CN114426980A - Lyase derived from vibrio alginolyticus phage and application thereof - Google Patents
Lyase derived from vibrio alginolyticus phage and application thereof Download PDFInfo
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Abstract
The invention discloses a co-expression vector of lyase genes holA and lysin of bacteriophage, a recombinant strain of the co-expression vector, a bacteriophage co-expression lyase holoA-lysin and a preparation method and application thereof, and the invention obtains the lyase gene of bacteriophage HH109 through PCR amplification and the likeholAAndlysincloning into an expression System and transformation into the target Strain to obtain recombinant DNAAnd (5) forming bacteria. The recombinant bacteria induce and express lyase gene in IPTG to realize efficient lysis of vibrio alginolyticus, colibacillus and other bacteria, and the lysis has no selectivity and wide application range. The lyase of the bacteriophage HH109 is suggested to have potential application value in preventing and treating vibrio infection such as vibrio alginolyticus.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a lyase derived from vibrio alginolyticus phage and application thereof.
Background
Vibrio alginolyticus is an important pathogenic bacterium for aquaculture, and causes great harm to the mariculture industry. In addition, the Vibrio may cause some human diseases, such as infections of five sense organs and gastrointestinal tract, otitis media and food poisoning. In recent years, diseases are frequently caused by rapid shift of aquaculture modes from extensive aquaculture modes to intensive aquaculture modes with high density. The traditional treatment methods using antibiotics and chemical disinfectants and the like generate a plurality of problems, such as drug resistance of bacteria, safety problems caused by pathogenic bacteria transmission, micro-ecological imbalance of aquaculture water environment, damage to human living environment and the like. These problems, in turn, make it difficult to effectively treat diseases of aquatic animals caused by bacterial pathogens such as vibrio, which has become a critical factor that restricts the sustainable development of aquaculture industry. Therefore, a safe and effective prevention and control strategy is urgently needed to be found so as to improve the success rate of aquaculture, the quality and the safety of aquatic products.
Although a bacteriophage is widely present in the environment and is a virus that uses microorganisms such as fungi and bacteria as a host, the bacteriophage has been widely noticed because of its advantages such as high specificity and no side effects on the human body, the bacteriophage, as an independent living body, generates an anti-bacteriophage strain when used as an antibacterial agent, and the biological safety has been questioned. Phage lyase is a hydrolase expressed by dsDNA phages at a later stage of infection with bacteria. Compared with bacteriophage, the lyase has the advantages of difficult generation of drug resistance of bacteria, capability of killing pathogenic bacteria planted on the surface of the mucous membrane, relatively wide host spectrum and the like. Therefore, the phage lyase has great application prospect.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a co-expression vector of lyase genes holA and lysin of a bacteriophage.
The technical problem to be solved by the invention is to provide a recombinant strain containing the co-expression vector.
The invention also aims to solve the technical problem of providing the phage lyase, namely the holA-lysin and a preparation method thereof.
The invention finally aims to solve the technical problem of providing the co-expression vector, the recombinant strain and the application of the phage lyase holoA-lysin in the lysis of vibrio alginolyticus.
The invention also provides an evaluation method of the cracking effect of the phage co-expression lyase holoA-lysin on the cracked escherichia coli and vibrio alginolyticus from different sources.
The invention aims to reduce and replace the use of antibiotics by searching for a novel phage lyase capable of inhibiting or killing pathogenic vibrios of aquatic products such as vibrio alginolyticus. The invention co-expresses the lyase holoA and lysin genes of the phage HH109 in escherichia coli and vibrio alginolyticus by a biotechnology means, proves that the expression of the genes can efficiently decompose the escherichia coli and the vibrio alginolyticus, and can be used as a potential novel biological preparation for preventing and treating aquatic animal diseases caused by pathogenic bacteria vibrio alginolyticus.
