CN117866930A - Salmonella phage lyase and application thereof - Google Patents

Abstract

The invention discloses a salmonella phage lyase and application thereof, belongs to the technical field of biology, and particularly relates to a phage lyase for salmonella and application thereof, wherein the lyase is a salmonella phage lyase Lys52, and the amino acid sequence of the lyase is SEQ ID No.2; the protein is subjected to prokaryotic expression and purification to obtain recombinant lyase, and the recombinant lyase can cleave salmonella of different serotypes, has a relatively wide cleavage spectrum and relatively good bactericidal activity, and can be used for preparing antimicrobial agents of salmonella and certain bacteria; the invention develops the high-efficiency lyase biological agent based on the biological engineering technology, and the agent can be used singly or in a compound way, thereby providing a safe and non-toxic enzyme preparation source for preventing and controlling the livestock and poultry salmonella disease and preventing and controlling the food salmonella pollution.

Description

Salmonella phage lyase and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a salmonella phage lyase and application thereof.

Background

At present, due to the widespread use and abuse of antibiotic drugs, the multidrug resistance of the whole salmonella has increased from 20% -30% of the 90 th year of the 20 th century to 70% of the beginning of the present century. With further exacerbation of antibiotic resistance, many salmonella produce severe multi-drug resistance (MRD) phenomena during long-term evolution, and MRD salmonella has been frequently reported. The salmonella infection is controlled by purifying means and antibiotics in farms and food chains, but the bacteria are spread in a vertical mode and a horizontal mode, so that the control measures are not ideal, and the control and solving of the salmonella pollution are always a problem to be overcome worldwide.

As a novel antibacterial agent and an antibiotic substitute, the bacteriophage lyase has the characteristics of rapid action, difficult resistance generation and synergistic sterilization with other antibiotics, and becomes a potential sterilization preparation which is more and more focused by scientists, and particularly the research on multi-drug resistant bacteria has become a current hot spot. Phage lytic enzymes are the core components of phage that perform the lytic function. It is a class of cell wall hydrolases that are characteristic of double-stranded DNA phages and are synthesized late in viral replication. Through sequence alignment, phage lyase has structural similarity, namely contains 2 structural domains, namely a relatively conserved N-terminal enzyme activity catalytic region and a choline binding region of C-terminal specific cell wall teichoic acid with a large difference. It was found that the N-terminus alone of the cell wall hydrolase is catalytically inactive, both in vitro and in vivo, and that the N-terminus catalytic activity is only exhibited after perfect binding of the N-and C-termini. In the aspect of sterilization, the phage lyase has the advantages of high-efficiency sterilization, short action time, safe use, no interference to normal flora of animal intestinal tracts and the like because of the species specificity.

As a special biological bactericide, the phage lyase has the advantages of high-efficiency sterilization, short action time, safe use, no interference to normal flora of animal intestinal tracts due to species specificity, and the like. The characteristics lay a foundation for the research and application of the salmonella in the fields of expression and activity, and develop a new idea for effectively preventing and treating diseases caused by salmonella infection or pollution.

Through analysis of the existing literature, the expression of foreign protein of interest in E.coli by recombinant genetic engineering technology is the main method for obtaining functional protein of interest in large quantity and economically at present. The invention utilizes a PCR method to amplify phage lyase genes, and the amplified products are subjected to prokaryotic expression to obtain the phage lyase which is high-efficiency and soluble in expression and has high-efficiency antibacterial activity, and the salmonella phage lyase is not reported in China.

Disclosure of Invention

The invention aims to solve the technical problem of providing a salmonella phage lyase and application thereof aiming at the defects of the background technology.

The invention adopts the following technical scheme for solving the technical problems:

an amino acid sequence of the salmonella phage lyase Lys52 is shown in SEQ ID No. 1.

A gene ORF52 encoding the salmonella phage lyase of claim 1, having a nucleotide sequence as shown in SEQ ID No. 2.

Preferably, the primers for amplifying the lyase nucleotides are:

F:3’-ACAGGATCCATGCAACTCTCAAGAA-5’；

R:3’-CGCAAGCTTCTTT GGATATACACTG-5’。

a plasmid containing the salmonella phage lyase gene ORF 52.

