CN116143886A - Gp21 protein encoded by pseudomonas aeruginosa phage and application thereof in inhibiting escherichia coli phage infection - Google Patents
Gp21 protein encoded by pseudomonas aeruginosa phage and application thereof in inhibiting escherichia coli phage infection Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and relates to a Gp21 protein encoded by pseudomonas aeruginosa phage and application thereof in inhibiting coliphage infection. The amino acid sequence of the Gp21 protein encoded by the pseudomonas aeruginosa phage is shown as SEQ ID NO. 1. The Gp21 protein can be expressed in non-original host bacterium escherichia coli BL21 (DE 3) to strengthen the resistance of escherichia coli to phage infection, shows potential of serving as a target spot to be used for modifying different bacteria to resist phage infection, and has the excellent characteristic of broad-spectrum resistance to phage infection.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a Gp21 protein encoded by pseudomonas aeruginosa phage and application thereof in inhibiting coliphage infection.
Background
Phage contamination has been a major potential hazard in the safe production of fermented foods. Phage (particularly "lytic phage") is a bacterial virus that infects specifically and kills fermenting strains with high efficiency and is not susceptible to resistance. Phages can affect the fermentation process of microorganisms using or using bacteria as a product, and once the fermentation strain is infected with phages, the fermentation performance will be lost, resulting in failure of the fermentation. However, phage contamination has been a worldwide technical problem that plagues the food fermentation industry, and light phage infection during fermentation results in reduced yield of fermentation products, and heavy phage infection results in plant shut down or even shut down, ultimately resulting in serious economic loss. Therefore, the control and elimination of phage contamination during fermentation is an urgent need for food fermentation industry production practices.
To avoid infection with phage, various defenses such as rational plant design, phage disinfection of production environment, transformation and optimization of fermentation strain, etc. are proposed and applied. However, food fermentation plants are not in a sterile environment, and phages are widely present, and regardless of the improvement in sanitary conditions and management levels of the laboratory or plant, the phenomenon of phage infection of the fermentation strain still occurs at times, so that the probability of complete elimination of phages is almost zero; although the alternate strains can reduce the risk of phage infection, the method has complicated operation and high input cost, so that the problem that the fermentation strain is frequently infected by phage is difficult to thoroughly solve. With the continuous and deep research, the breeding or transformation of the phage-resistant fermentation strain becomes a popular choice for scientists to study. Although it has been reported that bacteria can acquire resistance to phage through various mechanisms such as adsorption inhibition, injection blocking, restriction modification system, abortion infection, adaptive immunity, etc., strategies for acquiring phage resistance by constructing abortion infection system and CRISPR-Cas immune system using genetic engineering technology have been derived. However, since phages can rapidly adapt to these phage resistance systems by mutation; in addition, many of the discovered resistant functional elements are not validated for broad-spectrum phage resistance, limiting the engineering applications of the resistant functional elements in fermentation engineering bacteria.
Recently, studies have shown that phage proteins play an important role in promoting the host's progress in combating phage infection, such as the Aqs, BD24-4 and Tip proteins encoded by phage, etc., which have been reported. However, it is unclear whether the Aqs, JBD24-4 and Tip proteins reported are derived from lysophage, and whether lytic phage proteins can also mediate bacterial anti-phage infection. Lytic phages are generally capable of completing adsorption, invasion, proliferation, assembly and lysis in a short time, and are the main factors responsible for industrial fermentation failure. Therefore, the coding product of the lytic phage source is developed as a resistance element, and important theoretical basis and methodology basis are provided for developing and designing a novel anti-phage scheme in the future, stably, safely and long-acting for the food fermentation industry and breaking through the technical bottleneck of prevention and control of phage infection.
Disclosure of Invention
The invention provides a Gp21 protein coded by a lytic pseudomonas aeruginosa bacteriophage, the expression of the protein in non-original host bacterium escherichia coli BL21 (DE 3) can enhance the escherichia coli to resist the infection of different bacteriophage, the potential of being used as a target point for modifying different bacteria to resist the infection of the bacteriophage is shown, and the protein is a first discovered product coded by a lytic bacteriophage source at present, and has a resistance element with excellent characteristic of broad-spectrum anti-phage infection.
