Genetic manipulation method for large-scale DNA fragments of bacterial genome

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a genetic manipulation method for a large-scale DNA fragment of a bacterial genome. The technical problem to be solved by the invention is to provide a new choice for genetic manipulation of large-scale DNA fragments. According to the technical scheme, the vector suitable for genetic manipulation of the large-scale DNA fragment of the bacterial genome takes a suicide plasmid vector as a skeleton vector and carries the following structures: a conditional replication starting point, a homologous recombination element, a positive selection marker, a negative selection marker and a transferable element; the homologous recombination element is a deletion homologous recombination element or an insertion homologous recombination element. The invention also provides a bacterial genome large-scale DNA fragment genetic manipulation method based on the vector. According to the technology, large-scale DNA fragments can be accurately inserted into bacterial genomes, and traceless insertion mutation without any antibiotic selection marker can be realized.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for genetic manipulation of large-scale DNA fragments of bacterial genomes. Background Art

[0002] The outer membrane polysaccharides of many pathogens are good protective antigens for the body and have high scientific research and application value. The DNA sequence information responsible for encoding and synthesizing this polysaccharide structure mainly exists in the bacterial genome in the form of a "gene cluster", such as the common bacterial capsular polysaccharide and O antigen polysaccharide gene clusters. Generally speaking, the DNA size of these gene clusters is generally above 10Kbp, which is a "large-scale" DNA fragment. Therefore, if you want to obtain this polysaccharide substance, there are generally two ideas: one is to extract it directly from the pathogen itself; the other is to "clone and express" its entire gene cluster and then extract it indirectly from the host bacteria. The main problem of the former is that the pathogen itself may be difficult to culture, high culture cost and biosafety risks, while the main problem of the latter is the genetic manipulation of large-scale DNA fragments.

[0003] At present, it is easy to clone and express a single gene or a small DNA fragment with the help of recombinant plasmids, and the construction of such recombinant plasmids can be easily achieved through conventional molecular biological methods. However, there are various problems in the recombinant expression of large-scale DNA fragments (>10Kbp). For example, after inserting large DNA fragments, general expression plasmids will have genetic instability and other problems. Therefore, in order to overcome this instability problem, it is necessary to introduce a variety of "stabilization mechanisms", such as inserting some "partition system" DNA elements into the ordinary plasmid backbone. At present, only cosmids or fosmids plasmids with very few numbers and types can stably accommodate such large DNA fragments at the "gene cluster" level. Because these plasmids have different replicons, their copy numbers vary greatly between different host bacteria, and they are also affected by the growth state of the host bacteria, the specific components of the culture medium, and the differences between individual strains. In addition, recombinant plasmids have a universal disadvantage, that is, they need to rely on the selective pressure of antibiotics to maintain their own stable expression in the host bacteria. However, the use of antibiotics will bring the risk of allergies to biological products and increase the manufacturing cost during their production process.

[0004] The above problems do not exist when using bacterial genomes to accommodate large-scale exogenous gene clusters. This strategy has many advantages, such as good genetic stability, uniform expression levels, and no need to add additional antibiotics during cultivation. Existing genetic manipulation technologies can well insert small DNA sequences (such as 2-5Kbp) into bacterial genomes, but if you want to insert large-scale DNA fragments (>10Kbp) into bacterial genomes, there are still certain difficulties.

[0005] The patent document (WO2014057109A1) achieves the site-specific insertion of a large-scale gene cluster into the bacterial genome through the following key steps: 1) constructing two plasmids, pDOC and pTKRED, respectively; pDOC carries two adjacent Scel restriction enzyme sites; pTKRED can express Scel restriction endonuclease and λ-RED system recombinase; 2) between the two Scel sites of the pDOC plasmid, inserting the upstream homologous fragment sequence (upper homology arm) of the insertion site, the target gene cluster, the resistance screening gene with FRT characteristic sequences at both ends, and the downstream homologous fragment sequence (lower homology arm) of the insertion site in sequence, and finally obtaining to a new pDOC recombinant plasmid; 3) the pDOC recombinant plasmid and the pTKRED plasmid were successively transferred into Escherichia coli W3110, and the Scel restriction endonuclease "cut" the "upper homology arm + target gene cluster + resistance gene + lower homology arm" as a whole from the pDOC recombinant plasmid. Under the action of the recombinase, the "cut fragment" underwent homologous exchange at the genome level with Escherichia coli W3110, and a mutant strain in which the target gene cluster was site-specifically inserted into the genome of Escherichia coli W3110 was obtained; 4) the resistance selection marker was eliminated by the FRT enzyme, and finally a mutant strain with a gene cluster genome site-specific insertion without a resistance selection marker but with an FRT "scar" was obtained.