A recombinant bacterium comprising a plasmid containing the salmonella phage lyase gene ORF 52.

Realizes the application of the salmonella phage lyase Lys52,

1) The application of the lyase Lys52 in specific salmonella lysis;

2) Application of the lyase Lys52 in preparing biological antibacterial preparations;

3) The application of the lyase Lys52 in preparing a medicine combination for resisting salmonella infection;

4) The application of the lyase Lys52 as a food additive;

5) The application of the lyase Lys52 as a feed additive.

Compared with the prior art, the technical scheme provided by the invention has the following technical effects:

1. the salmonella phage lyase Lys52 provided by the invention has the characteristics of rapid sterilization, no resistance, high safety and no toxic or side effect, and is a potential sterilization preparation;

2. the invention utilizes the salmonella phage lyase Lys52 obtained in the invention, and can further study the effect of phage lyase as an effective bactericidal preparation in resisting salmonella infection, so as to make contribution in the prevention and control aspect of salmonella.

Drawings

FIG. 1 is an agarose electrophoresis chart of a PCR amplification product of a lysase lys52 gene;

FIG. 2 is a diagram of double agarose gel electrophoresis of recombinant expression plasmid pET-32a (+) -lys52;

FIG. 3 is an SDS-PAGE electrophoresis of recombinant lyase protein pET-32a (+) -Lys52;

FIG. 4 is an SDS-PAGE electrophoresis of purified recombinant protein pET-32a (+) -Lys52;

FIG. 5 is a recombinant protein Lys52 lyses Salmonella PSPu-116;

FIG. 6 is a graph showing the bacteriostatic effect of a biological bactericide comprising Lys52 as a main ingredient in chicken;

FIG. 7 is a graph showing the bacteriostatic effect of a biological bactericide comprising lyase Lys52 as a main ingredient in a cultivation environment.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to the accompanying drawings:

the present invention will be described in further detail with reference to the following specific examples, which will aid in understanding the present invention, but the scope of the present invention is not limited to the following examples.

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

An amino acid sequence of the salmonella phage lyase Lys52 is shown in SEQ ID No. 1.

Lys52 amino acid sequence:

MQLSRKGLEAIKFFEGLKLEAYEDSAGIPTIGYGTIRIDGKPVKMGMKITAEQAEQYLLADVEKFVAVVNKAIKVPTSQNEFDALVSETYNIGITAMQDSTFIKRHNAGNKVGCAEAMQWWNKVTVKGKKVTSNGLKNRRRMEADIYLDSVYPK。

a gene ORF52 encoding the salmonella phage lyase of claim 1, having a nucleotide sequence as shown in SEQ ID No. 2.

ORF52 lyase nucleotide sequence:

ATGCAACTCTCAAGAAAAGGTTTAGAGGCTATTAAGTTCTTCGAAGGTCTGAAGTTAGAGGCTTACGAAGACTCTGCCGGAATCCCAACAATTGGGTATGGTACAATCCGTATTGACGGAAAACCTGTTAAGATGGGTATGAAAATCACTGCCGAGCAAGCTGAACAGTATCTTCTTGCAGATGTTGAGAAGTTTGTTGCAGTAGTGAATAAAGCTATCAAGGTTCCAACTTCTCAGAATGAATTCGATGCACTTGTAAGTGAAACATACAACATCGGTATCACAGCTATGCAGGATTCTACATTTATCAAGCGCCACAATGCTGGTAATAAGGTAGGTTGTGCAGAAGCTATGCAGTGGTGGAACAAGGTCACAGTCAAGGGTAAGAAGGTCACTTCAAACGGCCTGAAAAACAGACGTAGAATGGAAGCTGACATTTATCTTGACAGTGTATATCCAAAG。

preferably, the primers for amplifying the lyase nucleotides are:

F:3’-ACAGGATCCATGCAACTCTCAAGAA-5’；

R:3’-CGCAAGCTTCTTT GGATATACACTG-5’。

a plasmid containing the salmonella phage lyase gene ORF 52.

A recombinant bacterium comprising a plasmid containing the salmonella phage lyase gene ORF 52.

