An aeromonas bacteriophage recombination system, expression vector, recombinant strain and application thereof

Abstract

The application discloses an Aeromonas phage recombinant system, an expression vector, a recombinant strain and application thereof, and the recombinant system comprises a recombinase operon 109TES derived from a salmonella Aeromonas phage vB_AsaM-56; the operon comprises at least one gene with exonuclease function and one single-stranded DNA annealing protein gene. Through function verification in Escherichia coli, 109TES can efficiently mediate linear circle recombination and linear linear recombination by using a short homologous arm (50 bp), and has stable and reliable recombination function in a heterologous host, indicating that the recombinase operon from a non-Escherichia coli phage can also exert efficient homologous recombination function in Escherichia coli, and can be used as a powerful candidate for constructing a new type of recombination tool, and provides a new choice for developing a multi-host available gene editing system.

Description

TECHNICAL FIELD

[0001] The present application relates to an Aeromonas spp. gene recombination technology, in particular to an Aeromonas spp. phage recombination system, an expression vector, a recombinant strain and application thereof. BACKGROUND

[0002] Aeromonas spp. is one of the main pathogens causing explosive infectious diseases in aquaculture, and has caused serious economic losses in fish farming. Although Aeromonas spp. has important research value in pathogenic mechanism and vaccine development, the genetic manipulation tools for Aeromonas spp. are still limited. This limitation seriously restricts the basic research and application development of Aeromonas spp.

[0003] The Red / ET homologous recombination system derived from E. coli phage has high gene editing function in E. coli. This system can mediate homologous recombination through exonuclease and single-stranded DNA annealing protein, and can realize gene editing using short homologous arms, greatly facilitating gene modification. However, a large number of studies have shown that the recombination efficiency of the Red / ET system in non-E. coli hosts is significantly reduced, especially in distant bacteria such as Aeromonas spp. It is almost impossible to apply. There is no report on high-efficiency recombination system for Aeromonas spp. so far. SUMMARY

[0004] The first technical problem to be solved by the present application is to provide an Aeromonas spp. phage recombination system to improve application efficiency.

[0005] The second technical problem to be solved by the present application is to provide an expression vector.

[0006] The third technical problem to be solved by the present application is to provide a recombinant strain.

[0007] The fourth technical problem to be solved by the present application is to provide a method for realizing genome modification in Aeromonas spp. or other strains using the Aeromonas spp. phage recombination system or the expression vector.

[0008] The fifth technical problem to be solved by the present application is to provide an application of the Aeromonas spp. phage recombination system or the expression vector in the research of functional genes of Aeromonas spp., the development of fish vaccines or the prevention and control of diseases in aquaculture.

[0009] The technical solution adopted by the present application to solve the first technical problem is an Aeromonas spp. phage recombination system, wherein the recombination system comprises an operon 109TES derived from Aeromonas salmonicida phage vB_AsaM-56, and the operon 109TES comprises:

[0010] An exonuclease gene;

[0011] A single-stranded DNA annealing protein gene;

[0012] A single-stranded binding protein gene;

[0013] At least one non-homologous protein gene.

[0014] Further, the nucleotide sequence of the operon 109TES is shown in SEQ ID No. 1.

[0015] The technical scheme adopted by the present application to solve its second technical problem is an expression vector, the vector comprising:

[0016] The aerobacter phage recombination system;

[0017] An origin of replication;

[0018] A resistance screening gene;

[0019] An inducible promoter.

[0020] Further, the origin of replication is pBBR1, the resistance screening gene is a kanamycin resistance gene, and the inducible promoter is a rhamnose inducible promoter.

[0021] Further, the expression vector is named pBBR1-Rha-109TES-Kan, and the nucleotide sequence thereof is shown in SEQ ID No. 2.

[0022] The technical scheme adopted by the present application to solve its third technical problem is a recombinant strain, the strain containing the expression vector.

