of Clostridium perfringens and wash it with KH2PO4 (50mM, pH 6.8), dilute it with KH2PO4 (50mM, pH6.8) to about 1.0×10 5 CFU / mL~9.0×10 5 CFU / mL bacterial suspension is ready for use.

[0041] The Clostridium perfringens phage CPS2 lyase and its mutant enzymes were diluted in a mixed solution containing 10 mg / mL trypsin, 50 mM KH2PO4, 10% Clostridium perfringens bacterial suspension, pH 6.8, so that the final concentration of the Clostridium perfringens phage CPS2 lyase and its mutant enzymes was 0.78 mg / mL. At the same time, a control group without the wild-type and mutant enzymes of the Clostridium perfringens phage CPS2 lyase was set up. All test samples and control samples were incubated at 36℃±1℃ for 1h, and then the plate colony counts were performed. The Clostridium perfringens plate was cultured for 24h, and the final results were observed, the inhibition rate was calculated, and the relative antibacterial effects of the wild-type and mutant enzymes of the Clostridium perfringens phage CPS2 lyase were compared. The results are shown in Tables 1 and Figure 2 shown.

[0042] The results show that different mutation sites have different effects on the antibacterial activity of Clostridium perfringens phage CPS2 lyase under the action of trypsin in the intestinal environment. The antibacterial activity of most sites decreased or was basically lost after mutation, while the antibacterial activity of some mutants was improved, and the antibacterial activity of some mutants was increased by 2.08 times or even 2.47 times, indicating that its anti-trypsin enzymatic performance was significantly enhanced.

[0043] Table 1

[0044]

Claims

1. A mutant of Clostridium perfringens bacteriophage CPS2 lytic enzyme having higher antibacterial activity than the wild-type enzyme under the action of trypsin in the intestinal environment, wherein the amino acid sequence thereof corresponds to the amino acid sequence shown in SEQ ID NO: 2, and only the following mutations are present: (1) Lysine 58 was mutated to proline; (2) mutation of leucine at position 91 to phenylalanine; (3) Lysine 214 is mutated to proline; or (4) Lysine at position 212 was mutated to glycine, and leucine at position 213 was mutated to proline.

2. The coding gene of the Clostridium perfringens phage CPS2 lytic enzyme mutant as claimed in claim 1.

3. A recombinant vector containing the coding gene as claimed in claim 2.

4. A recombinant bacterium containing the coding gene that can express the coding gene as claimed in claim 2.

5. A method for recombinant expression of the Clostridium perfringens phage CPS2 lytic enzyme mutant according to claim 1, characterized in that: It is obtained by expressing a recombinant bacterium containing a recombinant expression vector, wherein the recombinant expression vector contains the coding gene as described in claim 2.

6. The method according to claim 5, characterized in that The method also includes crushing the cells of the recombinant bacteria, collecting the supernatant, purifying the target protein with a Ni column, and eluting with a high concentration of imidazole buffer to obtain the target protein.

7. Use of the Clostridium perfringens phage CPS2 lytic enzyme mutant according to claim 1 in preparing a product for inhibiting Clostridium perfringens under the action of trypsin in the intestinal environment.

8. Use of the Clostridium perfringens phage CPS2 lytic enzyme mutant according to claim 1 in the preparation of a drug for preventing and / or treating Clostridium perfringens-induced related diseases.

9. The use according to claim 8, characterized in that The medicine is suitable for oral administration; the disease is food poisoning, necrotic enteritis or enterotoxemia caused by Clostridium perfringens.

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