CN109706126B - Method for site-specific integration of T7 phage genome with exogenous gene and application - Google Patents

Abstract

The invention relates to a method for introducing exogenous genes into T7 bacteriophage and application thereof, belonging to the technical field of biological engineering. The method adopts T7 bacteriophage, which is gene10, gene11 deficient T7 bacteriophage, and the genome thereof contains a positive gene downstream of gene 9

Integrase host integration into attB site; pIVOP-EXS plasmid, the genome of which contains the complete locus of the gp10A protein encoded by the above-mentioned T7 phage, and

an integrase protein coding region; a mini-loop DNA containing a foreign gene fragment to be introduced,

the integrase phage integrates into the attP site, as well as the coding region for the gp11 protein of the T7 phage described above. By adopting the integration strategy of the product, the introduction efficiency of the exogenous gene is greatly improved, the defect of low integration efficiency of the homologous recombination method is overcome, the technical advantage of directly carrying out in-vitro genetic engineering modification on the T7 phage genome is not needed, and the technical threshold and the economic cost of practical application are greatly reduced by combining with a simple experimental process similar to the homologous recombination method.

Description

Method for site-specific integration of T7 phage genome with exogenous gene and application

Technical Field

The invention relates to the technical field of biological engineering, in particular to a method for site-specific integration of a T7 bacteriophage genome with exogenous genes and application thereof.

Background

The T7 phage is a virulent phage using Escherichia coli (Escherichia coli) as a host, has the advantages of concise genome (39.937kbp) and short propagation period, and is currently applied to development of bactericidal drugs and display of protein libraries.

The application of T7 phage cannot be separated from genetic engineering modification, especially the introduction of exogenous genes. The traditional introduction method relies on homologous recombination, and is characterized in that an exogenous gene fragment with homologous sequences of about 50bp upstream and downstream of a target point on both sides is cloned to a donor plasmid, and the T7 bacteriophage continuously proliferates in a host bacterium with the plasmid to generate offspring carrying the exogenous fragment. With the development of the T7 phage in-vitro packaging technology, researchers can firstly carry out in-vitro genetic engineering transformation on T7 phage genome by adopting a molecular biology method, and then obtain phage particles with infection capacity by in-vitro packaging, namely an in-vitro cloning method.

Although the two methods described above have opened the way for the genetic engineering of the T7 phage, there are still respective shortcomings. The biggest disadvantage of the homologous recombination method is that the yield of the recombinant is not high, and a strict forward screening marker is needed, otherwise, the screening of the recombinant is time-consuming and labor-consuming. However, the difficulty in obtaining the recombinant is greatly reduced by means of an in vitro packaging technology, but the reagent components for in vitro packaging are complex, the commercial finished product is mainly relied on at present, the economic cost is higher, and the direct genetic engineering modification of the T7 phage genome is not as convenient and fast as the conventional molecular cloning, so the in vitro cloning method has the problem of higher technical threshold.

Disclosure of Invention

Therefore, it is necessary to overcome various application defects of the conventional T7 phage exogenous gene introduction method, such as low integration efficiency of the homologous recombination method, difficulty in mass introduction of exogenous genes, high economic cost and technical threshold of the in vitro cloning method, and inconvenience for popularization in conventional laboratories. The invention provides a product and a method for introducing exogenous genes into T7 bacteriophage, and the product and the method can ensure the high-efficiency introduction of the exogenous genes and reduce the overall economic cost and the practical technical threshold of an engineering T7 bacteriophage integration system. The in vivo integration strategy provided by the system greatly improves the introduction efficiency of the exogenous gene, makes up the defect of low integration efficiency of the homologous recombination method, does not directly carry out in vitro gene engineering modification on the T7 phage genome, combines a simple experimental process similar to that of the homologous recombination method, and greatly reduces the technical threshold and economic cost of practical application.

The T7 bacteriophage, wherein the T7 bacteriophage is gene10, gene11 lackThe trapped T7 phage has genome containing positive gene downstream of gene 9

The integrase host integrates the attB site.

The invention also discloses a host bacterium containing pIVOP-EXS plasmid, wherein the plasmid genome contains the complete locus of the T7 phage-coded gp10A protein, and

an integrase protein coding region; the gp10A protein is the protein encoded by the above-mentioned T7 phage gene 10.

It is understood that the above is not limited to the type of host bacterium, and may be any strain that meets the conditions of the T7 phage host. However, the selection of the above host bacteria has a good effect.

The invention also discloses a micro-ring DNA, which contains a foreign gene segment to be introduced,

the integrase phage integrates attP site, and the coding region of the above T7 phage gp11 protein; the gp11 protein is the protein encoded by the above-mentioned T7 phage gene 11.

The invention also discloses a product for introducing exogenous genes into the T7 phage, which comprises the T7 phage, the host bacterium containing pIVOP-EXS plasmid and the minicircle DNA.

Integrase is a temperate phage derived from Streptomyces

The site-directed recombinant enzyme of (2) has been widely used in the field of multi-species genetic engineering in view of its high efficiency and specificity. Site-directed recombinases can mediate the presence of both host integration site (attB) and phage integration site (attP)Leads to specific integration between attP and attB, has efficiency far higher than that of homologous recombination, and

the working environment condition of the integrase is not harsh, and the integrase is suitable for the introduction of the exogenous gene of the T7 phage.