[0023] Further, the strain is Escherichia coli GB 05 or aerobacter.

[0024] The technical scheme adopted by the present application to solve its fourth technical problem is a method for achieving genome modification in aerobacter or other strains by using the aerobacter phage recombination system or the expression vector, the method mediating gene knockout, insertion or replacement of short homologous arms (50 bp). Linear DNA and plasmid are recombined by using short homologous arms of 50 bp to achieve DNA site-directed modification

[0025] The technical scheme adopted by the present application to solve its fifth technical problem is an application of the aerobacter phage recombination system or the expression vector in functional gene research of aerobacter, fish vaccine development or disease prevention and control of aquaculture.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] (1) Through bioinformatics and functional verification, the present application first identifies the operon 109TES with recombination function in the Aeromonas salmonicida phage, and confirms that its recombination efficiency in Escherichia coli is significantly high.

[0028] (2) The recombination system can effectively mediate the homologous recombination of short homologous arms (50 bp), significantly improving the efficiency of gene knockout, insertion and replacement, and when part of the protein in the operon is deleted, the recombination efficiency is significantly reduced, proving the irreplaceability of the whole function of the operon.

[0029] (3) The Aeromonas recombination system or the expression vector constructed based on the recombination system provided by the present application provides a new genetic manipulation tool for functional gene research of Aeromonas, fish vaccine development and disease prevention and control in aquaculture, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is a schematic diagram of bioinformatics analysis results of 109TES operon.

[0031] Figure 2 It is a schematic diagram of recombination strategy.

[0032] Figure 3 It is a graph showing the influence of rhamnose induction on the fluorescence intensity driven by rhaR-rhaS-pRha promoter;

[0033] Among them, A is the fluorescence intensity without adding inducer, showing weak fluorescence signal;

[0034] B is the fluorescence intensity at 40 min of induction, showing medium fluorescence signal;

[0035] C is the fluorescence intensity at 80 min of induction, showing the strongest fluorescence signal.

[0036] Figure 4 It is a graph of the results of the drop plate experiment;

[0037] Among them, A is the GB05 (empty) without plasmid;

[0038] B is the GB05 carrying pBBR1-Rha-109TES-Kan;

[0039] C is the GB05 carrying pBBR1-Rha-TES-Kan (deletion of 109 protein);

[0040] D is the GB05 carrying pUC19 plasmid.

[0041] Figure 5 It is a graph of the electrophoresis results of the three substrates used in the recombination experiment.

[0042] Figure 6 Figures for colony morphology of transformants obtained under linear-linear recombination conditions for 109TES and TES two groups of different recombination strategies.

[0043] Figure 7 Bar chart for quantitative comparison of transformation efficiency of four groups of experiments (expressed in cfu / μg DNA);

[0044] Note: "****" means P < 0.0001; "***" means P < 0.001.

[0045] Figure 8 Figure for verification results of recombination transformants;

[0046] A is the colony PCR verification results of linear-linear recombination transformants obtained under two recombination modes;

[0047] B is the colony PCR verification results of linear-circular recombination transformants obtained under two recombination modes. DETAILED DESCRIPTION

[0048] The present application will be further described below in conjunction with the drawings and specific examples. It should be understood that the examples are only used to illustrate the technical solutions of the present application, and not to limit the present application. Any equivalent replacement or deformation according to the technical essence of the present application shall fall within the protection scope of the present application.

[0049] The recombination system expression strains E. coli GB05, GB05(pBBR1-Rha-109TES-Kan) and GB05(pBBR1-Rha-TES-Kan) described in the present example are all preserved in the Hunan Provincial Key Laboratory of Microbial Molecular Biology. The gene sequencing and gene synthesis required in the process of plasmid construction are all entrusted to Nanjing Kingsway company to complete. Except for special instructions, the plasmids used are all commercially available conventional plasmids, and the electroporation of the recipient bacteria is carried out according to the general method in the art.

