CN117802127B - Salmonella phage lyase and gene, gene recombinant expression vector and application thereof - Google Patents

Abstract

The invention discloses salmonella phage lyase Lys20059, and a coding gene, an expression vector and application thereof. The amino acid sequence of the salmonella phage lyase Lys20059 is shown as SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1. The salmonella phage lyase protein has a bacteriostasis ring on 42 strains of salmonella, has a salmonella lysis rate of 84%, shows a good salmonella lysis effect on salmonella, has a lysis effect on various salmonella serotypes, and has a wide lysis spectrum. The salmonella phage lyase Lys20059 has the effect of inhibiting the growth of salmonella, and has obvious high-concentration effect. The antibacterial agent is used for industrial production, aims at reducing cost, and has good antibacterial effect on salmonella under the action of a mixed preparation of a lower amount of bacteriophage and lyase.

Description

Salmonella phage lyase and gene, gene recombinant expression vector and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a high-activity salmonella lyase, gene expression and application.

Background

Salmonella (Salmonella pullorum) belongs to the Enterobacteriaceae family, and gram-negative Enterobacter is the causative agent of salmonellosis. Among Salmonella are human pathogenic bacteria, veterinary pathogenic bacteria, and human and veterinary pathogenic bacteria. After salmonella infection, clinical symptoms such as gastrointestinal inflammation, fever, diarrhea, septicemia and the like generally occur. After infection with salmonella, animals may be affected in fertility and even have increased symptoms and mortality.

At present, antibiotics are mainly used in a method for treating salmonellosis, but due to unreasonable use of the antibiotics, the problems of bacterial multi-drug resistance, drug residues, food safety, unbalanced environmental flora and the like are caused, and the health of human beings and animals is seriously threatened.

Thus, in view of the reality of antibiotic resistance and policy requirements, there is a need to find effective methods for treating salmonellosis to alleviate or even solve the problems caused by antibiotic abuse.

In recent years, polypeptide antibacterial substances are becoming important points of research, because of their green and safe properties, and phage lytic enzymes are also widely used, among them, as antibacterial substances for polypeptides.

Phages replicate and reproduce bacteria as hosts, where mature particles are released by the synthesis of a class of proteolytic enzymes that lyse the bacterial cell walls. Such hydrolases are called phage lyases, also known as lyases, endolysins, lywallases, the molecular target of which is highly conserved peptidoglycan structures at the strain or genus level, which are critical for bacterial survival. During the co-evolution of phage and bacterial hosts for several billion years, lytic enzymes have also evolved and can attack molecular targets that are not easily altered by bacteria, and phages that do not attack these key targets will be naturally selected for elimination. Therefore, the case where bacteria develop resistance to lyase is extremely rare. The natural phage lyase can destroy bacterial biofilm, and has the advantages of green safety, difficult drug resistance and the like.

Therefore, providing a phage lyase and its use in the treatment of salmonellosis is a problem that those skilled in the art are urgent to address.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides salmonella phage lyase, a gene thereof, a gene recombination expression vector and application thereof.

The technical scheme of the invention is as follows:

In a first aspect, the application provides a coding gene of salmonella phage lyase Lys20059, wherein the nucleotide sequence of the coding gene of salmonella phage lyase Lys20059 is shown as SEQ ID NO. 1.

ATGTCAAACCGAAACATCAGTGACAACGGGTTACACTTCACCGCCGCGTTCGAGGGGTTCCGAGGAACCGCGTACAAGGCAACGAAGAACGAGAAGTACCTTACTATTGGTTATGGTCACTATGGTGCCGATGTGAAAGAAGGTCAGAAGATTACCGAAGGCCAGGGTCTTCTGCTGCTGCACAAGGATATGGCTAAGGCCGTAGCTGCGGTAGACACCGTTGCGCATCCGTCTCTAAATCAGTCACAGTTCGACGCCGTGTGTGACCTTGTGTATAACGCTGGTGCAGGTGTGATTGCCGCTTCTACCGGTACAGGTCAGGCGCTGCGCAAAGGCGATGCATCTACACTGCGTAATAAGTTAACTCAGTTCCATTATCAGAACGGCAAATCACTCCTCGGATTACGTCGTCGTGCCGCCGGTCGTGTTGCGCTGTTCGATGGCATGCTGTGGCAACAGGCCGAAGCTATCGGCCGTGGTGCAAAGTAG

Further, primers for amplifying the genes are as follows:

Lys-20059-F：CGGAATTCTCAAACCGAAACATCAGTG,SEQ ID NO.3;Lys-20059-R：CCCAAGCTTCTTTGCACCACGGCCGATAGCT,SEQ ID NO.4.

in a second aspect, the application provides a recombinant expression vector comprising a gene encoding the above-described salmonella phage lyase Lys 20059.