[0006] The above scheme also has some disadvantages: 1) It may be difficult to transfer the super-large pDOC recombinant plasmid into E. coli, and it will be more difficult to transfer both pDOC and pTKRED plasmids into E. coli; 2) The "cutting" of large-scale DNA fragments requires the help of SceI restriction endonuclease, and the copy number of pDOC recombinant plasmid itself in E. coli is limited. Therefore, the "effective release amount" of large-scale DNA fragments in the cytoplasm of E. coli will be limited, and the success rate of insertion mutation may be low. 3) This scheme will eventually leave a "scar" of the FRT characteristic sequence on the genome, and it is impossible to achieve scarless insertion mutation. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a new option for genetic manipulation of large-scale DNA fragments.

[0008] The technical solution of the present invention is a vector suitable for genetic manipulation of large-scale DNA fragments of bacterial genomes, which uses a suicide plasmid vector as a backbone vector and carries the following structures: a conditional replication origin, a homologous recombination element, a positive screening marker, a negative screening marker and a transferable element; the homologous recombination element is a deletion homologous recombination element or an insertion homologous recombination element.

[0009] The conditional origin of replication, i.e., the self-replication of the vector, requires special transcription factors. For example, the pRE112 plasmid contains the R6κori conditional origin of replication, and its self-replication ability depends on the special Pir factor. If the host bacteria has this factor, the plasmid can self-replicate, otherwise it cannot. The pir gene is not expressed in general host bacteria, but only in some special engineered bacteria (such as χ7232 and χ7213).

[0010] Furthermore, the structure of the deletion homologous recombination element is an upstream homologous arm of the target segment to be deleted-a downstream homologous arm element of the target segment to be deleted, and the target segment is a gene or a gene cluster.

[0011] Furthermore, the structure of the inserted homologous recombination element is an element of upstream homologous arm of the operating site-target fragment to be inserted-downstream homologous arm of the operating site, the operating site is a predetermined insertion site for the target fragment to be inserted in the background strain, and the target fragment is a gene or a gene cluster.

[0012] Wherein, the target fragment length is 10 to 30 Kbp.

[0013] Specifically, the gene cluster is an Escherichia coli O antigen polysaccharide gene cluster, a Salmonella typhimurium (S.Typhimurium) O antigen polysaccharide gene cluster, a Salmonella Enteritidis (S.Enteritidis) O antigen polysaccharide gene cluster, a Salmonella Paratyphi (S.Paratyphi A) O antigen polysaccharide gene cluster, an Escherichia coli ECA gene cluster, an Escherichia coli CA gene cluster or a Salmonella Typhi (S.Typhi) Vi capsular polysaccharide gene cluster.

[0014] Specifically, the positive selection marker is an antibiotic marker.

[0015] Preferably, the antibiotic marker is chloramphenicol Cm, kanamycin Kan, spectinomycin Spec, gentamicin Gm or ampicillin Amp.

[0016] Wherein, the negative screening marker is sacB, lacY or pheS.

[0017] Furthermore, the backbone vector is pRE112.

[0018] Specifically, during scar insertion, the structure of the inserted homologous recombination element is: homologous arm upstream of the operating site-target fragment to be inserted-FRT-resistance gene-FRT-element of the homologous arm downstream of the operating site; and the resistance gene is different from the positive selection marker.