[0050] Some technical terms appearing in the present application and their definitions are as follows:

[0051] 1. λ-Red α / β / γ system: a classic recombination system derived from λ phage, composed of Red α (Exo, an exonuclease), Red β (SSAP, a single-strand annealing protein) and Red γ (a RecBCD inhibitor), which can mediate homologous recombination between linear-circular DNA fragments, and is one of the most mature gene editing tools in E. coli.

[0052] 2. RecE / RecT system: a recombination system derived from E. coli Rac prophage, composed of RecE (5'→3' exonuclease) and RecT (single-strand annealing protein), which functions similarly to the lambda-Red system and also performs linear-linear homologous recombination, and is a representative system of the SSAP (single-strand annealing protein) family.

[0053] 3. 109 genes: refer to three encoding genes, i.e., orf19, orf20, and orf21, in the 109TES operon identified in the genome of Aeromonas phage vB_AsaM-56 by the present application, numbered in the order of phage genome annotation. It is presumed that the three genes are involved in recombination function.

[0054] 4. 109 proteins: refer to the proteins translated from the three 109 genes. Experimental results show that deletion of the three genes can significantly reduce the overall homologous recombination efficiency of the 109TES operon, suggesting that they play an important role in the recombination process.

[0055] 5. 109TES operon: refers to a six-gene structural module identified from Aeromonas phage vB_AsaM-56, containing orf18, orf17, orf16, and three unknown function genes (orf19, orf20, orf21). Among them, orf18, orf17, and orf16 have homology with RecT, Reda, and SSB proteins, respectively, and are considered to constitute a "exonuclease-SSAP type linear homologous recombination system" derived from phage.

[0056] 6. Linear-linear recombination: refers to a homologous recombination method between two linear DNA fragments mediated by homologous sequences.

[0057] 7. Linear-circular recombination: refers to a homologous recombination method between a linear DNA fragment and a circular plasmid mediated by homologous sequences.

[0058] Unless otherwise defined, other technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art.

[0059] Example 1: Identification and expression vector construction of phage 109TES operon

[0060] (1) Screening and identification of 109TES operon

[0061] With the reported amino acid sequences of lambda-Red α / β / γ system and RecE / RecT system as reference templates, BLASTP alignment analysis was performed on the genomes of Aeromonas spp. and their phages in the NCBI database to screen potential recombinase-encoding genes. As a result, a group of candidate genes with high homology was found in the Aeromonas salmonicida phage vB_AsaM-56 (NCBI accession number: NC_019527.1), the nucleotide sequence of which is shown as SEQ ID No. 1, and the group was named as 109TES operon. The phage has host specificity for Aeromonas salmonicida.

[0062] The intergenic relationship is shown as Figure 1 The overall structure is highly similar to the RecE / RecT system. The 109TES operon structure identified from the genome of the Aeromonas salmonicida phage vB_AsaM-56 is shown in the figure, and the operon comprises six genes, Where orfl8, orfl7, orfl6 encode proteins similar to RecT, Redα and SSB respectively, the remaining three proteins are unknown functional proteins The 109TES operon comprises six open reading frames (ORFs), specifically as follows:

[0063] ①orf18 (T protein): presumed to be a single-stranded annealing protein (SSAP), 292 aa in length, and the similarity with the RecT protein amino acid sequence is 30.94%;

[0064] ②orf17 (E protein): presumed to be a 5'-3' exonuclease, 227 aa in length, and the similarity with lambda-Red α is 44.55%;

[0065] ③orf16 (S protein): presumed to be a single-stranded DNA binding protein (SSB), 160 aa in length, and the similarity with E. coli SSB is 31.02%;

[0066] ④orf19, orf20, orf21: the functions are not clear, but considering the functional correlation between the genes within the operon, they are also retained.