The technical scheme is as follows: in order to solve the technical problem, the invention provides a co-expression vector of lyase genes holA and lysin of a bacteriophage, which is obtained by taking bacteriophage genome DNA as a template, respectively carrying out PCR amplification by using a primer pair of the gene holA and a primer pair of the gene lysin to obtain a gene holA fragment and a gene lysin fragment, fusing the gene holA fragment and the gene lysin fragment, and then connecting the gene holA fragment and the gene lysin fragment with the vector.
Wherein the bacteriophage is bacteriophage HH 109.
Wherein the primer pair of the gene holoA comprises an upstream primer holoA-F1 and a downstream primer holoA-R1; or an upstream primer holoA-F2 and a downstream primer holoA-R2; wherein the sequence of the upstream primer holoA-F1 is shown as SEQ ID NO: 1, the sequence of the downstream primer holoA-R1 is shown as SEQ ID NO: 2, the sequence of the upstream primer holoA-F2 is shown as SEQ ID NO: 5, the sequence of the downstream primer holoA-R2 is shown as SEQ ID NO: and 6.
Wherein the primer pair of the gene lysin comprises an upstream primer lysin-F1 and a downstream primer lysin-R1; or an upstream primer lysin-F2 and a downstream primer lysin-R2, wherein the sequence of the upstream primer lysin-F1 is shown as SEQ ID NO: 3, the sequence of the downstream primer lysin-R1 is shown as SEQ ID NO: 4, the sequence of the upstream primer lysin-F2 is shown as SEQ ID NO: 7, the sequence of the downstream primer lysin-R2 is shown as SEQ ID NO: shown in fig. 8.
Wherein, the vector is pET32a or pMMB 207.
The invention also comprises a recombinant strain containing the co-expression vector.
Wherein the recombinant bacteria is one of Escherichia coli BL21, Vibrio alginolyticus E110 and insensitive Vibrio alginolyticus E601 or HN 198.
The invention also comprises phage lyase holA-lysin which is obtained by performing induction expression on the recombinant strain.
The invention also provides a preparation method of the phage lyase holoA-lysin, which comprises the following preparation steps: the recombinant strain is obtained after induction expression.
The invention also comprises the co-expression vector, the recombinant strain and the application of the phage lyase holoA-lysin in bacterial lysis.
Wherein the bacteria is Escherichia coli or Vibrio alginolyticus.
The invention also comprises the application of the co-expression of phage lyase genes holA and lysin in the evaluation of the cracking effect of escherichia coli and vibrio alginolyticus, and the method comprises the following steps:
1) amplifying lyase holA and lysin genes of a bacteriophage HH109, connecting with an expression plasmid, and respectively transferring into escherichia coli and vibrio alginolyticus to obtain a bacterial strain with co-expression of the perforin holA and the lysin genes;
2) culturing the Escherichia coli or vibrio alginolyticus containing the holA and lysin recombinant plasmids to OD of the bacterial liquid6000.6-0.8, inducing with 0.5mM IPTG for 8h, and measuring bacterial liquid OD with spectrophotometer600Drawing a bacterial growth curve;
3)0.5mM IPTG was used to induce Escherichia coli containing plasmid pET32a-holA-lysin for 4h, and after centrifugation, 500. mu.L of the culture supernatant was added with 100. mu.L of 20mM ONPG, reacted in a water bath at 45 ℃ for 30min, followed by 600. mu.L of 0.5M Na2CO3Stopping the reaction;
4) after the induction of Escherichia coli containing plasmid pET32a-holA-lysin with IPTG for 4 hours, the cells were collected and the morphological changes of Escherichia coli were observed using a transmission electron microscope.
5) The fluorescent dye of the GeneCopoeia company is used for dyeing the pMMB 207-holA-lysin-containing vibrio alginolyticus 4h after IPTG induction, NucViewGreen can dye live cells to be green, Propidiumiodide can dye dead cells to be red, and the strain color is observed by a fluorescence microscope to judge the cracking efficiency of the strain.