The technical scheme adopted for solving the technical problems is as follows:
the amino acid sequence of the Gp21 protein encoded by the pseudomonas aeruginosa phage is shown as SEQ ID NO. 1. The Gp21 protein is encoded by lytic phage and can confer resistance to phage infection to different host strains.
The Gp21 protein coded by the pseudomonas aeruginosa phage can endow the escherichia coli with the capability of resisting phage infection, and has the excellent characteristic of broad-spectrum phage infection resistance. The invention expands the knowledge of the existing anti-phage system or anti-phage functional element and provides a new idea for anti-phage pollution in biotechnology development and fermentation process.
A nucleotide sequence of the gene encoding the protein is shown as SEQ ID NO. 2.
The invention relates to application of a Gp21 protein coded by pseudomonas aeruginosa phage in inhibiting coliphage infection.
Preferably, the coliphage is a long tail phage or a myotail phage. Further, the myophagidae phages may be myophagics such as T4, vb_eco m_ime281, vb_eco m_ime338, vb_eco m_ime339, vb_eco m_ime340, vb_eco m_ime341, and the like, and the long-tail phages may be myophagics such as T1, vb_eco s_ime18, vb_eco s_ime253, vb_eco s_ime347, JMPW1, and the like.
The invention relates to application of a Gp21 protein coded by pseudomonas aeruginosa phage in inhibiting coliphage infection.
A method for improving the anti-phage capability of escherichia coli, which comprises the following steps: the gene with the nucleotide sequence shown as SEQ ID NO.2 is overexpressed in the escherichia coli,
or transferring an expression vector containing a gene with a nucleotide sequence shown as SEQ ID NO.2 into escherichia coli, or integrating a gene with a single copy or multiple copies of the nucleotide sequence shown as SEQ ID NO.2 on the genome of escherichia coli.
Preferably, the E.coli is E.coli BL21 (DE 3).
Preferably, the escherichia coli uses a pET series as an expression vector, and the pET series vector comprises pET28a (+), pET21a and pET30a.
The application of the escherichia coli BL21 (DE 3) obtained by the method in the invention in the aspect of inhibiting escherichia coli phage infection in industrial fermentation.
The gene provided by the invention is used as a target spot in screening medicaments related to inhibition of coliphage infection.
Compared with the prior art, the invention has the beneficial effects that:
the invention discovers for the first time that the Gp21 protein coded by the lytic phage can protect different hosts against infection of phage. Expression of Gp21 protein in the original host Pseudomonas aeruginosa PAO1 can help PAO1 resist infection by Pseudomonas aeruginosa phage. In non-original host bacterium escherichia coli BL21 (DE 3), the expression of Gp21 can also help escherichia coli resist phage infection, namely Gp21 has the performance of broad-spectrum phage resistance, is hopeful to be used as a target point for transformation of chassis cells of fermentation engineering strains in the future, and solves the technical bottleneck of frequent phage infection in industrial fermentation production.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic representation of plasmid pET28a (+) containing the gp21 gene encoded by the phage genome from P.aeruginosa in lytic form;
FIG. 2 SDS-PAGE of E.coli BL21 (DE 3) (gp 21) protein expression. M: a Marker;1, pET28a-gp21 plasmid of escherichia coli BL21 (DE 3) is not induced by IPTG; 2 pET28a-gp21 plasmid of colibacillus BL21 (DE 3), IPTG induction. Wherein the arrow indicates the target strip;
FIG. 3 is a graph showing the results of experiments on the resistance of E.coli BL21 (DE 3) (gp 21) to phage at 37℃without IPTG induction and at 37℃with 0.5mM IPTG induction for 30min, respectively.
Detailed Description
The technical scheme of the invention is further specifically described by the following specific examples. It should be understood that the practice of the invention is not limited to the following examples, but is intended to be within the scope of the invention in any form and/or modification thereof.
In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment, materials, etc. used are commercially available or are conventional in the art. The methods in the following examples are conventional in the art unless otherwise specified.