[0067] (2) Construction of expression vector

[0068] To verify the recombination function of the operon, the full-length 109TES operon was artificially synthesized and cloned into the broad-host-range plasmid pBBR1 backbone, and the expression thereof was regulated by the rhaR-rhaS pRha promoter, thereby obtaining the recombinant plasmid pBBR1-Rha-109TES-Kan, the nucleotide sequence of which is shown as SEQ ID No. 2.

[0069] Meanwhile, a control plasmid pBBR1-Rha-TES-Kan was constructed by deleting the gene encoding 109 protein in 109TES operon, the nucleotide sequence of which is shown as SEQ ID No. 3, to verify the functional role of 109 protein in the recombination process. The nucleotide sequence of the control plasmid pBBR1-Rha-TES-Kan is shown as SEQ ID No. 4.

[0070] The above two plasmids were introduced into E. coli GB05 by electroporation to obtain recombinant strains GB05(pBBR1-Rha-109TES-Kan) and GB05(pBBR1-Rha-TES-Kan), respectively.

[0071] (3) Verification of promoter activity

[0072] To confirm the induction characteristics of rhaR-rhaS pRha promoter, a control strain GB05(pBBR1-Rha-eGFP-Kan) was constructed. The nucleotide sequence of the control strain GB05(pBBR1-Rha-eGFP-Kan) is shown as SEQ ID No. 5. Figure 3 It can be seen that:

[0073] ①The fluorescence signal of the control group without adding L-rhamnose inducer was significantly lower than that of the experimental group with adding inducer;

[0074] ②In the induction group, the fluorescence intensity gradually increased with the extension of the induction time.

[0075] The results show that prolonging the induction time can enhance the promoter activity and improve the protein expression level. In the subsequent experiment, L-rhamnose induction for 80 min was used as the best induction condition.

[0076] Example 2: Construction and preliminary functional verification of the recombination system

[0077] The present embodiment provides a method for constructing a 109TES operon recombination system based on a rhamnose inducible promoter (pRha) and its functional verification in E. coli.

[0078] The strains used include E. coli DH5α and GB05, which were cultured in LB medium at 37℃. The antibiotics used in the experiment include ampicillin (Amp), kanamycin (Kan) and gentamicin (Gen), which were purchased from Shanghai Generay Biotech Co., Ltd.; restriction enzymes and other molecular biology reagents were purchased from Baobioengineering Co., Ltd. The plasmids and their functions are as follows:

[0079] ① pBBR1-Rha-109TES-Kan: containing 109TES operon;

[0080] ② pBBR1-Rha-TES-Kan: control plasmid with 109 gene deletion;

[0081] ③ pBBR1-Rha-eGFP-Kan: control plasmid for verifying the inducible activity of pRha promoter;

[0082] ④ pUC19 and pBBR1-Gen-Kan: used as the source of circular plasmid template and linearized fragment, respectively.

[0083] The above plasmids were synthesized by Nanjing Kingsway.

[0084] Construction of recombinant strains

[0085] pBBR1-Rha-109TES-Kan and pBBR1-Rha-TES-Kan were electroporated into E. coli GB05, respectively, to obtain recombinant strains GB05(pBBR1-Rha-109TES-Kan) and GB05(pBBR1-Rha-TES-Kan).

[0086] LB plates containing X-gal (purchased from Beijing Coolaber) and IPTG (purchased from Shanghai Generay) were used for the drop plate experiment to verify the background expression of the recombinant system. As shown in FIG. 2, no blue colonies appeared on the plates for both recombinant strains, while the positive control group transformed with pUC19 produced obvious blue colonies, and the GB05 control group that was only electroporated but not added with plasmid was white. Figure 4

[0087] The results show that the constructed recombinant plasmid does not cause false positive signals, the system has low background expression, and is suitable for subsequent functional verification experiments.