In one embodiment, the genome of the T7 phage retains the upstream T7 promoter and the downstream gene10 gene

A transcription termination signal coding region;

in the genome of pIVOP-EXS plasmid, the complete locus of gp10A protein comprises upstream T7 promoter, ribosome binding region and protein coding region; the complete locus of the T7 phage-encoded gp10A protein and

the integrase protein coding region is inserted with a T7 early transcription terminator (T7Te transcription terminator);

the genome of the micro-ring DNA also comprises a T7 promoter,

A transcription terminator, wherein each coding region comprises the following components in sequence from upstream to downstream:

integrase phage integration of attP site, T7 promoter,

Transcription terminator, coding region of T7 bacteriophage gp11 protein and exogenous gene segment.

In one embodiment, the entire locus of gp10A protein in the genome of pIVOP-EXS plasmid

The integrase protein coding region gene is in the form of polycistronicThe expression of the formula is driven by the T7 promoter.

In one embodiment, the genome of the minicircle DNA contains a p15A moderate copy number replicon; the host bacterium containing pIVOP-EXS plasmid is MC1061F containing pIVOP-EXS plasmid^-A host bacterium. Specifically, the medium copy number refers to the average copy number of the plasmid in a unit cell body, and the average copy number is in the range of 10-50. And MC1061F is selected^-The host bacteria are caused by two reasons, one of which is MC1061F^-The strain is an excellent engineering bacterium with higher transformation efficiency, and is beneficial to the uptake of micro-ring DNA; second, MC1061F^-Is a normal host for the T7 bacteriophage.

The invention also discloses a method for site-specific integration of the T7 phage genome by using the product, which comprises the following steps:

1) preparation of minicircle DNA: taking pMCB as a template, obtaining a micro-ring DNA precursor through PCR amplification, and cyclizing the micro-ring DNA precursor to obtain micro-ring DNA;

2)MC1061F^-the transformation of (2):

MC1061F containing pIVOP-EXS plasmid was taken^-Host strain, and the micro-ring DNA is transformed into the MC1061F containing pIVOP-EXS plasmid by a heat shock method or an electric transformation method^-A host strain;

3) MCB integration in vivo:

adding the T7 bacteriophage into the recovered host strain for in vivo integration, and performing thallus lysis to obtain a lysate;

4) isolation of MCB integrants: and (3) plating the lysate on an agarose LB (lysogeny broth) culture medium for culture until plaques appear, taking a plaque monoclonal, carrying out PCR (polymerase chain reaction) detection on an integrant, and collecting a target integrant.

In one embodiment, step 1) of preparing the micro-loop DNA is performed by

II One Step Cloning Kit circularizes the minicircle DNA precursor.

In one embodiment, step 2) MC1061F^-In the transformation of (1), the MC1061F^-Host computerThe genotype of the strain is araD139 delta (araA-leu)7697 delta (lac) X74 galK16 galE15(GalS) lambda-e14-mcrA0 relA1 rpsL150 (Str)^R)spoT1 mcrB1 hsdR2。

In one embodiment, step 2) MC1061F^-In the transformation, the heat shock method comprises the following specific operation steps:

the minicircle DNA was reacted with MC1061F^-After mixing the host strains, carrying out ice bath for 30min, carrying out water bath at 42 ℃ for 30sec, carrying out ice bath for 2min for cooling, finally adding 900 mu l of 37 ℃ non-resistant LB culture medium, and carrying out shake culture at 37 ℃ and 220rpm for 1h to recover the strains.

In one embodiment, step 3) MCB in vivo integration comprises the following specific steps:

after the thalli are recovered, centrifuging at room temperature of 2000g for 1min to collect thalli, discarding supernatant, suspending in 500 mul 37 ℃, adding the T7 phage into the heavy suspension according to MOI (equal to 0.1), transferring the mixed solution to a sterilized centrifuge tube, and performing shake culture at 37 ℃ and 220rpm until the thalli are completely cracked; centrifuging the lysate at room temperature at 10000g for 5min, and collecting the supernatant.

The MOI (multiplicity of infection) is the multiplicity of infection, which means the ratio of the number of phage to bacteria at the time of infection, i.e., the average number of phage per bacteria infected.

In one embodiment, in the step 4) of separating the MCB integron, the specific operation steps are as follows:

taking a proper amount of the lysate supernatant, uniformly mixing with a host bacterium solution and a 0.7% agarose LB culture medium, inverting the mixture in an LB culture dish, placing the LB culture dish in an incubator at 37 ℃ for 3-5 hours until plaques appear, selecting a display plate with the plaque number less than 100, inoculating 48 plaque monoclonals into the bacterial solution, standing and culturing at 37 ℃ for 2 hours until the bacterial solution is clear, carrying out PCR detection on an integron, transferring the PCR positive monoclonals to a new centrifuge tube, carrying out PCR again, recycling a product, and then sequencing to complete the confirmation of the integron.

In one example, in step 4) isolation of the MCB integrants, PCR was performed using Int-SP3 and G11-SR as specific primers to detect the integrants;

Int-SP3：5’-TTTTCAGAGCAAGAGATTACGC-3’(SEQ ID No.1)

G11-SR：5’-GAAGGATGTCAGGCTGTTGG-3’(SEQ ID No.2)。

the system for introducing the exogenous gene into the T7 phage comprises 3 modules of the T7 phage, host bacteria containing pIVOP-EXS plasmid and donor micro-ring DNA, and is matched with an experimental flow with both practicability and repeatability on the basis.

The T7 phage (T7 delta G10G11-attB) mainly has the following characteristics: first, it contains a forward sense in its genome

The integrase host integrates an attB site (attB). Secondly, most of the gene10 and gene11 loci in the genome of the engineered phage have been deleted, and the engineered phage is a gene10 and gene11 deficient T7 phage.