[0019] Preferably, the resistance gene is a gene for resistance to chloramphenicol Cm, kanamycin Kan, spectinomycin Spec, gentamicin Gm or ampicillin Amp.

[0020] The invention also provides an expression system containing the vector.

[0021] Preferably, the expression system further comprises a vector for expressing the FRT site-specific recombinase.

[0022] Specifically, the vector expressing the FRT site-specific recombinase is a pCP20 plasmid.

[0023] The technical solution of the present invention is a genetic manipulation method for seamless deletion of large-scale DNA fragments in bacterial genomes, comprising the following steps:

[0024] S1, constructing a deletion homologous recombination element; the structure of the deletion homologous recombination element is the upstream homologous arm of the target fragment to be deleted-the downstream homologous arm of the target fragment to be deleted;

[0025] S2, connecting the missing homologous recombination element obtained in step S1 to the linearized suicide plasmid backbone vector, transforming the engineering bacteria, and screening out positive strains; the suicide plasmid backbone vector carries a positive screening marker, a negative screening marker and a transferable element;

[0026] S3, combining and transferring the positive strain obtained in step S2 with the background strain of the target fragment to be deleted, screening out the positive strain, and completing the genetic operation of the target fragment.

[0027] Specifically, in step S1, the construction process of the deletion homologous recombination element is as follows: amplify the upstream homologous arm of the target fragment to be deleted and the downstream homologous arm of the target fragment to be deleted, and fuse the two by fusion PCR to obtain.

[0028] Specifically, the operation of step S2 is: using the circular PCR method, the missing homologous recombination elements are combined with the linearized suicide plasmid vector, transformed into Escherichia coli χ7232, positive strains are screened out, the recombinant suicide plasmid is extracted, and Escherichia coli χ7213 is transformed again to screen out positive strains.

[0029] Wherein, in step S2, the engineered bacteria is Escherichia coli.

[0030] Specifically, the positive selection marker is an antibiotic marker.

[0031] Preferably, the antibiotic is labeled Cm, Kan, Spec, Gm or Amp.

[0032] Wherein, the negative selection marker on the suicide plasmid vector in step (a) is sacB, lacY or pheS.

[0033] Furthermore, in step S2, the suicide plasmid backbone vector is pRE112.

[0034] Wherein, in step S1, the target fragment length is 10 to 30 Kbp.

[0035] Specifically, in step S1, the target fragment is a gene or a gene cluster.

[0036] Specifically, in step S1, the gene cluster is an Escherichia coli O antigen polysaccharide gene cluster, a Salmonella typhimurium (S.Typhimurium) O antigen polysaccharide gene cluster, a Salmonella Enteritidis (S.Enteritidis) O antigen polysaccharide gene cluster, a Salmonella Paratyphi (S.Paratyphi A) O antigen polysaccharide gene cluster, an Escherichia coli ECA gene cluster, an Escherichia coli CA gene cluster or a Salmonella Typhi (S.Typhi) Vi capsular polysaccharide gene cluster.

[0037] The present invention also provides a genetic manipulation method for seamless insertion of large-scale DNA fragments in bacterial genomes, comprising the following steps:

[0038] S4, obtaining the target fragment to be inserted;

[0039] S5, constructing a suicide plasmid vector containing an upstream homology arm of the operation site-a downstream homology arm of the operation site, inserting the target fragment to be inserted between the upstream homology arm of the operation site and the downstream homology arm of the operation site; obtaining a suicide plasmid vector containing an inserted homologous recombination element; transforming engineering bacteria, and screening out positive strains; the suicide plasmid vector carries a conditional replication origin, a positive screening marker, a negative screening marker and a transferable element; the structure of the inserted homologous recombination element is the upstream homology arm of the operation site-the target fragment to be inserted-the downstream homology arm of the operation site, and the operation site is a predetermined insertion site for the target fragment to be inserted in the background strain;

[0040] S6, combining and transferring the positive strain obtained in step S5 with the background strain to screen out the positive strain, thus completing the genetic operation on the target fragment.