[0088] Verification of homologous recombination efficiency

[0089] To evaluate the recombination efficiency of the 109TES recombination system under different lengths of homologous arms, homologous arms of 50 bp, 100 bp, and 150 bp were designed to connect the gentamicin resistance gene (Gen) fragment, and were introduced into the recombinant strain for recombination experiments. The results are shown in Table 1,

[0090] Table 1

[0091]

[0092] The experimental results show that positive clones can be obtained under the three conditions, and the recombination efficiency is significantly improved with the increase of the length of the homologous arm; even under the condition of a 50 bp homologous arm, positive transformants can be stably obtained.

[0093] Therefore, the minimum effective homologous arm length of the system is determined to be 50 bp, indicating that the 109TES operon can mediate efficient recombination of short homologous arms.

[0094] ​Example 3: Different recombination mode validation of 109 recombination system in E. coli and 109 gene function analysis

[0095] This example aims to validate the functional performance of 109 TES recombination system in E. coli under different recombination modes (linear-linear, linear-circular, as shown in Figure 2 Figure 1) and further analyze the role of 109 gene in the system. The three substrates used in the recombination experiment are shown in Figure 5 Figure 1, lane 1 is the circular pUC19 plasmid, lane 2 is the linearized pUC19 plasmid, and lane 3 is the Gen-QLacZα fragment carrying the gentamicin resistance gene.

[0096] The specific steps are as follows:

[0097] (1) Preparation of plasmids and fragments

[0098] pUC19 plasmid was extracted from E. coli DH5α (pUC19) culture using the column plasmid mini-extraction kit from Shanghai Biosciences. Part of the plasmid was linearized by double digestion with NdeI and BamHI for linear-linear (linear-linear) recombination experiment; the other part remained circular for linear-circular (linear-circular) recombination experiment.

[0099] Plasmid pBBR1-Gen-Kan was extracted from GB05 (pBBR1-Gen-Kan) strain and double-digested with PstI and NcoI to obtain the Gen template fragment. Then, using this as a template, the Gen resistance fragment with 50 bp homologous arms was amplified using primers Gen-QLacZα-F and Gen-QLacZα-R.

[0100] (2) Induction of recombination system and electroporation

[0101] To induce the expression of 109 TES operon and verify its recombination activity, a single GB05 (pBBR1-Rha-109TES-Kan) colony was inoculated in 1 mL LB medium and incubated at 37°C overnight.

[0102] The next day, 20 μL of bacterial solution was inoculated into 1 mL of fresh LB medium and incubated at 37°C until the OD 600 was 0.4-0.6, 25 μL of 100 mg / mL L-rhamnose solution was added, and induction was performed for 40 min. After induction, the cells were washed twice with pre-cooled sterile water, centrifuged at 8,000 rpm for 1 min, the supernatant was discarded, and about 100 μL of the suspension was retained as the bacterial solution for electroporation.

[0103] During the electroconversion process: cyclic pUC19 and Gen-QLacZ fragments were added to electroconversion cuvette 1 for linear-cyclic recombination; linearized pUC19 and Gen fragments were added to electroconversion cuvette 2 for linear-linear recombination.

[0104] The cells were electroporated once at 1,250 V using an Eppendorf 2510 electroporator, followed immediately by 1 mL of LB broth for 2 h of recovery. The recovery solution was plated onto LB agar plates containing 30 μg / mL Gen and X-gal and incubated at 37 °C for 24 h. White colonies were picked for colony PCR to verify positive recombination events.

[0105] (3) Quantification and analysis of experimental results

[0106] On selection plates containing X-gal, positive colonies that successfully underwent recombination appeared white, while colonies that did not undergo recombination appeared blue. The number of transformants obtained under each recombination condition and the distribution of blue and white colonies are shown below. Figure 6 As shown.