T7 gene10A and contained in engineering plasmid (pIVOP-EXS)

The integrase encoding genes are jointly expressed by the driving of a T7 promoter in a polycistronic mode, the expressed gp10A protein can support the multiplication of a gene10 function-deleted T7 phage, and

the integrase can mediate the integration of exogenous circular DNA into the engineered T7 phage without affecting the proliferation of the T7 phage.

The T7 phage gp11 protein coding region contained in the matched micro-loop DNA is positioned at the downstream of the T7 promoter, and an insertion is arranged between the two

A transcription termination signal. Secondly, upstream of the T7 promoter is

The integrase phage integrates into the attP site (attP). Besides, the minicircle DNA contains p15A moderate copy number replicons, which can ensure the stable inheritance of the minicircle DNA in host bacteria.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a method for site-specific integration of a T7 phage genome by exogenous genes and application thereof.A specific T7 phage, a host bacterium containing pIVOP-EXS plasmid and 3 modules of donor micro-ring DNA are matched, so that the high-efficiency introduction of the exogenous genes can be ensured, and the overall economic cost and practical technical threshold of an engineering T7 phage integration system can be reduced. The in vivo integration strategy provided by the system greatly improves the introduction efficiency of the exogenous gene, makes up the defect of low integration efficiency of the homologous recombination method, does not directly carry out in vitro gene engineering modification on the T7 phage genome, combines a simple experimental process similar to that of the homologous recombination method, and greatly reduces the technical threshold and economic cost of practical application.

Drawings

FIG. 1 is a schematic of a precursor for the construction of T7 Δ G10G11-attB (T7 Δ G9).

FIG. 2 is a pRF-C1 map.

FIG. 3 is a schematic diagram of the construction of T7 Δ G10G 11-attB.

FIG. 4 is a pRF-C2 map.

FIG. 5 is a structural diagram of the major portion of T7 Δ G10G11-attB gene.

FIG. 6 is a pIVOP-EXS plasmid map.

FIG. 7 is a pMCB plasmid map.

FIG. 8 is a MCB minicircle DNA map.

FIG. 9 is a diagram showing the theoretical gene structure of the MCB integrant.

FIG. 10 is a MCDX1 micro-loop DNA map.

FIG. 11 shows the results of the T7 Δ G10G11-attB infestation experiments.

Wherein, BL 21-C1011: BL21 strain capable of providing gp10A and gp11 proteins; BL 21-C10: BL21 strain which can only provide gp 10A; BL 21-C11: only BL21 strain providing gp 11.

FIG. 12 is a gel electrophoresis image of the integrant-specific primer PCR amplification product of the in vivo integration reaction product in the examples;

FIG. 13 is a gel electrophoresis image of the PCR amplification product of detecting integrants of 48 single clones in example.

FIG. 14 is a gel electrophoresis chart of the PCR amplification products of the 8 positive integrants isolated in the example and then identified.

FIG. 15 is a gel electrophoresis image of PCR amplification products of integrated reaction products of different sizes of exogenous fragments.

FIG. 16 shows the result of PCR for identifying 47 monoclonals of the integrant MCB-I1.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The starting materials used in the following examples, unless otherwise specified, are all commercially available; the molecular cloning or genetic engineering techniques employed, unless otherwise specified, are conventional.

Example 1

First, T7 phage (T7 Δ G10G11-attB) was prepared.

The T7 phage (T7 Δ G10G11-attB) parent was derived from the T7 phage cDNA display library, which was previously generated by

10-3Cloning Kit (from Merck, USA). Thus, the T7 phage in the library possessed the characteristics of the product described above, as follows: the upstream expression regulatory element of the gene10 locus is modified, the expression level of the downstream gene is reduced, and the gene must be in a host bacterium additionally provided with gp10A protein to be normally propagated.

The T7 phage (T7 Δ G10G11-attB) was constructed as follows:

1. a precursor of T7 Δ G10G11-attB (T7 Δ G9) was constructed.

The construction of T7 delta G9 adopts a homologous recombination method, firstly a donor plasmid (pRF-D1) and a complementation plasmid (pRF-C1) for homologous recombination are constructed by a conventional molecular cloning technology, the two sides of a recombination region in the recombination plasmid contain a homologous region of T7 bacteriophage, an upstream homologous region sequence of pRF-D1 is positioned upstream of a gene 9 coding region, a downstream homologous region sequence is positioned at the carboxyl terminal of a gene10 coding region, and a middle target fragment is a complete gp10A protein coding sequence, as shown in figure 1. pRF-C1 contains the gene 9 upstream promoter and the downstream entire protein coding region, as shown in FIG. 2.

After pRF-D1 and pRF-C1 were obtained, BL21 competent cells were transformed, respectively, to obtain a host bacterium BL21(pRF-D1) for homologous recombination and a host bacterium BL21(pRF-C1) for maintenance and selection of recombinants.

Library T7 phage infection and lysis of BL21(pRF-D1), the resulting lysate supernatant diluted 10³After doubling, 1. mu.l was taken for plaque formation experiment of BL21 (pRF-C1). Theoretically, only recombinants were able to form plaques on BL21 (pRF-C1). And (3) separating a plaque monoclonal, carrying out PCR (polymerase chain reaction) and sequencing to identify a positive recon, and finally obtaining T7 delta G9.