[0041] Preferably, in step S5, the suicide plasmid vector containing the upstream homology arm of the operation site and the downstream homology arm of the operation site is obtained as follows: amplify the upstream homology arm of the operation site and the downstream homology arm of the operation site, fuse the two by fusion PCR to obtain a fusion fragment, and use a circular PCR method to connect the fusion fragment to the linearized suicide plasmid backbone vector.

[0042] Specifically, in step S4, the method of obtaining the target segment to be inserted is one of the following:

[0043] a. The target fragment to be inserted is divided into an upstream fragment to be inserted and a downstream fragment to be inserted, and the fragments are amplified separately; or the target fragment to be inserted can be divided into multiple fragments, and each fragment is amplified separately;

[0044] b. Long-range PCR amplification was used to obtain the full length of the target fragment;

[0045] c. Total chemical synthesis.

[0046] Specifically, the operation of step S5 is: linearizing the suicide plasmid vector containing the homology arm upstream of the operation site-the homology arm downstream of the operation site; connecting the upstream fragment of the target fragment to be inserted, the downstream fragment of the target fragment to be inserted, and the linearized suicide plasmid vector containing the homology arm upstream of the operation site-the homology arm downstream of the operation site using DNA seamless cloning technology; transforming Escherichia coli χ7232, screening out positive strains, extracting the recombinant suicide plasmid, and transforming Escherichia coli χ7213 again to screen out positive insertion mutants.

[0047] DNA seamless cloning technology, that is, commercial kits based on DNA seamless cloning (Gibson Assembly or Golden Gate, etc.) can be used; for example, NEB high-fidelity DNA large fragment assembly kit, Gibson CloningKit and Golden Gate Assembly Kit(BsaI- v2) etc.

[0048] Wherein, in step S5, the engineered bacteria is Escherichia coli.

[0049] Specifically, the positive selection marker is an antibiotic marker.

[0050] Preferably, the antibiotic is labeled Cm, Kan, Spec, Gm or Amp.

[0051] Wherein, the negative selection marker on the suicide plasmid vector in step (a) is sacB, lacY or pheS.

[0052] Furthermore, in step S2, the suicide plasmid backbone vector is pRE112.

[0053] Wherein, in step S1, the target fragment length is 10 to 30 Kbp.

[0054] Specifically, in step S1, the target fragment is a gene or a gene cluster.

[0055] Specifically, in step S1, the gene cluster is an Escherichia coli O antigen polysaccharide gene cluster, a Salmonella typhimurium (S.Typhimurium) O antigen polysaccharide gene cluster, a Salmonella Enteritidis (S.Enteritidis) O antigen polysaccharide gene cluster, a Salmonella Paratyphi (S.Paratyphi A) O antigen polysaccharide gene cluster, an Escherichia coli ECA gene cluster, an Escherichia coli CA gene cluster or a Salmonella Typhi (S.Typhi) Vi capsular polysaccharide gene cluster.

[0056] The present invention also provides a genetic manipulation method for inserting a large-scale DNA fragment with a scar in a bacterial genome, comprising the following steps:

[0057] S7, obtaining the upstream homology arms and downstream homology arms on both sides of the insertion site of the background strain, and constructing them into a linearized E. coli-yeast shuttle vector by circular PCR to obtain an E. coli-yeast shuttle vector carrying the upstream homology arms and the downstream homology arms;

[0058] S8, obtaining a resistance gene fragment FRT-resistance gene-FRT with FRT sites at both ends; using a circular PCR method, connecting FRT-resistance gene-FRT with a linearized E. coli-yeast shuttle vector with upstream homology arm-downstream homology arm; obtaining an E. coli-yeast shuttle vector carrying upstream homology arm-FRT-resistance gene-FRT-downstream homology arm;

[0059] S9, linearizing the E. coli-yeast shuttle vector carrying upstream homology arm-FRT-resistance gene-FRT-downstream homology arm; obtaining the target fragment to be inserted; connecting the first two using yeast transformation-mediated homologous recombination technology (TAR); obtaining the E. coli-yeast shuttle vector carrying upstream homology arm-target fragment-FRT-resistance gene-FRT-downstream homology arm;