Another object of the invention is to achieve the use of the salmonella phage lyase Lys52;

1) The application of the lyase Lys52 in specific salmonella lysis;

2) Application of the lyase Lys52 in preparing biological antibacterial preparations;

3) The application of the lyase Lys52 in preparing a medicine combination for resisting salmonella infection;

4) The application of the lyase Lys52 as a food additive;

5) The application of the lyase Lys52 as a feed additive.

The application of the salmonella phage lyase Lys52 is realized, and the method is characterized by comprising the salmonella phage lyase and excipients.

The excipient comprises one or more of buffer solution, metal ion and penetrant.

The lytic salmonella phage PSPu-4-116 for the test is preserved in China center for type culture Collection (China center for type culture Collection) on 2 months and 21 days 2011, and has the address of university of Wuhan, wu Changou, and the preservation number of: cctccc M2011042.

Example 1 preparation of a recombinant lyase protein:

1. phage particles were prepared and phage genomes were extracted:

centrifuging the amplified lysate of phage, removing precipitate, filtering supernatant with 0.22 μm filter membrane, adding DNase I and RNase A with final concentration of 1 μg/mL, and incubating at 37deg.C for 30min; sequentially adding 5.8g NaCl and 10% PEG 8000, shaking to dissolve, and ice-bathing for 1 hr; centrifuging at 10000rpm at 4deg.C for 30min, and removing supernatant; adding 5mL of SM solution to fully dissolve phage; adding chloroform with the same volume for extraction, and oscillating for 30s; centrifuging at 5000rpm for 15min at 4 ℃, recovering the aqueous phase containing the phage particles, obtaining purified phage particles. The phage genome of the obtained phage particles is extracted by using a lambda phage genome rapid extraction kit, and the kit is specifically carried out according to the specification of the kit.

2. Cloning of phage lyase Lys52 and construction of expression vector:

the whole genome of the lytic salmonella phage PSPu-4-116 was sequenced by high throughput sequencing, and the phage lyase-encoding gene was determined by comparison of the whole genome splicing software and the Genebank database BLASTN sequence, which was 462bp in length. A pair of specific primers is designed according to the gene sequence of the lyase,

(BamHⅠ)

F:3’-ACAGGATCCATGCAACTCTCAAGAA-5’

R:3’-CGCAAGCTTCTTTGGATATACACTG-5’

(HindⅢ)

the whole length sequence of Lys52 gene is amplified by PCR procedure using the extracted phage genome as template and the specific primer, and the size of amplified fragment is identified by 1% agarose electrophoresis, and the result is shown in FIG. 1, the size of PCR amplified fragment is correct and is 462bp. FIG. 1 is an agarose electrophoresis chart of PCR amplification products of lysase lys52 gene.

Lane 1.DNA Maker DL2000; PCR product; DNA marker 1Kb

Firstly cutting glue, purifying and recovering amplified fragment Lys52 through a glue recovery kit, connecting the fragment with an expression vector pET-32a (+) at 16 ℃ overnight, converting competent cells of escherichia coli BL21 (DE 3) the next day, coating the converted bacterial liquid on a flat plate containing ampicillin (50 mug/mL), sequencing and identifying the converted positive clone, and naming the positive clone as ORF52 gene, wherein the nucleotide sequence is Seq ID No.1;

positive clones were picked and inoculated into LB liquid medium containing ampicillin (50. Mu.g/mL), after incubation at 37℃overnight, plasmids were extracted by alkaline lysis, and double restriction identification was performed with BamH I and Hind III, while the BamH I single restriction pET-32a (+) empty plasmid was used as a control. The plasmid identified as correct was designated pET-32a (+) -Lys52 and the recombinant strain was designated BL21 (DE 3) -pET-32a (+) -Lys52.

As shown in FIG. 2, after double digestion of the recombinant plasmid pET-32a (+) -Lys52 with Bam H I and Hind III, pET-32a (+) vector and Lys52 coding gene appeared at 5.4kb and 462bp, respectively, and the sizes were consistent with expectations, indicating that the recombinant plasmid was constructed correctly.