In a third aspect, the present application provides a host cell comprising a recombinant expression vector as described above.

In a fourth aspect, the invention also provides a salmonella phage lyase Lys20059, wherein the phage lyase Lys20059 is encoded by the encoding gene of the salmonella phage lyase Lys20059 in the first aspect, and the amino acid sequence of the salmonella phage lyase Lys20059 is shown in SEQ ID NO. 2.

MSNRNISDNGLHFTAAFEGFRGTAYKATKNEKYLTIGYGHYGADVKEGQKITEGQGLLLLHKDMAKAVAAVDTVAHPSLNQSQFDAVCDLVYNAGAGVIAASTGTGQALRKGDASTLRNKLTQFHYQNGKSLLGLRRRAAGRVALFDGMLWQQAEAIGRGAK

In a fifth aspect, based on the same invention, the invention also provides the use of the salmonella phage lyase according to the fourth aspect for inhibiting salmonella of different serotypes, in a biological formulation.

The invention has the following advantages:

1. The invention provides a salmonella phage lyase Lys20059 with an amino acid sequence shown in SEQ ID NO.2 and a nucleotide sequence of a coding gene shown in SEQ ID NO. 1. The phage lyase obtained by purification can well inhibit salmonella infection by constructing a recombinant vector and expressing in a genetically engineered strain escherichia coli BL21 (DE 3), and has potential clinical application value.

2. The salmonella phage lyase protein has a bacteriostasis ring for 42 strains of salmonella, has a salmonella lysis rate of 84%, shows a good salmonella lysis effect, has a salmonella lysis effect on various salmonella serotypes, and has a relatively wide lysis spectrum.

3. The salmonella phage lyase Lys20059 has the effect of inhibiting the growth of salmonella, and has obvious high-concentration effect. The antibacterial agent is used for industrial production, aims at reducing cost, and has good antibacterial effect on salmonella under the action of a mixed preparation of a lower amount of bacteriophage and lyase.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is apparent that the drawings in the following description are only one embodiment of the present invention, and that other embodiments of the drawings may be derived from the drawings provided without inventive effort for a person skilled in the art.

Fig. 1: the phage lyase Lys-20059 fragment agarose electrophoresis detection result of the invention;

fig. 2: the phage lyase Lys-20059 expression vector agarose electrophoresis detection result;

Fig. 3: SDS-PAGE detection result of the phage lyase Lys-20059;

fig. 4: the invention relates to a bacteriostasis difference curve of phage lyase Lys-20059 and phage preparation combination on salmonella with different concentrations.

Detailed Description

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 fall within the scope of the invention. In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

EXAMPLE 1 extraction of Salmonella phage genome

1.1 Salmonella phage RDP-SA-20059

Salmonella phage RDP-SA-20059 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2021 and 6 months and a preservation code of CGMCC No.22490.

1.2 Preparation of the genome of Salmonella phage RDP-SA-20059

Extracting salmonella phage RDP-SA-20059 by using a viral genome DNA extraction kit according to the method described in the application specification, and finally obtaining the extracted product, namely the genome DNA of the salmonella phage RDP-SA-20059, and storing at the temperature of minus 20 ℃ for standby.

Example 2 functional alignment search of salmonella phage RDP-SA-20059 lyase Gene

2.1 Phage genome sequencing

Phage whole genome sequencing and analysis: constructing a library by using an Illumina TruSeq ^TM Nano DNA SAMPLE PREP KIT method; the method comprises the following specific steps:

(1) Library construction with an initial amount of 1. Mu.g of phage genomic DNA;

(2) The Covaris M220 breaks the DNA to 300-500bp by ultrasound;

(3) Filling in the 3' end, adding A, and connecting index joint (TruSeq ^TM Nano DNA SAMPLE PREP KIT);

(4) Library enrichment, PCR amplification of 8 cycles;

(5) Recovering the target band (CERTIFIED LOW RANGE ULTRA AGAROSE) from the 2% agarose gel;

(6) Quantitative TBS380 (PicoGreen), mixing and loading according to the data proportion;

(7) carrying out bridge PCR amplification on cBot solid-phase carriers to generate clusters;

(8) The Illumina Hiseq sequencing platform was subjected to 2X 150bp sequencing.

2.2 Functional alignment analysis of the salmonella phage RDP-SA-20059 lyase Gene

By analyzing the sequencing annotation result, the predicted amino acid sequence with the function of cracking the cell wall is compared with public data, and is identified as salmonella phage lyase, which is named as Lys20059. The nucleotide sequence of the coding gene of the salmonella phage lyase Lys20059 is shown as SEQ ID NO. 1; the amino acid sequence of the salmonella phage lyase Lys20059 is shown as SEQ ID NO. 2.