2. Construction of T7 Δ G10G11-attB

The construction of T7 delta G10G11-attB adopts a homologous recombination method, firstly a donor plasmid (pRF-D2) and a complementation plasmid (pRF-C2) for homologous recombination are constructed by a molecular cloning technology, the upstream homologous region sequence of the pRF-D2 is positioned in a gene 9 promoter region, the downstream homologous region sequence is positioned at the carboxyl end of a gene11 protein coding region, an intermediate target segment contains a gp9 complete protein coding region, and attB (A), (B) and (D) are combined to form a new plasmid

Integrase host integration site), the T7 promoter and

transcription termination signals, as shown in FIG. 3. pRF-C2 contains the gene10A upstream promoter and the downstream complete protein coding region,

transcription termination signals and the entire gene11 protein coding region are shown in FIG. 4.

After pRF-D2 and pRF-C2 were obtained, BL21 competent cells were transformed, respectively, to obtain a host bacterium BL21(pRF-D2) for homologous recombination and a host bacterium BL21(pRF-C2) for maintenance and selection of recombinants. T7 delta G9 infects and lyses BL21(pRF-D2), and after the supernatant of the lysate is diluted 10^3 times, 1 mu l of the lysate is taken for BL21(pRF-C2) plaque formation experiment. Theoretically, only recombinants were able to form plaques on BL21 (pRF-C2). And (3) separating a plaque monoclonal, carrying out PCR (polymerase chain reaction) and sequencing to identify a positive recon, and finally obtaining T7 delta G10G 11-attB.

II, MC1061F containing pIVOP-EXS plasmid^-And (4) preparing host bacteria.

1. Construction of pIVOP-EXS plasmid

Enhancement by molecular cloning techniques

The integrase coding sequence is cloned to the downstream of gene10 gene locus, the gene10 gene locus includes complete upstream expression regulation and control original and protein coding region, the gene10 gene locus and enhancement type

The integrase coding region contains a T7 early transcription termination signal.

2. pIVOP-EXS transformed MC1061F^-Commercializing competent cells (geoOnly, Shanghai, China), MC1061F was obtained^-(pIVOP-EXS)。

MC1061F^-Competent cells were obtained from Shanghai Diego Biotech, Inc. by heat shock method using pIVOP-EXS for MC1061F^-Transformation of competent cells, the procedure was as follows: the mixture of competent cells and plasmids is ice-bathed for 30min, then heat shock treatment is carried out in 42 ℃ water bath for 30sec, the mixture is cooled in 90sec ice bath, 900 mul of 37 ℃ non-resistant LB culture medium is added, shaking culture is carried out at 37 ℃ and 220rpm for 1h to recover the thalli, and finally the transformation product is coated on Amp⁺LB dishes, 37 ℃ incubator overnight.Selecting single clone to carry out colony PCR and sequencing on the next day to identify positive transformant, and finally obtaining MC1061F^-(pIVOP-EXS)。

3. Preparation of MC1061F by Inore method^-(pIVOP-EXS) competent cells.

Mixing MC1061F^-(pIVOP-EXS) streaking (37 ℃, overnight) and the next day picking single clones to 10ml of 37 ℃ SOB complete medium (Mg addition before use)²⁺Making Mg²⁺Final concentration of 20mM), shaking culture at 37 deg.C and 220rpm until OD600 absorbance reaches 0.6, ice-cooling for 20min, and entering into competent cell preparation. Competent cell preparation buffer (TB buffer) was prepared according to the Inore method, the suspension was centrifuged at 4 ℃ for 5min at 2500g, the supernatant was discarded and resuspended in 10ml of precooled (4 ℃) TB buffer. The resuspension was centrifuged at 4 ℃ and 2500g for 5min, the supernatant discarded and resuspended in 10ml of precooled (4 ℃) TB buffer in an ice bath for 10 min. The resuspension was centrifuged at 2500g for 5min at 4 ℃ and the supernatant discarded, resuspended in 1ml of precooled (4 ℃) TB buffer, 930. mu.l of resuspension was mixed with 70. mu.l DMSO and dispensed into precooled (4 ℃)1.5ml centrifuge tubes (100. mu.l/tube).

And thirdly, preparing the plasmid pMCB.

Using molecular cloning technique to sequentially add attP (A), (B), (C) and (C)

Integrase phage integration site), the T7 promoter,

a transcription termination signal and a gp11 protein coding sequence are cloned to a plasmid with p15A as a replication origin to obtain a plasmid which simultaneously contains an attP site and a T7 promoter,

the p15A plasmid containing the transcription termination signal and the gp11 protein coding region is the pMCB plasmid.

Example 2

A product for introducing an exogenous gene into a T7 bacteriophage, comprising:

1) genetically engineered T7 phage (T7 Δ G10G11-attB) for site-directed integration of exogenous DNA.

The T7 phage is gene10, gene11 deficient T7 phage, and the genome contains a positive gene downstream of gene 9

The integrase host integrates an attB site (attB).

Specifically, the T7 phage genome retains the gene10 upstream T7 promoter and downstream

The transcription termination signal, its coding region and most of the gene11 segment are deleted.

First, it contains a forward sense in its genome

Integrase host integration site (attB). Secondly, most of the gene10 and gene11 loci in the genome of the engineered phage have been deleted, and the engineered phage is a gene10 and gene11 deficient T7 phage. The main gene structure of T7 Δ G10G11-attB is shown in FIG. 5.

2) A host bacterium comprising an artificially constructed plasmid (pIVOP-EXS) for mediating the integration of exogenous circular DNA into the above engineered T7 phage.