Has the beneficial effects that: compared with the prior art, the invention has the following remarkable advantages: the lyase genes holA and lysin of the phage HH109 are obtained through PCR amplification and the like, cloned to an expression system and transferred to a target strain to obtain a recombinant strain. The recombinant bacteria induce and express lyase genes in IPTG to realize efficient cracking of bacteria such as vibrio alginolyticus, escherichia coli and the like, the cracking effect has no selectivity, and a broad-spectrum application range is shown, so that the lyase of the phage HH109 has potential application value in preventing and treating vibrio infection such as vibrio alginolyticus, and the cracking rate of the vibrio alginolyticus phage HH109 lyase of the invention on the basis of holo-lysin co-expression is up to more than 95%.
Drawings
FIG. 1 shows construction of pET32 a-holoA-lysin co-expression vector referred to in example 1, and PCR amplification to obtain the products of the bacteriophages perforin (holoA) and endolysin (lysin).
FIG. 2 shows the construction of pMMB207-holA-lysin co-expression vector, the amplification products of the bacteriophages perforin (holA) and endolysin (lysin) involved in example 1.
FIG. 3 is a graph showing the effect of perforin (holA) and endolysin (lysin) expression involved in example 2 on the growth of E.coli, wherein OD is shown on the abscissa600The absorbance values measured, the ordinate, are for each treatment group, and the control is indicated in the legend.
FIG. 4 is a graph showing the effect of perforin (holA) and endolysin (lysin) expression on E.coli cell membrane permeability as referred to in example 2.
FIG. 5 is a graph showing the effect of perforin (holA) and endolysin (lysin) expression on E.coli morphology as referred to in example 2.
FIG. 6 is a graph showing the effect of perforin (holA) and endolysin (lysin) expression on Vibrio alginolyticus growth as referred to in example 3.
FIG. 7 is a graph showing the effect of perforin (holA) and endolysin (lysin) expression on the lytic effect of Vibrio alginolyticus, which is described in example 3.
Detailed Description
The invention is further described below with reference to specific embodiments. The following embodiments are only for more clearly illustrating the technical solutions of the present invention, and the protection scope of the present invention is not limited thereby.
Example 1 construction of a bacterium having a co-expression of the perforin holA and endolysin genes
The co-expression vector of the holoA gene and the lysin gene is constructed based on a molecular cloning technology, and comprises the construction of a co-expression vector pET32 a-holoA-lysin and pMMB 207-holoA-lysin.
1) pET32a-holA-lysin vector clone construction: purifying the phage HH109 (phage with the preservation number of CCTCC NO: M2020397 in the patent application with the application number of 202011154028.2), obtaining high-concentration virus particles, extracting the genome DNA of the phage HH109, designing reading frame amplification primers of perforin holA and endolysin genes according to the HH109 genome information, wherein the gene holA primers are holA-F1: GCCATGGCTGATATCGGATCCGTGAGTGAAAAGATTGTAAAAGCAG, respectively; holoA-R1: AGAATTGCTTCAACGAACATAATCCCCTTTCTCCACTTAT; the gene lysin primer is lysin-F1: ATAAGTGGAGAAAGGGGATTATGTTCGTTGAAGCAATTCT, respectively; lysin-R1: TGCGGCCGCAAGCTTGTCGACCAGGTCTTCCTCCATTATCATAG are provided. And (3) performing PCR amplification by using phage genome DNA as a template and using holoA-F/R and lysin-F/R as primers respectively, and purifying and recovering PCR products. Then, the two sections of the holoA and the lysin are fused by overlap extension PCR, the holoA and the lysin gene sequences are used as templates, and the holoA-F and the lysin-R are used as primers for amplification. The empty pET32a plasmid (BamHI/SalI cleavage site) was inserted using an In-fusion cloning kit; heat shock transformed Escherichia coli DH5 alpha, 37 ℃ culture for 12h, picking single clone sequencing verification, pET32a-holA-lysin vector plasmid (figure 1) is obtained. 100ng of pET32a-holA-lysin plasmid is transformed into an escherichia coli BL21 strain through heat shock to obtain a recombinant expression escherichia coli strain, namely an engineering strain BL 21-holA-lysin.