Example 3: cloning of salmonella phage RDP-SA-20059 lyase gene, recombinant expression vector and construction of expression strain

3.1 PCR amplification of phage RDP-SA-20059 lyase Gene

The primers were designed according to the sequence of the lyase:

Lys-20059-F：CGGAATTCTCAAACCGAAACATCAGTG,SEQ ID NO.3;Lys-20059-R：CCCAAGCTTCTTTGCACCACGGCCGATAGCT,SEQ ID NO.4; The cleavage sites were EcoRI and HindIII, respectively.

Adding double enzyme cutting sites and protecting bases, taking phage genome as a template, and amplifying a lyase gene by using PCR (polymerase chain reaction), wherein the PCR procedure is shown in the following table 1; and (5) performing agarose gel electrophoresis detection on the PCR product.

Double digestion of the cleavage enzyme gene fragment (reaction system is shown in Table 2), phosphorylation and recovery of agarose gel fragment cleavage gel, and agarose gel electrophoresis detection of PCR products is carried out, and the results are shown in FIG. 1.

TABLE 1 PCR procedure for amplifying phage lyase genes

TABLE 2 double cleavage reaction System for PCR products

3.2 Construction of recombinant plasmid for phage lyase

(1) Preparation of Linear plasmids

The expression vector was constructed by selecting plasmid pET-28a, the vector was subjected to double digestion (Table 3), dephosphorylation (Table 4) and agarose gel fragment gel digestion recovery, and the plasmid enzyme digestion product was subjected to agarose gel electrophoresis detection.

Table 3 plasmid double enzyme cleavage reaction system

TABLE 4 phosphorylation reaction System of lyase fragments

(2) Construction of expression vectors

The cleavage enzyme fragment and the plasmid after cleavage were ligated, the ligation reaction system is shown in Table 5, and the construction result of the expression vector is shown in FIG. 2.

TABLE 5 ligation reaction System of cleavage enzyme fragments and restriction enzyme plasmids

3.3 Construction of Salmonella phage lyase Lys 20059-expressing Strain

1) Mu.l of the above ligation product was added to 50-100. Mu.l of competent cell BL21 (DE 3) and left to stand on ice for 30min. Then heat-shocking in a water bath at 42 ℃ for 90s, and placing the mixture on ice for 2-3min. 1ml of LB liquid medium was added thereto, and the culture was resumed by shaking at 220rpm at 37℃for 1 hour.

2) The bacterial liquid is coated on LB (antibiotic) plates, the plates are placed forward for about 2 hours at 37 ℃ to allow excessive liquid to permeate into the culture medium, and then the culture medium is inverted and cultured overnight.

3) Single colonies grown on LB+ resistant plates were picked and inoculated into 5-7ml of LB (antibiotic) liquid medium. Shaking table overnight shake culture at 37 ℃;

4) Plasmid extraction and sequencing were performed using the plasmid miniprep kit.

EXAMPLE 4 Induction, purification, preparation of recombinant lyase proteins

1) Transforming the expression plasmid into E.coli BL21 (DE 3), picking the transformant into 5mL of liquid LB medium added with ampicillin, and culturing at 37 ℃ with shaking overnight;

2) The overnight cultured bacterial liquid is transferred into LB liquid medium containing antibiotics according to the proportion of 1 percent, and is cultured at 200rpm and 37 ℃ until the OD600 is 0.6-0.8. 1mL of the bacterial liquid is centrifuged at 12,000rpm for 10min, 100. Mu.L of lysis buffer (50 mM Tris-HCl, 150mM NaCl, pH 8.0) is added to resuspend the bacterial cells, an equal volume of 2X protein loading buffer is added, and the bacterial cells are treated in a boiling water bath for 10min; meanwhile, adding IPTG to a bacterial liquid with the OD600 reaching 0.6-0.8 and the final concentration of 0.6-1.0mM into an inducer (isopropyl-beta-D-thiogalactoside), carrying out induced expression for 4 hours, taking 1mL of bacterial liquid, centrifuging to collect bacteria, adding a proper amount of lysis buffer to resuspend the bacteria, adding an equal volume of 2X protein loading buffer, and carrying out boiling water bath treatment for 10 minutes; SDS-PAGE detection proteins are soluble expressed;

3) Preparing a lyase protein expression thallus by the same method, adding a lysia buffer with the volume of 1/10 of the bacterial liquid, performing ultrasonic crushing, centrifuging at 12000rpm and 4 ℃ for 30min, and collecting a supernatant;