[0060] S10, using long-range PCR to amplify the upstream homology arm-target fragment-FRT-resistance gene-FRT-downstream homology arm fragment, and connecting it to the linearized suicide plasmid backbone vector; transforming the engineering bacteria to obtain a positive strain; the suicide plasmid vector carries a conditional replication origin, a positive screening marker, a negative screening marker and a transferable element; and the positive screening marker is different from the resistance gene;

[0061] S11, combining and transferring the positive strain obtained in step S10 with the background strain, screening out the positive insertion mutant strain, transferring the vector expressing the FRT site-specific recombinase into the positive strain, eliminating the resistance gene in the middle of the FRT, and completing the genetic operation of the target fragment.

[0062] Wherein, in step S10, the engineered bacteria is Escherichia coli.

[0063] Specifically, the positive selection marker is an antibiotic marker.

[0064] Preferably, the antibiotic is labeled Cm, Kan, Spec, Gm or Amp.

[0065] Wherein, the negative selection marker on the suicide plasmid vector in step (a) is sacB, lacY or pheS.

[0066] Furthermore, in step S10, the suicide plasmid backbone vector is pRE112.

[0067] Wherein, in step S9, the target fragment length is 10 to 30 Kbp.

[0068] Specifically, in step S9, the target fragment is a gene or a gene cluster.

[0069] Specifically, in step S9, the gene cluster is an Escherichia coli O antigen polysaccharide gene cluster, a Salmonella typhimurium (S.Typhimurium) O antigen polysaccharide gene cluster, a Salmonella Enteritidis (S.Enteritidis) O antigen polysaccharide gene cluster, a Salmonella Paratyphi (S.Paratyphi A) O antigen polysaccharide gene cluster, an Escherichia coli ECA gene cluster, an Escherichia coli CA gene cluster or a Salmonella Typhi (S.Typhi) Vi capsular polysaccharide gene cluster.

[0070] Specifically, in step S7, the yeast vector is pYES1L-URA.

[0071] Specifically, in step S8, FRT-resistance gene-FRT is connected to a linearized E. coli-yeast shuttle vector with upstream homology arms-downstream homology arms, and the connection product is transferred into E. coli ER2566, and positive clones with successful connection are screened.

[0072] In particular, in step S9, the E. coli-yeast shuttle vector carrying the upstream homology arm-FRT-resistance gene-FRT-downstream homology arm is linearized; the target fragment to be inserted is obtained; the first two are connected using yeast transformation-mediated homologous recombination technology (TAR); the connection product is transferred into yeast mav203, and the positive clones with successful connection are screened.

[0073] Specifically, in step S10, the linearized suicide plasmid backbone vector and the upstream homology arm-target fragment-FRT-resistance gene-FRT-downstream homology arm fragment are connected using DNA seamless cloning technology, and the connection product is electroporated into Escherichia coli χ7232 to screen out positive clones; the plasmid of the positive clone is provided and transferred into Escherichia coli χ7213 to screen out positive insertion mutants; the resistance gene is different from the positive screening marker.

[0074] Wherein, in step S11, the vector expressing the FRT site-specific recombinase is a pCP20 plasmid.

[0075] Preferably, in step S8, the resistance gene is a gene resistant to Cm, Kan, Spec, Gm or Amp.

[0076] Preferably, the specific operation of obtaining FRT-resistance gene-FRT is: amplifying FRT-Cm-FRT using pKD3 plasmid as template.

[0077] During scar insertion, when the suicide plasmid carrying two resistance genes is combined and transferred with the background strain, the first screening is performed using the resistance on the suicide plasmid backbone; then the second screening is performed using sucrose + the resistance in the middle of the homologous arm, and finally a mutant strain with the target gene + FRT + resistance gene + FRT as a whole inserted at the site can be obtained. Finally, the pCP20 plasmid is transferred into the mutant strain to eliminate the resistance gene in the middle, and finally a non-resistance insertion mutant strain with no resistance but a FRT sequence is obtained.