Construction of a vector clone of pMMB 207-holA-lysin: the specific method refers to the construction of a co-expression vector pET32 a-holoA-lysin, and the primer of the perforin gene holoA is holoA-F2: TTCACACAGGAAACAGAATTCGTGAGTGAAAAGATTGTAAAAGCAG, respectively; holoA-R2: AGAATTGCTTCAACGAACATAATCCCCTTTCTCCACTTAT, respectively; the primer of the endolysin gene lysin is lysin-F2: ATAAGTGGAGAAAGGGGATTATGTTCGTTGAAGCAATTCT, respectively; lysin-R2: CAGGTCGACTCTAGAGGATCCAGGTCTTCCTCCATTATCATAG are provided. The restriction sites of the empty plasmid pMMB207-holA-lysin inserted are EcoRI and BamHI respectively. Heat shock transformed Escherichia coli S17, 37 ℃ culture for 12h, picking monoclonal sequencing verification, obtain pMMB207-holA-lysin vector plasmid (figure 2). 100ng pMMB207-holA-lysin plasmid is conjointly transformed into Vibrio alginolyticus E110 sensitive to bacteriophage HH109 and insensitive Vibrio alginolyticus E601 and HN198 strains, thus obtaining engineering strains E110+ holA + lysin, E601+ holA + lysin and HN198+ holA + lysin.
Example 2: observation of phenotype of cleaved Escherichia coli by co-expression of holA and lysin genes
The part comprises the influence of the co-expression of the holoA and lysin genes on the growth rate of escherichia coli, the change of cell membrane permeability and the observation of the phenotype of strain morphological change.
1) Identification of growth rate of inhibiting escherichia coli: culturing Escherichia coli BL21(DE3) bacterial solution carrying pET32a-holA-lysin to logarithmic growth period, adding 0.5mM IPTG to induce protein expression, after 8h, measuring bacterial solution OD with spectrophotometer600DrawingBacterial growth curves. Compared with the control group with no load (BL 21: pET32(+)), BL21 and pET32a-holA-lysin Escherichia coli which is not induced by IPTG, the OD value of the Escherichia coli carrying pET32a-holA-lysin is lower after the induction by IPTG, and the growth of the Escherichia coli can be obviously inhibited by the co-expression of holA and lysin (figure 3).
2) Identification of cell membrane permeability changes: picking a single colony of escherichia coli BL21 carrying a recombinant plasmid of pET32a-holA-lysin into 5mL of Amp-containing liquid LB culture medium, and carrying out overnight culture at 37 ℃ at 200/min; transferring the culture medium into a new 5mL liquid LB culture medium according to the ratio of 1: 100, and culturing the culture medium to a logarithmic phase; adding IPTG to a final concentration of 0.5mmol/L, and performing shaking culture at 37 deg.C and 200r/min for 30 min; centrifuging at 12,000 Xg for 5min, and collecting supernatant; collecting 500 μ L extracellular supernatant, adding 100 μ L20 mM ONPG, reacting in water bath at 45 deg.C for 30min, and adding 600 μ L0.5M Na2CO3The reaction was terminated. Coli harboring pET32a-holA-lysin detected nearly 100Miller units of β -galactosidase when compared to the control group, which was empty. These data indicate that expression of the phage lysis-associated genes affects the integrity of the cell membrane (fig. 4).