The strain involved in the test of the invention:
the coliphage vB_EcoM_IME338 is preserved in China general microbiological culture Collection center (CGMCC) at 30 days of 8 months of 2018, and the preservation number is CGMCC No.16292; the accession number of the collection center of the coliphage vB_EcoM_IME281 is CGMCC No.16291; the accession number of the collection center of the coliphage vB_EcoM_IME339 is CGMCC No.16293;
coli BL21 (DE 3) was purchased from North Nanopsis under the product number BNCC353806.
LB liquid medium, qingdao sea Bo biotechnology Limited liability company;
isopropyl-beta-D-l thiogalactoside (IPTG), beijing Soy Bao technology Co.
In one embodiment of the invention, genes related to the production of a target product are connected to an expression vector, the expression vector is transferred into a host cell to obtain recombinant bacteria, and the recombinant bacteria are tested for phage infection resistance after induced expression of the recombinant protein.
The present invention provides a recombinant host cell comprising the above expression vector or a chromosome having integrated therein one or more copies of the exogenous gp21 gene.
The invention provides a recombinant bacterium, or application of the expression vector, or application of a method for improving the anti-phage capability of escherichia coli in production of target products.
EXAMPLE 1 construction of pET28a plasmid containing gp21 Gene
The gp21 gene sequence with homology arm ends was synthesized, with homology arm sequence ACAGCAAATGGGTCGCGGATCC upstream (containing BamHI restriction sites) (SEQ ID NO. 3) and homology arm sequence GTGGTGGTG GTGGTGCTCGAGT downstream (containing XhoI restriction sites) (SEQ ID NO. 4) on the gp21 gene. Chemical synthesis of gp21 gene sequence was performed by Shanghai Biotechnology. The fragment gp21 was ligated to the linear vector pET28a obtained by BamHI and XhoI digestion by In-Fusion splicing to give the recombinant plasmid pET28a-gp21 (FIG. 1).
Example 2 heterologous expression of the anti-phage defenses Gp21 in E.coli
Transferring the recombinant plasmid pET28a-gp21 into escherichia coli DH5 alpha to perform screening of positive clones, wherein the screening resistance is the kanamicin; the obtained positive monoclonal is activated and inoculated into 10mL LB broth culture medium (containing 50 mug/mL kanamycin) for overnight shaking culture at 37 ℃ for 200r/min, after bacterial liquid is collected, recombinant plasmid is extracted by using a small amount extraction kit of the engineering EZ-10 column type plasmid, and the recombinant plasmid pET28a-gp21 is further transformed into escherichia coli BL21 (DE 3). E.coli BL21 (DE 3) bacteria transformed with expression plasmid pET28a-gp21 were inoculated in a tube containing 5mL of LB at 37℃and cultured overnight at 200rpm, the following day at 1:100 proportion was transferred to fresh LB medium, cultured at 30℃and 200rpm until OD600nm was 0.6, then IPTG was added to a final concentration of 0.5mM and induced at 25℃for 12 hours. After centrifugally collecting cells, crushing the cells at 4 ℃ by a high-pressure crusher, purifying the protein by using a Ni column, dialyzing to remove free imidazole, finally storing the protein in a desalting Buffer containing 10% of glycerol, and performing subsequent SDS-PAGE analysis to confirm that the Gp21 protein can successfully realize heterologous expression.
The results are shown in FIG. 2: SDS-PAGE separation shows that there is obvious difference band after IPTG induction, the size of the protein band position is similar to the theoretical molecular weight (11 kDa) of Gp21 protein, which shows that Gp21 protein realizes heterologous expression successfully in colibacillus BL21DE 3.
Example 3 application of Gp21 protein to helping E.coli BL21 (DE 3) to resist phage infection
Single colonies of the expression strain BL21 (DE 3) (gp 21) were picked up and grown overnight with shaking at 37℃under 200r/min in tubes containing 5mL LB medium (containing 50. Mu.g/mL kanamycin). The following day, the cultured overnight bacteria were transferred to 100mL of fresh LB medium at 1% inoculum size, and the culture was continued at 37℃and 200rpm for 3 hours. At this time, bacterial cells grown to log phase were aliquoted into two treated differently, i.e., the control group without inducer IPTG and the experimental group induced for 30min with 0.5mM IPTG. Binding phage plaque assay the Gp21 element was tested to confer resistance to phage infection on the host E.coli. The method comprises the following specific steps: taking 100 mu L of the treated BL21 bacterial liquid (an induction group and a non-induction group), respectively adding into 5mL of LB semisolid culture medium, uniformly mixing, spreading on a lower LB solid culture medium plate, and airing at room temperature for 5min; coli phages vB_EcoM_IME338, vB_EcoM_IME340, vB_EcoM_IME341, vB_EcoM_IME347 and vB_EcoM_IME39 were subjected to gradient dilution, 3. Mu.L of the resulting stock solution was dropped onto a bacterial layer medium, and after culturing at 37℃for 8 hours, plaque transparency was observed.