The complete locus of the T7 phage-encoded gp10A protein is contained in the plasmid genome, and

an integrase protein coding region; the gp10A protein is the protein encoded by the above-mentioned T7 phage gene 10.

The complete locus of the gp10A protein includes the upstream T7 promoter, ribosome binding region and protein coding region; the complete locus of the T7 phage-encoded gp10A protein and

the early transcription terminator of T7 was inserted between coding regions of the integrase protein.

Among them, T7 gene10A and

the integrase encoding genes are jointly expressed by the driving of a T7 promoter in a polycistronic mode, the expressed gp10A protein can support the multiplication of a gene10 function-deleted T7 phage, and

the integrase can mediate the integration of exogenous circular DNA into the engineered T7 phage without affecting the proliferation of the T7 phage.

The map of pIVOP-EXS plasmid is shown in FIG. 6.

3) Minicircle dna (minicircle) capable of expressing T7 bacteriophage gp11 protein in the presence of the T7 promoter.

The minicircle DNA contains a foreign gene fragment to be introduced,

the integrase phage integrates attP site, and the coding region of the above T7 phage gp11 protein; the gp11 protein is the protein encoded by the above-mentioned T7 phage gene 11.

Specifically, the minicircle DNA contains one

The integrase phage integrates an attP site (attP) capable of being at

Integrase in the presence of host integration sites (attB), used in combination with the engineered T7 phage described above, was able to integrate into the engineered T7 phage host binding sites (attB) under the action of the plasmids described above, resulting in T7 phage that was able to proliferate normally in host bacteria that provide only gp10A, but not gp11 protein.

Furthermore, the coding region of the T7 phage gp11 protein contained in the minicircle DNA is located downstream of the T7 promoter, and an intervening region is provided between the two

A transcription termination signal. Secondly, T7 startMover upstream is

Integrase phage integration site (attP). Besides, the minicircle DNA contains p15A moderate copy number replicons, which can ensure the stable inheritance of the minicircle DNA in host bacteria.

In this example, the parent of the minicircle DNA was from the plasmid pMCB, whose map is shown in FIG. 7. This plasmid differs from the minicircle DNA by additionally containing an ampicillin resistance gene. The micro-loop DNA precursor is obtained by PCR amplification using pMCB as a template, and in this example, the micro-loop DNA precursor is a linear DNA segment of 1549bp in size. It is understood that the size of the resulting minicircle DNA precursor varies depending on the size of the foreign gene fragment to be introduced.

Cyclization of the mini-circle DNA precursor was carried out by Novonzan

II One Step Cloning Kit, according to the instruction manual, 0.015pmol of micro-ring DNA precursor was added to the system, the volume of the micro-ring DNA precursor cyclization reaction was set to 10. mu.l, and the cyclization product was used directly in the subsequent experiment. The map of the minicircle DNA is shown in FIG. 8.

The core operation object, engineering T7 phage (T7 delta G10G11-attB), is transformed by genetic engineering technology, gene10 and gene11 loci are deleted and inserted simultaneously

Host integration site (attB) recognized by integrase. Deletion of the gene10 locus helps to increase the capacity of the T7 phage genome, thereby allowing insertion of larger exogenous fragments, and experiments have shown that engineered T7 phage can introduce exogenous DNA fragments of greater than 3 kb. The gene11 deletion is used for forward screening of the integrants, and the matched minicircle DNA has a gene11 locus, so that the integrants obtain functional complementation of the gene11 and can normally proliferate in host bacteria which do not provide gp11 protein (gene 11 coding protein). Integration of the micro-loop DNA is dependent on

The integrase functions, another core module of the invention, the integration driver plasmid (pIVOP-EXS) provides

Integrase and gp10A packaging protein for complementing T7 delta G10G11-attB gene10 functional defects.

The expression of integrase and gp10A is controlled by T7 promoter in polycistronic form, so that the normal proliferation of host bacteria carrying pIVOP-EXS is not affected by the expression of exogenous genes. The structure of the integration theoretical gene is shown in FIG. 9.

In particular pIVOP-EXS coded

The integrase is enhanced integrase, and the mediated site-directed recombination efficiency is improved by 1 time on the basis of keeping the original site specificity. The 3 rd module of the integration system has both stability and simplicity in design of the micro-loop DNA, the contained p15A replicon can ensure the stable inheritance of the micro-loop DNA in a host bacterium body, and the gene11 locus is used as an efficient defective T7 phage forward screening marker, so that the integrants can be further proliferated and enriched, and the difficulty in screening and separating the integrants is greatly reduced.

Example 3

A method for introducing a foreign gene into a T7 bacteriophage, comprising the steps of:

1) and preparing micro-ring DNA.

The miniloop DNA was originally derived from the plasmid pMCB, which differs from the miniloop DNA in that it additionally contains an ampicillin resistance gene. The micro-loop DNA precursor is obtained by PCR amplification using pMCB as a template, and in this example, the micro-loop DNA precursor is a linear DNA segment of 1549bp in size. It is understood that the size of the resulting minicircle DNA precursor varies depending on the size of the foreign gene fragment to be introduced. If the size of MCDX1 micro-loop DNA after the introduction of the foreign gene fragment is 2801bp, it is shown in FIG. 10.

Cyclization of the mini-circle DNA precursor was carried out by Novonzan

II One Step Cloning Kit, according to the instruction manual, 0.015pmol of micro-ring DNA precursor was added to the system, the volume of the micro-ring DNA precursor cyclization reaction was set to 10. mu.l, and the cyclization product was used directly in the subsequent experiment.