3) And E, identifying the morphological change of the escherichia coli: taking a single colony of escherichia coli BL21 carrying pET32a-holA-lysin recombinant plasmid to 5mL of liquid LB culture medium containing Amp, and carrying out overnight culture at 37 ℃ at 200 r/min; transferring the culture medium into a new 5mL liquid LB culture medium according to the ratio of 1: 100, and culturing the culture medium at 37 ℃ and 200r/min to a logarithmic phase; adding IPTG to a final concentration of 0.5mmol/L, and performing shaking culture at 37 deg.C and 200r/min for 30 min; the cells were collected and observed for morphological changes in E.coli using a transmission electron microscope. After IPTG induction, control cells exhibited typical strain morphology, intact cell structure and few dead cells in the field of view (fig. 5A-B, panels C and D are magnified images of panels a and B, respectively). And the contraction and separation phenomena of the cell wall and the cell membrane of the Escherichia coli carrying pET32a-holA-lysin occur, the density of substances in the cells is reduced, the color of the strain is lightened, the volume of the cytoplasm is reduced, the structure in the cytoplasm is disordered, and the obvious hole phenomenon occurs, which indicates that the expression of holin and lysin protein causes cell lysis and death to a certain extent by changing the permeability of the bacterial cell membrane.
Example 3: identification and application of holoA and lysin gene coexpression for cracking vibrio alginolyticus from different sources
Vibrio alginolyticus E110: capable of being cleaved by bacteriophage HH109, vibrio alginolyticus E601 and HN 498: cannot be cleaved by the bacteriophage HH 109.
1) Identification of growth rate of vibrio alginolyticus: respectively culturing bacterial solutions of the vibrio alginolyticus E110, E601 and HN498 carrying the pMMB207-holA-lysin to the logarithmic growth period, adding 0.5mM IPTG to induce the expression of protein, and measuring the OD of the bacterial solution by a spectrophotometer after 8h600And drawing a bacterial growth curve. IPTG-induced OD values of pMMB 207-holA-lysin-carrying Vibrio alginolyticus E110, E601 and HN498 were significantly lower compared to control no-load, and thus holA and lysin co-expression inhibited the growth of heterologous Vibrio alginolyticus (FIG. 6).
2) Picking single colonies of vibrio alginolyticus E110, E601 and HN498 carrying pMMB207-holA-lysin recombinant plasmids into 5mL of Amp-containing liquid LB respectively, and culturing at 37 ℃ and 200r/min overnight; transferred into 5mL LB liquid medium containing Amp according to the ratio of 1: 100, and cultured for 2h at 37 ℃ and 200r/min with shaking until OD600At about 0.6; adding IPTG to a final concentration of 0.5mmol/L, and carrying out shake culture lh at 37 ℃ at 200 r/min; the cells were collected and resuspended in PBS for use in the following staining experiments. Prepare 100 Xstaining mixture. Mixing 1.5 μ L NucViewGreen and 1.5 μ L Propidiumodide (Miyajie, China), adding 7 μ L0.85% NaCl solution, and mixing; adding 1 mu L of 100 Xstaining mixed solution into 100 mu L of the induced bacteria sample, and uniformly mixing; incubating at room temperature for 15min, and keeping away from light; respectively absorbing 5 mu L of bacterial staining suspension liquid onto a clean glass slide, performing microscopic examination under a fluorescence microscope, wherein live bacteria are green, dead bacteria are red, and compared with a control group with no load, the proportion of live bacteria of the vibrio alginolyticus E110, E601 and HN498 which are induced by IPTG and carry pMMB207-holA-lysin is only 3-5 percent, which shows that the co-expression of holA and lysin leads to the cracking of heterologous vibrio alginolyticus, the cracking rate of the heterologous vibrio alginolyticus reaches more than 95 percent (figure 7), and is higher than the cracking effect of the co-expression of the same phage holA and lysin on a host.