[0107] In the GB05 (pBBR1-Rha-TES-Kan) control group lacking the 109 gene, recombination efficiency was significantly reduced, with the number of positive colonies being only about 5%–10% of that in the intact operon group. Figure 7 This indicates that the 109 protein plays an important auxiliary role in recombination, possibly participating in the stabilization, annealing, or formation of the recombination complex of single-stranded DNA. Randomly selected positive colonies were subjected to PCR amplification, and the correctly inserted Gen fragment was detected in all cases. The colony PCR verification results of the recombinant transformants obtained under six recombination conditions are shown in the agarose gel electrophoresis results. Figure 8 As shown, they include: 109TES line-to-line recombination ( Figure 8 A, lanes 1–5), 109 TES line-loop recombination ( Figure 8 B, lanes 1–5), TES line-to-line reconstruction ( Figure 8 A, lanes 6–10), TES line-loop recombination ( Figure 8 B, lanes 6–10). Specific bands of approximately 1004 bp were detected in all samples, indicating successful recombination (M is the DNA Marker).

[0108] The above results prove that the 109TES operon can mediate efficient homologous recombination in E. coli and has stable recombination function. In this study, E. coli is used as a chassis to verify the 109TES recombination system, but it should be pointed out that the 109TES operon is derived from Aeromonas phage vB_AsaM-56. The recombination module carried by the phage can usually function in its natural host or closely related bacterial genera. Therefore, from the perspective of evolutionary origin and protein functional conservation, it is speculated that the 109TES system also has potential application value in Aeromonas.

[0109] On the one hand, the core genes orf18 (RecT class SSAP protein), orf17 (Red alpha / RecE class exonuclease) and orf16 (SSB protein) in 109TES have high structural and functional conservation with the homologous recombination module widely existing in gram-negative bacteria, and can theoretically realize the homologous recombination of linear DNA fragments in Aeromonas.

[0110] On the other hand, 109TES is derived from Aeromonas phage, indicating that the system is naturally adapted to the cellular environment of Aeromonas (such as nuclease system, replication mechanism and DNA repair system), so it is biologically reasonable for it to function in Aeromonas.

[0111] The above is only the preferred embodiment of the present application, and the protection scope of the present application is not limited to the above-mentioned embodiments. Any technical solutions falling within the concept of the present application shall fall within the protection scope of the present application. It should be noted that, for ordinary researchers in the technical field, some improvements and refinements without departing from the principles of the present application shall also be considered as the protection scope of the present application.

Claims

1. An Aeromonas bacteriophage recombinant system, characterized by, The recombinant system includes bacteria derived from Aeromonas salmonidus (… Aeromonas salmonicida ) Operator 109TES of bacteriophage vB_AsaM-56, said operator 109TES comprising: a exonuclease gene; a single-stranded DNA annealing protein gene; a single-stranded binding protein gene; at least one non-homologous protein gene; the nucleotide sequence of the operon 109TES is shown as SEQ ID No.

1.

2. An expression vector, characterized by, the vector comprises: the Aeromonas bacteriophage recombination system of claim 1; an origin of replication; a resistance screening gene; an inducible promoter.

3. The expression vector of claim 2, wherein, the origin of replication is pBBR1, the resistance screening gene is kanamycin resistance gene, and the inducible promoter is rhamnose inducible promoter.

4. The expression vector of claim 3, wherein, the expression vector is named pBBR1-Rha-109TES-Kan, and the nucleotide sequence thereof is shown as SEQ ID No.

2.

5. A recombinant bacterial strain, characterized in that, the strain contains the expression vector of any one of claims 2-4.

6. The recombinant bacterial strain of claim 5, wherein, the strain is Escherichia coli GB 05 or Aeromonas.

7. A method for achieving genome modification in Aeromonas or Escherichia coli using the Aeromonas phage recombination system of claim 1 or the expression vector of any one of claims 2-4, characterized in that, the method mediates gene knockout, insertion or replacement of short homologous arms; the method is a non-disease diagnosis or treatment method.

8. Use of the Aeromonas bacteriophage recombination system of claim 1 or the expression vector of any one of claims 2-4 in functional gene research of Aeromonas or fish vaccine development.

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