FIG. 2 recombinant expression plasmid pET-32a (+) -lys52 double enzyme agarose electrophoresis

Lane 1, double cleavage pET32a (+) -lys52; lane 3.DNA Marker DL2000

3. Inducible expression and purification of phage lyase Lys52 protein

Inoculating recombinant BL21 (DE 3) -pET-32a (+) -Lys52 into LB culture solution containing ampicillin (50 mu g/mL), and culturing at 37 ℃ with shaking overnight; the next day, transfer to 1000mL LB medium according to the ratio of 1:100, shake culture at 37 ℃ to OD ₆₀₀ When the value is about 0.3-0.5, IPTG with the final concentration of 1.0mM is added and the induction is carried out for 16h at 26 ℃. Collecting bacterial cells and culture supernatant, crushing bacterial cells by ultrasonic waves, centrifuging at 4 ℃ and 10000rpm for 10min, collecting bacterial cell supernatant, and analyzing protein expression conditions in the culture supernatant and the bacterial cell supernatant by SDS-PAGE. The cleavage supernatant was purified by nickel column affinity chromatography (Ni-NTA, GE Healthcare) and the procedure was specifically followed as described in the kit. Finally, the purified soluble protein was designated as lyase Lys52 and the protein concentration was determined using the BCA protein concentration determination kit (Thermo).

SDS-PAGE analysis results are shown in FIG. 3, after recombinant BL21 (DE 3) -pET-32a (+) -Lys52 is induced by IPTG, a thicker protein band is arranged at a position of about 37kD in culture supernatant and thallus supernatant, and the size accords with expected size, so that the recombinant BL21 (DE 3) -pET-32a (+) -Lys52 is correctly constructed, and the expressed lyase protein product Lys52 is soluble protein. As shown in FIG. 4, after the bacterial supernatant was purified by nickel column affinity chromatography, a coarser protein band was present at about 37kD, and no other protein remained, indicating a better purification effect.

FIG. 3 is a SDS-PAGE electrophoresis of recombinant lyase protein pET-32a (+) -Lys52;

lanes: protein Maker; BL21 (DE 3); 3. lys52 of the culture supernatant; 4. thallus supernatant Lys52

FIG. 4 is an SDS-PAGE electrophoresis of purified recombinant protein pET-32a (+) -Lys52;

lanes: protein Maker;2. thallus supernatant Lys52

Example 2 high-efficient bactericidal effect of phage lyase Lys52 on Salmonella PSPu-116;

in this example, salmonella PSPu-116 was selected to verify the bactericidal effect of the lyase.

After the logarithmic phase of salmonella PSPu-116 was collected by centrifugation and washed 2 times with PBS, the cells were resuspended with PBS and the absorbance OD was adjusted ₆₀₀ About 1.0; adding 100. Mu.L recombinant lyase protein Lys52 with concentration of 100. Mu.g/mL and EDTA with final concentration of 5mM into 100. Mu.L bacterial solution, culturing at 37deg.C, and measuring absorbance OD ₆₀₀ The experiment was set up to 3 replicates with PBS as negative control.

As shown in FIG. 5, 10. Mu.g of lyase was shown to be capable of making the bacterial liquid OD in 2 hours under the synergistic effect of EDTA as compared with the PBS control group ₆₀₀ Reduces the time to about 0.12, and when the action time reaches 5 hours, the lyase can lead the bacterial liquid OD ₆₀₀ The result shows that the lyase Lys52 can specifically cleave the salmonella PSPu-116 with high efficiency.

FIG. 5 recombinant lyase protein Lys52 lyses Salmonella PSPu-116;

example 3 sterilization experiments of biological bactericides with phage lyase Lys52 as the main ingredient in chicken breast this example selected Salmonella PSPu-116 to verify the sterilizing effect of the lyase.

Dividing chicken into two groups, and inoculating Salmonella 10 to Lys52 group ⁴ CFU, and 100. Mu.L of lyase Lys52 at a concentration of 100. Mu.g/mL and EDTA at a final concentration of 5mM were added; adding 10 to the negative control group ⁴ CFU salmonella and 100 μl PBS. And (3) respectively placing the prepared samples at 4 ℃ for 8 hours, and detecting the quantity of salmonella every 2 hours.