Claims (10)
1. The co-expression vector of the lyase genes holA and lysin of the phage is characterized in that the co-expression vector is obtained by taking phage genome DNA as a template, respectively carrying out PCR amplification by using a primer pair of the gene holA and a primer pair of the gene lysin to obtain a gene holA fragment and a gene lysin fragment, fusing the gene holA fragment and the gene lysin fragment, and then connecting the fused gene holA fragment and the gene lysin fragment with the vector.
2. The co-expression vector of lyase genes holA and lysin of bacteriophage of claim 1, wherein the bacteriophage is bacteriophage HH109, and the vector is pET32a or pMMB 207.
3. The co-expression vector of lyase genes holoA and lysin of bacteriophage as claimed in claim 1, wherein the primer pair of the gene holoA comprises an upstream primer holoA-F1 and a downstream primer holoA-R1; or an upstream primer holoA-F2 and a downstream primer holoA-R2; wherein the sequence of the upstream primer holoA-F1 is shown as SEQ ID NO: 1, the sequence of the downstream primer holoA-R1 is shown as SEQ ID NO: 2, the sequence of the upstream primer holoA-F2 is shown as SEQ ID NO: 5, the sequence of the downstream primer holoA-R2 is shown as SEQ ID NO: and 6.
4. The co-expression vector of lyase genes holA and lysin of bacteriophage according to claim 1, wherein the primer pair of the gene lysin comprises an upstream primer lysin-F1 and a downstream primer lysin-R1; or an upstream primer lysin-F2 and a downstream primer lysin-R2, wherein the sequence of the upstream primer lysin-F1 is shown as SEQ ID NO: 3, the sequence of the downstream primer lysin-R1 is shown as SEQ ID NO: 4, the sequence of the upstream primer lysin-F2 is shown as SEQ ID NO: 7, the sequence of the downstream primer lysin-R2 is shown as SEQ ID NO: shown in fig. 8.
5. A recombinant strain comprising the co-expression vector of any one of claims 1 to 4.
6. The recombinant strain of claim 5, wherein the recombinant strain is one of E.coli BL21, Vibrio alginolyticus E110, and insensitive Vibrio alginolyticus E601 or HN 198.
7. A phage co-expression lyase, holoa-lysin, characterized in that, the phage lyase is obtained by inducing and expressing the recombinant strain of claim 5 or 6.
8. The method for preparing the phage co-expression lyase, holoa-lysin, as claimed in claim 7, comprising the following preparation steps: the recombinant strain of claim 5 or 6 is induced to express.
9. Use of the co-expression vector of any one of claims 1 to 4, the recombinant strain of claim 5 or 6, the phage co-expression lyase, holoa-lysin, of claim 7 for bacterial lysis.
10. The use according to claim 9, wherein the bacteria are escherichia coli or vibrio alginolyticus.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060040393A1 (en) * | 2004-08-17 | 2006-02-23 | Xiyu Jia | Controlled lysis of bacteria |
| CN108410884A (en) * | 2018-03-07 | 2018-08-17 | 中国科学院南海海洋研究所 | The application of bacteriophage Vp670 perforin genes holA |
| CN112301021A (en) * | 2020-10-30 | 2021-02-02 | 西南大学 | Enlysin and perforin composition for resisting escherichia coli phage expression and preparation method and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060040393A1 (en) * | 2004-08-17 | 2006-02-23 | Xiyu Jia | Controlled lysis of bacteria |
| CN108410884A (en) * | 2018-03-07 | 2018-08-17 | 中国科学院南海海洋研究所 | The application of bacteriophage Vp670 perforin genes holA |
| CN112301021A (en) * | 2020-10-30 | 2021-02-02 | 西南大学 | Enlysin and perforin composition for resisting escherichia coli phage expression and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| XIXI LI等: "Bactericidal activity of a holin-endolysin system derived from Vibrio alginolyticus phage HH109", 《MICROBIAL PATHOGENESIS》, vol. 159, pages 3 - 5 * |
| 周勤: "《医药分子生物学实验教程》", 中山大学出版社, pages: 2 - 3 * |
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