2)MC1061F^-(pIVOP-EXS).

MC1061F^-(pIVOP-EXS) is MC1061F containing pIVOP-EXS plasmid^-Competent cells, genotype of araD 139. delta. (araA-leu) 7697. delta. (lac) X74 galK16 galE15(GalS) lambda. e14-mcrA0 relA1 rpsL150 (Str)^R) spoT1 mcrB1 hsdR 2. Micro-ring DNA transformation MC1061F^-The method of (pIVOP-EXS) is a heat shock method, and the steps are briefly as follows: micro-circle DNA and MC1061F^-(pIVOP-EXS) are mixed and then are subjected to ice bath for 30min, water bath at 42 ℃ for 30sec, ice bath for 2min for cooling, 900 mul of LB culture medium with 37 ℃ and no resistance is finally added, and shaking culture is carried out at 37 ℃ and 220rpm for 1h for recovering the thalli.

3) MCB integration in vivo.

MC1061F^-(pIVOP-EXS) after recovery of the transformation product, RT, 2000g centrifugation for 1min to collect the bacterial cells, discard the supernatant, resuspend in 1ml 37 ℃ Amp⁺LB medium (ampicillin concentration 100. mu.g/ml), 1. mu. l T7. delta.G 10G11-attB (2X 10) was added to the resuspension at an MOI of 0.1⁷pfu), and the mixture was transferred to a sterilized 15ml centrifuge tube and shake-cultured at 37 ℃ and 220rpm until the lysis of the cells was complete. The lysate was centrifuged at 10000g for 5min at RT, and the supernatant was collected into a new 1.5ml centrifuge tube and stored at 4 ℃.

In the experiment, the inventors infected different strains with T7 Δ G10G11-attB, the results of which are shown in FIG. 11. The result shows that when a BL21 strain (BL21-C1011) which can provide gp10A and gp11 proteins is infected by T7 delta G10G11-attB, the OD600 light absorption value of the bacterial liquid is sharply reduced after infection for two hours, and T7 delta G10G11-attB can normally proliferate and crack host thalli; when a BL21 strain (BL21-C10) which only can provide gp10A is infected by T7 delta G10G11-attB, the OD600 light absorption value of the bacterial liquid continuously rises within 160min after infection, and the phenomenon that the OD600 light absorption value is sharply reduced according with the thallus lysis characteristic does not occur, so that T7 delta G10G11-attB is difficult to normally proliferate in host bacteria which do not provide gp11 protein; when a BL21 strain (BL21-C11) which only can provide gp11 is infected with T7 delta G10G11-attB, the absorbance value of the bacterial liquid OD600 continuously rises after infection, which indicates that T7 delta G10G11-attB is difficult to normally proliferate in a host bacterium which does not provide gp10A protein.

And (3) carrying out PCR detection on the integrants of the in-vivo integration reaction product, wherein the theoretical size of the PCR amplification product of the specific primer of the integrants is 518bp, the positions of the primers and the theoretical amplification product are shown in figure 1, a forward primer Int-SP3 is positioned in the micro-loop DNA, a reverse primer G11-SR is positioned in a T7 delta G10G11-attB genome, the regions covered by the two primers contain an integrated symbolic sequence attR, and the target product is difficult to amplify by using a non-integrant as a template. The gel electrophoresis pattern is shown in FIG. 12, and each lane is designed as follows:

TABLE 1 design of the experimental conditions in each lane

	1	2	3	4	5
						MCB	﹢	-	﹢	-	-
MCDX1	-	-	-	﹢	-
						T7ΔG10G11-attB	﹢	﹢	-	﹢	-

Wherein: MCB: micro-loop DNA without foreign fragment insertion;

MCDX 1: inserting micro-ring DNA of 1267bp exogenous segment;

m: and (5) DNA size identification.

As can be seen from the figure, the target bands appeared in the PCR products using the lysates of experiment groups 1 and 4 as templates, indicating that the lysates of experiment groups 1 and 4 providing the minicircle DNA contain the integrants.

4) Isolation of MCB integrants.

And (3) performing gradient treatment on the lysate supernatant obtained in the step (3), and respectively taking 100 mu l of dilution from 10^ 6X, 10^ 7X and 10^ 8X for plating. The plate laying refers to an experimental method of uniformly mixing a solution containing phage, a host bacterium liquid and a 0.7% agarose LB culture medium and then pouring the mixture on an LB solid culture dish. After plating, the plates were placed in an incubator at 37 ℃ for 3-5h, or at RT overnight until obvious plaques appeared. The isolated host cell suspension (BL21-gp10A, OD600 about 0.6) was added to a 96-well plate at 100. mu.l/well for a total of 50 wells. Selecting a display plate with the plaque number less than 100, inoculating 48 plaque monoclonals into the bacterial liquid, and standing and culturing at 37 ℃ for 2h until the bacterial liquid is clear. Int-SP3 (5'-TTTTCAGAGCAAGAGATTACGC-3', SEQ ID No.1) and G11-SR (5'-GAAGGATGTCAGGCTGTTGG-3', SEQ ID No.2) are used as specific primers to carry out PCR detection on the integron, and the theoretical size of a target fragment is 518 bp. And transferring the PCR positive monoclonal to a new centrifugal tube, carrying out PCR again, recovering the product, and then sequencing to complete the confirmation of the integron.