As shown in FIG. 6, after 2 hours of action at 4 ℃, the number of salmonella is reduced to below 1000CFU, and as the action time of the lyase is prolonged, the number of salmonella is continuously reduced, after 8 hours, the number of salmonella is lower than 100CFU, and the number of salmonella is obviously different from that of a PBS control group, and the result shows that the biological bactericide taking phage lyase Lys52 as a main component has high-efficiency sterilization effect in food.

FIG. 6 shows the bacteriostatic effect of the biological bactericide comprising phage lyase Lys52 as the main component in chicken.

EXAMPLE 4 biological Bactericide with phage lyase Lys52 as the main ingredient to control Salmonella contamination in culture Environment

EDTA treated at a concentration of 10 ⁴ cfu/mL of salmonella is smeared on the surface of a slaughterhouse trough, then spraying and killing are carried out on the trough by using a lyase Lys52 biological bactericide with the concentration of 100 mug/mL, after 2 hours, the detection of the salmonella is started, and a PBS group is used as a control.

The detection result is shown in fig. 7, after spraying the biological bactericide for 2 hours, the number of salmonella on the surface of the trough is reduced to below 500CFU, and after 6 hours, the number of salmonella on the surface of the trough is reduced to below 100CFU, which indicates that the biological bactericide can effectively kill salmonella in the culture environment.

FIG. 7 is a graph showing the bacteriostatic effect of a biological bactericide comprising phage lyase Lys52 as a main component in a cultivation environment.

Example 5 biological bactericides based on phage lyase Lys52 for treatment of Salmonella infection;

infection experiments were performed using 20 chicks of 1 day old. Firstly, spraying and attacking experiments are carried out by using salmonella PSPu-16, all the 3 rd day of attacking chickens are poor and have some diarrhea symptoms, then 20 infected chickens are equally divided into 2 groups, 10 chickens in each group are treated by intramuscular injection, and the chickens infected with salmonella are observed and recorded continuously for 24 hours by using a lyase Lys52 biological bactericide and PBS respectively.

The results show that: the chicks in the PBS group are listless and severe in diarrhea; however, after treatment with Lys52 biocide, the diarrhea symptoms began to be relieved around 24 hours in the chicks, and the chicks slowly recovered, and the results showed that Lys52 biocide was an effective means of controlling salmonella infection.

EXAMPLE 6 analysis of the cleavage spectrum of phage lyase Lys52

The test selects 24 strains of bacteria (including Salmonella, E.coli) for analysis of the host profile of lyase Lys52. Firstly, respectively preparing 24 bacterial lawn, then equally dividing each flat lawn into two areas, taking 10 mu L of lyase Lys52 to be dripped on the surface of the lawn, dripping 10 mu L of PBS to be used as a control in the other area, inverting the liquid drops to be cultured for 12-16 hours at 37 ℃ after the liquid drops are dried, and observing the results.

As a result, as shown in Table 1, the region to which lysase Lys52 was added dropwise had a lysis effect on 18 strains out of the 24 strains collected, and the lysis rate was 75%. The prepared lyase Lys52 has the property of cross-genus cleavage and is a broad host spectrum lyase. The results of the phage lyase Lys52 cleavage spectrum are shown in Table 1;

TABLE 1

The invention provides a salmonella phage lyase and application thereof, and the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims (6)

1. A salmonella phage lyase Lys52, comprising: the amino acid sequence is shown as SEQ ID No. 1.

2. A gene ORF52 encoding the salmonella phage lyase of claim 1, wherein: the nucleotide sequence is shown as SEQ ID No. 2.

3. The salmonella phage lyase Lys52 of claim 1, wherein primers for amplifying the lyase nucleotides are:

F:3’-ACAGGATCCATGCAACTCTCAAGAA-5’；

R:3’-CGCAAGCTTCTTT GGATATACACTG-5’。

4. a plasmid comprising the salmonella phage lyase gene ORF52 of claim 2.

5. A recombinant bacterium comprising the plasmid according to claim 4.

6. The use of a bacteriophage lytic enzyme Lys52 according to claim 1,

1) The application of the lyase Lys52 in specific salmonella lysis;

2) Application of the lyase Lys52 in preparing biological antibacterial preparations;

3) The application of the lyase Lys52 in preparing a medicine combination for resisting salmonella infection;

4) The application of the lyase Lys52 as a food additive;

5) The application of the lyase Lys52 as a feed additive.