[0078] With the help of yeast's homologous recombination ability, it is easier to construct a target large plasmid; only E.coliχ7213 has the ability of conjugation transfer. When the target sequence is too large, you can first use yeast to perform the "component assembly construction" of upper homology arm + target fragment + FRT + resistance gene + FRT + lower homology arm. After the early "parts" are assembled, they can be moved to pRE112 at one time, which has a higher success rate. pRE112 then uses the conjugation transfer ability of E.coliχ7213 to transfer and integrate into the recipient bacterial genome, and finally achieve the site-specific insertion of large-scale DNA fragments into the bacterial genome.

[0079] Beneficial effects of the present invention: The present invention provides a new genetic manipulation technology for large-scale DNA fragments of bacterial genomes, which can accurately insert large-scale DNA fragments (10-30Kbp) into bacterial genomes, and can achieve traceless insertion mutations without any antibiotic screening markers. This method solves the problem that it is difficult for general plasmids to carry and express large-scale gene clusters. At the same time, the Escherichia coli engineered bacteria constructed based on this method are easy to culture, easy to extract, low cost and no biosafety risk when used to obtain polysaccharide antigens on the outer membrane surface of certain pathogenic strains, so it has high scientific research and application value. The significant feature of this technology is that the super-large suicide plasmid is transferred into bacteria by combining transfer; then the suicide plasmid carrying the target gene cluster is integrated into the bacterial genome by homologous recombination, and finally a mutant strain with the exogenous gene cluster inserted into the bacterial genome is obtained by positive screening and negative screening. This technology does not require the introduction of SceI restriction enzyme cutting sites and the expression of SceI restriction endonucleases throughout the process, and does not require the use of λ-RED recombinase. The operation process is simpler and the success rate is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 , schematic diagram of the structure of related gene clusters.

[0081] Figure 2 , Schematic diagram of the structure of the recombinant suicide plasmid pRE112-Cm-D_OAg in Example 1.

[0082] Figure 3 , PCR identification results of gene cluster deletion in Examples 1 to 3. M: 4500bp marker (Tiangen Marker III, MD103); P1, P2 and P3: positive controls for ΔCA, ΔOAg and ΔECA gene cluster deletion, using pRE112-Cm-D_CA, pRE112-Cm-D_OAg and pRE112-Cm-D_ECA as PCR templates, respectively; 1 and 2: E. coli W3110ΔCA positive mutant; 3: E. coli W3110ΔOAg positive mutant; 4, 5: E. coli W3110ΔECA positive mutant.

[0083] Figure 4 , Schematic diagram of the structure of the recombinant suicide plasmid pRE112-Cm-D_ECA in Example 2.

[0084] Figure 5 , Schematic diagram of the structure of the recombinant suicide plasmid pRE112-Cm-D_CA in Example 3.

[0085] Figure 6, Schematic diagram of the structure of the recombinant suicide plasmid pRE112-Cm-D_OAg-viaB in Example 4.

[0086] Figure 7 , Colony PCR identification of E. coli W3110ΔOAg::viaB_locus mutant strain in Example 4. M: 5000bp marker (sangon, B500351); the positive bands in the figure are all E. coli W3110ΔOAg::viaB_locus positive mutant strains.

[0087] Figure 8 , Schematic diagram of the structure of the recombinant plasmid pYES1L-D_OAg in Example 5.

[0088] Fig. 9 , Schematic diagram of the structure of the recombinant plasmid pYES1L-D_OAg-Cm_FRT2 in Example 5.

[0089] Fig.10 , Schematic diagram of the structure of the recombinant plasmid pYES1L-D_OAg-ST_OAg-Cm_FRT2 in Example 5.

[0090] Fig.11 , Schematic diagram of the structure of the recombinant plasmid pRE112-Kan-D_OAg-ST_OAg-Cm_FRT2 in Example 5.

[0091] Fig.12 , partial genome sequence alignment of E. coli W3110 parent strain and E. coli W3110ΔOAg::ST_OAg mutant strain.