The results show that 8 integrants were selected from 48 plaques in this experiment, and the gel electrophoresis pattern is shown in FIG. 13. FIG. 13 is the gel electrophoresis image of the PCR detection of the 48 single clone integrants, the theoretical size of PCR amplification product of the integrant specific primer is 518bp, and it can be seen from the image that 8 of the 48 single clone PCR reaction products have the target fragment amplified.

The 8 integrants were again subjected to PCR and the gel electrophoresis pattern is shown in FIG. 14. Sequencing results show that the fragments are positive integrants, and PCR amplification fragments clearly contain attR sites of integration products of attB and attP and exogenous sequences which are not originally present in T7 delta G10G 11-attB.

In the process of the experimental method:

the micro-circle DNA prepared in stage 1 usually has a size of 0.01pmol (i.e., approximately 15ng), MC1061F^-(pIVOP-EXS) conversion efficiency of 10⁵～10⁶cfu/. mu.g, therefore MC1061F^-(pIVOP-EXS) the number of starting transformants was about 10³～10⁴cfu。

MC1061F^-(pIVOP-EXS) competent cells were prepared autonomously in the laboratory. Firstly, the MC1061F is transformed by pIVOP-EXS^-Commercializing competent cells, MC1061F was obtained^-(pIVOP-EXS) and then preparing competent cells, wherein the competent cells are prepared by an Inoue method. MC1061F is selected^-The host bacteria are caused by two reasons, one of which is MC1061F^-The strain is an excellent engineering bacterium with higher transformation efficiency, and is beneficial to the uptake of micro-ring DNA; second, MC1061F^-Is a normal host for the T7 bacteriophage.

In the in vivo integration step, attention needs to be paid to the dosage of T7 Δ G10G11-attB, too low dosage can cause the bacterial liquid to be difficult to clarify, or the bacterial cell is completely lysed for too long dosage, and too high dosage can seriously affect the yield of integron, thus being not beneficial to the subsequent processSeparation of (4). Competent cells prepared at an MOI of about 0.1 with addition of T7 Δ G10G11-attB, 100. mu.l generally contain n 10⁸cfu, therefore add 10⁷pfu T7 Δ G10G 11-attB. Gene11 from T7 Δ G10G11-attB⁺The T7 phage contamination rate was 1/2500, 10⁷pfu T7 Δ G10G11-attB doped with about 10³～10⁴pfu gene 11⁺T7 bacteriophage, gene11⁺The T7 phage can also be found in MC1061F^-(pIVOP-EXS) is a non-integrant root source.

The final integrant isolation step is primarily carried out by MC1061F^-(pIVOP-EXS), the quality and concentration of the micro-circular DNA and the amount of T7 Δ G10G11-attB, the yield of integron and MC1061F^-(pIVOP-EXS) transformation efficiency, quality of the micro-circle DNA and concentration are in positive correlation. Trace endogenous gene11 contained in T7 delta G10G11-attB solution⁺The T7 phage resulted in a high proportion of gene11 in the lysate after the integration reaction⁺T7 bacteriophage, gene11 experimentally detected⁺The proportion of T7 phage reaches 50% -90%. To overcome gene11⁺Interference by the T7 phage, the integrant screen number was at least 48 monoclonals.

The minicircle DNA carrying the foreign gene is also prepared by recombinant cloning. Firstly, PCR amplification is carried out to obtain a micro-ring DNA linear skeleton and a target fragment, both positive and negative primers for amplifying the target fragment are provided with homologous sequence joints overlapped with both ends of the micro-ring DNA linear skeleton, and an amplification primer pair of the micro-ring DNA linear skeleton is fixed and is irrelevant to an exogenous fragment. Then, according to

II One Step Cloning Kit instructions that the micro-loop DNA linear skeleton and the target fragment are subjected to recombination cyclization reaction, and the product can be directly used for transforming MC1061F^-(pIVOP-EXS) competent cells. The subsequent experimental procedures were as described above. If the exogenous fragment has adverse biological effects on the T7 bacteriophage or the host bacterium, the integrant yield will be significantly reduced, in which case it is necessary to increase the amount of minicircle DNA used for transformation or to improve the design of the exogenous fragment.

Example 4

In this example, the method according to example 2 integrates exogenous gene fragments of different sizes, and the selected exogenous gene fragments are respectively: the length of the I-SceI coding region is 699 bp;

the integrase partial coding area is 1815bp in length; SP-Cas9 part of the coding region, 3104bp in length.

The method for preparing the micro-ring DNA comprises the following steps: designing amplification primers according to the required exogenous fragment, wherein the forward primers and the reverse primers respectively contain a section of adaptor sequence besides the binding region of the specific sequence, and are used for carrying out recombination reaction cyclization with the micro-ring DNA skeleton part to obtain the micro-ring DNA carrying the exogenous gene. The forward primer adapter sequence was 5'-ctggtctactgactcgctaa-3' and the reverse primer adapter sequence was 5'-cccgaaggtgagccaggtga-3'. The micro-ring DNA skeleton part is also obtained by PCR amplification, the PCR template is pMCB plasmid, the forward primer is 5'-TCACCTGGCTCACCTTCGG-3', the reverse primer is 5'-TTAGCGAGTCAGTAGACCAGAAGTG-3', and the size of the amplified product is 1531 bp. Recombination reaction of the exogenous fragment with the purified product of the minicircle DNA backbone moiety was performed (as above, using

II One Step Cloning Kit), the recombinant product was used directly in MC1061F^-(pIVOP-EXS) transformation of competent cells. The subsequent flow is the same as the previous flow.