The results are shown in FIG. 3: although there was a difference in infection efficiency or plaque formation efficiency of the different phages on host E.coli BL21 (DE 3), in general, the phage plaques formed without IPTG induction were clear and transparent, whereas the phage plaques formed with IPTG induction in the experimental group were small and opaque compared with the control group, and there was a clear difference in plaque formation ability between the two groups, indicating that expression of Gp21 could significantly enhance E.coli BL21 (DE 3) resistance to infection by different phages.
In this specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, so that the same or similar parts between the embodiments are referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.
The Gp21 protein coded by the pseudomonas aeruginosa phage and the application thereof in inhibiting coliphage infection are described in detail above. The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.
Claims (10)
1. The amino acid sequence of the Gp21 protein encoded by the pseudomonas aeruginosa phage is shown as SEQ ID NO. 1.
2. A nucleotide sequence of the gene encoding the protein is shown as SEQ ID NO. 2.
3. Use of a Gp21 protein encoded by a pseudomonas aeruginosa phage according to claim 1 for inhibiting infection with an escherichia coli phage.
4. A use according to claim 3, characterized in that: the coliphage is a long tail phage or a myotail phage.
5. Use of a Gp21 protein encoded by a pseudomonas aeruginosa phage according to claim 1 for inhibiting infection with an escherichia coli phage.
6. A method for improving the phage resistance of escherichia coli, which is characterized by comprising the following steps: the gene with the nucleotide sequence shown as SEQ ID NO.2 is overexpressed in the escherichia coli,
or transferring an expression vector containing a gene with a nucleotide sequence shown as SEQ ID NO.2 into escherichia coli, or integrating a gene with a single copy or multiple copies of the nucleotide sequence shown as SEQ ID NO.2 on the genome of escherichia coli.
7. The method according to claim 6, wherein: the escherichia coli is escherichia coli BL21 (DE 3).
8. The method according to claim 6, wherein: the escherichia coli takes a pET series as an expression vector, and the pET series vector comprises pET28a (+), pET21a and pET30a.
9. Use of the escherichia coli BL21 (DE 3) obtained by the method of claim 6 for inhibiting escherichia coli phage infection in industrial fermentation.
10. Use of the gene of claim 2 as a target in screening drugs related to inhibition of coliphage infection.
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Citations (2)
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|---|---|---|---|---|
| US20020090678A1 (en) * | 2000-08-15 | 2002-07-11 | Kordyum Vitaliy A. | Phage-dependent superproduction of biologically active protein and peptides |
| CN111849848A (en) * | 2020-07-23 | 2020-10-30 | 江南大学 | Construction and application of bacteriophage-resistant escherichia coli chassis cells |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020090678A1 (en) * | 2000-08-15 | 2002-07-11 | Kordyum Vitaliy A. | Phage-dependent superproduction of biologically active protein and peptides |
| CN111849848A (en) * | 2020-07-23 | 2020-10-30 | 江南大学 | Construction and application of bacteriophage-resistant escherichia coli chassis cells |
Non-Patent Citations (2)
| Title |
|---|
| GUANHUA XUAN等: "xpression of a Phage-Encoded Gp21 Protein Protects Pseudomonas aeruginosa against Phage Infection", 《J VIROL》, vol. 96, no. 5, 9 March 2022 (2022-03-09), pages 2 * |
| 贡嘉澳等: "细菌抗噬菌体防御***研究进展", 《生命科学仪器》, vol. 20, no. 02, 30 April 2022 (2022-04-30), pages 17 - 26 * |
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