4) Balancing Ni column (His tag protein specific filler), mixing the supernatant sucked and collected by the filler, and incubating at 4 ℃ for 2h; adding the mixture of the supernatant and the filler into the empty column, and collecting the effluent supernatant for further detection;

5) Subjecting to column chromatography with a lysia buffer containing 10mM, 20mM, 50mM, 100mM, 250mM, 300mM, 400mM, and 500mM imidazole, eluting the hetero protein, collecting eluate to be detected, detecting protein concentration during detection, and detecting elution effects of imidazole at different concentrations to prepare lyase protein;

6) 500mM imidazole-eluted protein was collected, assayed for protein concentration, and subjected to thrombin treatment. The N-terminal 6 XHis tag was removed and the treatment conditions are shown in Table 6:

TABLE 6 thrombin treatment system and reaction conditions

7) Dialysis (dialysate: 50mM Tris-HCl, 150mM NaCl, 5% glycerol, pH 7.9) except imidazole: mixing the correct protein sample, adding into the treated dialysis bag, placing into dialysate, and treating at 4deg.C overnight; centrifuging at 12000rpm for 30min if precipitation occurs, and collecting supernatant; adding the dialyzed protein sample into an ultrafiltration tube, concentrating to a proper concentration, sub-packaging into small tubes, and preserving to-80 ℃;

SDS-PAGE detection of lyase Lys-20059 is shown in FIG. 3.

EXAMPLE 5 application of Salmonella phage lyase Lys20059

5.1 Antibacterial spectrum of Salmonella phage lyase Lys20059 against Salmonella

Salmonella 50 strains of different serotypes were selected for 17 Salmonella serotypes in total. The specific operation is as follows: spreading solid LB on a flat plate with the thickness of 3mm, placing a sterile oxford cup on a solid culture medium, mixing 100 mu l of salmonella bacteria liquid with 5ml of 0.7% LB semi-solid culture medium, pouring the mixture on the solid plate with the oxford cup, taking out the oxford cup by using sterile forceps after solidification, and forming a semi-solid culture medium hole. Filtering and sterilizing the prepared lyase protein solution, adding 20 mu l of protein solution into oxford cup hole seeds, culturing overnight at 37 ℃, and observing whether a bacteria inhibition zone exists.

The results are shown in Table 7, the cleavage enzyme protein has a bacteriostasis zone on 42 strains of salmonella, the cleavage rate on salmonella is 84%, the cleavage effect on salmonella is shown, the cleavage effect on salmonella of various serotypes is shown, and the cleavage spectrum is wide.

TABLE 7 Salmonella serotype 50 and cleavage of Salmonella by lytic enzymes

5.2 Use of Salmonella phage lyase Lys20059 in the preparation of a biological preparation for lysing Salmonella

Different concentrations (5, 10, 20, 30 and 40 mug) of lyase proteins are mixed with salmonella phage to form preparations, and bacteriostasis of the phage, the lyase and the mixed preparations of the phage and the lyase on salmonella is detected, wherein the phage performs bacteriostasis experiments with MOI of 1.0.

As shown in FIG. 4, the antibacterial effect of the low-concentration lyase on the phage is similar to that of the phage, and the antibacterial effect of the high-concentration lyase on salmonella is superior to that of the phage alone; the inhibition of salmonella growth by the experimental group of lytic enzymes and phage cocktails is superior to the inhibition of salmonella growth by phage or lytic enzymes alone.

In conclusion, the salmonella phage lyase Lys20059 has a growth inhibition effect on salmonella, and the high concentration effect is obvious. The antibacterial agent is used for industrial production, aims at reducing cost, and has good antibacterial effect on salmonella under the action of a mixed preparation of a lower amount of bacteriophage and lyase.

It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the accompanying claims.

Claims (6)

1. The coding gene of the salmonella phage lyase Lys20059 is characterized in that the nucleotide sequence of the coding gene of the salmonella phage lyase Lys20059 is shown as SEQ ID NO. 1.

2. A recombinant expression vector comprising a gene encoding the salmonella phage lyase Lys20059 of claim 1.

3. A host cell comprising the recombinant expression vector of claim 2.

4. A salmonella phage lyase Lys20059 is characterized in that phage lyase Lys20059 is encoded by the encoding gene of salmonella phage lyase Lys20059 of claim 1, and the amino acid sequence of salmonella phage lyase Lys20059 is shown in SEQ ID NO. 2.

5. Use of salmonella phage lyase Lys20059 of claim 4 for inhibiting salmonella of different serotypes, wherein the use is not diagnostic, therapeutic or prophylactic in relation to a disease.

6. A biological agent, wherein the active ingredient of the biological agent comprises salmonella phage lyase Lys20059 of claim 4.