[0092] Fig.13 , Schematic diagram of the structure of the recombinant plasmid pYES1L-D_OAg-SE_OAg-Cm_FRT2 in Example 6.

[0093] Fig.14 , Schematic diagram of the structure of the recombinant plasmid pRE112-Kan-D_OAg-SE_OAg-Cm_FRT2 in Example 6.

[0094] Fig.15 , Partial genome sequence alignment of E. coli W3110 parent strain and E. coli W3110ΔOAg::SE_OAg mutant strain

[0095] Fig.16 , Schematic diagram of the structure of the recombinant plasmid pRE112-Kan-D_OAg-Cm_FRT2 in Example 7.

[0096] Fig.17, Schematic diagram of the structure of the recombinant plasmid pRE112-Kan-D_OAg-SA_OAg-Cm_FRT2 in Example 7.

[0097] Fig.18 , partial genome sequence alignment of E. coli W3110 parent strain and E. coli W3110ΔOAg::SA_OAg mutant strain.

[0098] Fig.19 , schematic diagram of the structure of recombinant plasmid pET9a-wzy_ST.

[0099] Fig. 20 , Schematic diagram of the structure of recombinant plasmid pET9a-wzy_SE.

[0100] Fig.21 , schematic diagram of the structure of recombinant plasmid pET9a-wzy_wzzB_ST.

[0101] Fig. 22 , schematic diagram of the structure of recombinant plasmid pET9a-wzy_wzzB_SE.

[0102] Fig.23 , schematic diagram of silver staining and Western-blot results of heterologous expression of Salmonella Typhimurium O antigen polysaccharide. Left: Silver staining results; Right: Western-blot (anti-O4) results; 1: Salmonella Typhimurium wild strain; 2: E. coli W3110ΔwecA mutant; 3: E. coli W3110ΔwecAΔOAg::ST_OAg mutant; 4: E. coli W3110ΔwecAΔOAg::ST_OAg (pET9a-wzy_ST) monoclonal mutant 1; 5: E. coli W3110ΔwecAΔOAg::ST_OAg (pET9a-wzy_ST) monoclonal mutant 2; 6: E. coli W3110ΔwecAΔOAg::ST_OAg (pET9a-wzy_wzzB_ST) monoclonal mutant 1; 7: E. coli W3110ΔwecAΔOAg::ST_OAg(pET9a-wzy_wzzB_ST) monoclonal mutant strain 2.

[0103] Fig.24, schematic diagram of silver staining and Western-blot results of heterologous expression of Salmonella Enteritidis O antigen polysaccharide. Left: Silver staining results; Right: Western-blot results (anti-O9); 1: Wild-type Salmonella Enteritidis (S. Enteritidis); 2: E. coli W3110ΔwecA mutant; 3: E. coli W3110ΔwecAΔOAg::SE_OAg mutant; 4: E. coli W3110ΔwecAΔOAg::SE_OAg (pET9a-wzy_SE) monoclonal mutant 1; 5: E. coli W3110ΔwecAΔOAg::SE_OAg (pET9a-wzy_SE) monoclonal mutant 2; 6: E. coli W3110ΔwecAΔOAg::SE_OAg (pET9a-wzy_wzzB_SE) monoclonal mutant 1; 7: E. coli W3110ΔwecAΔOAg::SE_OAg(pET9a-wzy_wzzB_SE) monoclonal mutant strain 2.

[0104] Fig.25 , schematic diagram of silver staining and Western-blot results of heterologous expression of Salmonella Paratyphi O antigen polysaccharide. Left: Silver staining results; Right: Western-blot results (anti-O2); 1: Wild-type Salmonella Paratyphi A (S. Paratyphi A); 2: E. coli W3110ΔwecA mutant; 3: E. coli W3110ΔwecAΔOAg::SA_OAg mutant; 4: E. coli W3110ΔwecAΔOAg::SA_OAg (pET9a-wzy_ST) monoclonal mutant 1; 5: E. coli W3110ΔwecAΔOAg::SA_OAg (pET9a-wzy_SA) monoclonal mutant 2; 6: E. coli W3110ΔwecA ΔOAg::SA_OAg (pET9a-wzy_wzzB_SA) monoclonal mutant 1; 7: E. coli W3110ΔwecAΔOAg::SA_OAg(pET9a-wzy_wzzB_SA) monoclonal mutant strain 2. DETAILED DESCRIPTION

[0105] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0106] Unless otherwise specified, the reagents and materials used in the following examples can be obtained from commercial sources.