TABLE 2 exogenous fragment Length parameters for each lane

Lane lane	1	2	3	4	5
						Insert size (bp)	0(MCB)	699	1815	3064	0(Blank)

The gel electrophoresis image of the integrated reaction product after PCR amplification is shown in FIG. 15, the theoretical size of the target amplification product is 518bp, and it can be seen from the figure that the product and method for introducing exogenous gene into T7 bacteriophage of the present invention can introduce exogenous DNA fragment larger than 3 kb.

Similarly, a positive integrant was isolated and identified from the corresponding lysate of MCB-I1 (integrated with the 699bp foreign fragment of the I-SceI coding region). FIG. 16 shows the result of PCR for identifying 47 monoclonals of the integrant MCB-I1. "+" indicates positive monoclonal integrants, P indicates positive control, and PCR was performed using MCB-I1 lysate as template. As shown, there was a PCR positive integrant in 47 single clones, which identified the presence of the band of interest in the PCR product, which was of the same size as the positive control amplified fragment.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Zhongshan university

<120> method for site-specific integration of T7 phage genome by exogenous gene and application thereof

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<170> SIPOSequenceListing 1.0

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<213> Artificial Sequence (Artificial Sequence)

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ttttcagagc aagagattac gc 22

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<213> Artificial Sequence (Artificial Sequence)

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gaaggatgtc aggctgttgg 20

Claims (10)

1. A T7 phage, wherein the T7 phage is gene10, gene11 deficient T7 phage, and the genome contains positive gene downstream of gene 9

The integrase host integrates the attB site.

2. A host bacterium comprising pIVOP-EXS plasmid, wherein pIVOP-EXS plasmid genome comprises the complete locus of gp10A protein encoded by the T7 phage of claim 1, and

an integrase protein coding region; the gp10A protein isThe protein encoded by the T7 phage gene10 was calculated as 1.

3. A minicircle DNA comprising a foreign gene fragment to be introduced,

an integrase phage integration attP site, and the coding region of the T7 phage gp11 protein of claim 1; the gp11 protein is the protein encoded by the T7 bacteriophage gene11 of claim 1.

4. A product for introducing a foreign gene into a T7 bacteriophage, comprising the T7 bacteriophage of claim 1, the pIVOP-EXS plasmid-containing host bacterium of claim 2, and the minicircle DNA of claim 3.

5. The product of introducing a foreign gene into a T7 bacteriophage according to claim 4,

the genome of the T7 phage retains the upstream T7 promoter and the downstream gene10 gene

A transcription termination signal coding region;

in the genome of pIVOP-EXS plasmid, the complete locus of gp10A protein comprises upstream T7 promoter, ribosome binding region and protein coding region; the complete locus of the T7 phage-encoded gp10A protein and

the T7 early transcription terminator is inserted between coding regions of the integrase protein;

the genome of the micro-ring DNA also comprises a T7 promoter,

A transcription terminator, wherein each coding region comprises the following components in sequence from upstream to downstream:

integrase phage integration of attP site, T7 promoter,

Transcription terminator, coding region of T7 bacteriophage gp11 protein and exogenous gene segment.

6. The product of introducing foreign genes into T7 phage of claim 5, wherein the pIVOP-EXS plasmid has the complete locus of gp10A protein in its genome

The integrase protein coding region genes are expressed together in polycistronic form driven by the T7 promoter.

7. The product of introducing foreign genes into T7 phage of claim 4, wherein the genome of said minicircle DNA contains a p15A moderate copy number replicon; the host bacterium containing pIVOP-EXS plasmid is MC1061F containing pIVOP-EXS plasmid^-A host bacterium.

8. A method for site-directed integration of a T7 phage genome with a foreign gene, comprising the steps of using the product of claim 7:

1) preparation of minicircle DNA: taking pMCB as a template, obtaining a micro-ring DNA precursor through PCR amplification, and cyclizing the micro-ring DNA precursor to obtain micro-ring DNA;

2)MC1061F^-the transformation of (2):

MC1061F containing pIVOP-EXS plasmid was taken^-Host strain, and the micro-ring DNA is transformed into the MC1061F containing pIVOP-EXS plasmid by a heat shock method or an electric transformation method^-A host strain;

3) MCB integration in vivo:

adding the T7 bacteriophage into the recovered host strain for in vivo integration, and performing thallus lysis to obtain a lysate;

4) isolation of MCB integrants: and (3) plating the lysate on an agarose LB (lysogeny broth) culture medium for culture until plaques appear, taking a plaque monoclonal, carrying out PCR (polymerase chain reaction) detection on an integrant, and collecting a target integrant.

9. The method for site-directed integration of an exogenous gene into the T7 phage genome of claim 8, wherein the step 2) comprises MC1061F^-In the transformation of (1), the MC1061F^-The genotype of the host strain was araD 139. delta. (araA-leu) 7697. delta. (lac) X74 galK16 galE15(GalS) lambda. e14-mcrA0 relA1 rpsL150 (Str)^R)spoT1 mcrB1 hsdR2。

10. The method for site-directed integration of an exogenous gene into the T7 phage genome of claim 8, wherein the step 2) comprises MC1061F^-In the transformation, the heat shock method comprises the following specific operation steps:

the minicircle DNA was reacted with MC1061F^-After mixing the host strains, carrying out ice bath for 30min, carrying out water bath at 42 ℃ for 30sec, carrying out ice bath for 2min for cooling, finally adding 900 mu l of 37 ℃ non-resistant LB culture medium, and carrying out shake culture at 37 ℃ and 220rpm for 1h to recover the strains.

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