[0107] (I) Experimental materials and reagents

[0108] Macklin: glycerol (G810575), glacial acetic acid (A801295), anhydrous ethanol (E809056), ammonia (A801005), periodic acid (P815693), sodium hydroxide (S835850), formaldehyde (F809702), citric acid (C805019), Tween-20 (T6335), sodium chloride (S805275), potassium chloride (P816348), potassium dihydrogen phosphate (P815662), disodium hydrogen phosphate (767482), methanol (M813895) and spectinomycin (S6106).

[0109] Aladdin: Silver nitrate (S116265), (A665714), ammonium persulfate (AP) (A112450), NNN',N-tetramethylethylenediamine (TEMED) (T105496), sodium dodecyl sulfate (SDS) (S105389), bromophenol blue (B109642), β-mercaptoethanol (M301573) and glycine (A110752) and kanamycin (K331597).

[0110] White shark: 30% Acr-Bis (29:1) (BL513B), 1 M Tris-HCl solution pH 6.8 (BL514B), 1.5 M Tris-HCl solution pH 8.8 (BL516B) and ECL chemiluminescent substrate (BL523B).

[0111] Shanghai Sangon Biotech: arabinose (A610071), sucrose (A610498), chloramphenicol (A600118), kanamycin (A600286), ampicillin (A600064), spectinomycin (A600901) and proteinase K (B600452) solutions, etc. Solarbio: LB liquid (L1010) or solid (L1015) medium and YPD complete medium (LA0220).

[0112] Merrill: phenol-chloroform-isoamyl alcohol (25:24:1) (N09169), nucleic acid extraction reagent (24:1) (M76752).

[0113] Ningbo Tianrun Biopharmaceuticals: Various species of Salmonella O antigen antiserum and Vi capsular polysaccharide antiserum (TR103).

[0114] Bioworlde: HRP-labeled goat anti-rabbit IgG secondary antibody (BS13278).

[0115] TianGen: DNAmarker III (MD103), plasmid extraction kit (DP103), DNA purification and recovery kit (DP204) and 2×Taq PCR Mix (KT201), etc.

[0116] TaKaRa: 2× PrimeSTAR Max Premix (R045A) and PrimeSTAR GXL DNA Polymerase (R050A). New England Biolabs (NEB): NEB High-Fidelity DNA Large Fragment Assembly Kit (E2623S).

[0117] Thermo Fisher: GeneArt Advanced Genetic Assembly System (A13285), Mav203 yeast competent vector (A13292), E. coli-yeast shuttle vector pYES1L (A13287).

[0118] Bio-Tech: Escherichia coli ER2566 competent cells (D1039S).

[0119] Beijing Coolaber: Yeast plasmid extraction kit (PE052) and SD / -Trp selection medium (PM2251).

[0120] Sigma Aldrich: lysozyme (10837059001), RNase (10109142001), DNase (DN25), bovine serum albumin (V900933) and 2,6-diaminopimelate (DAP) (33240), PVDF membrane 0.22 μm (ISEQ00005), etc.

[0121] BGI: conventional primer synthesis and sequencing.

[0122] The bacterial culture medium involved in the present invention is mainly Luria-Bertani (LB) liquid or solid culture medium, which is cultured statically or shaken at 220rpm under aerobic conditions at 37°C, unless otherwise specified. If necessary, antibiotics, arabinose, sucrose or DAP, etc. will be added to the LB basal medium, and the added concentrations are as follows: chloramphenicol 25μg / mL, kanamycin 25μg / mL, ampicillin 25μg / mL, DAP 50μg / mL, sucrose 12.5%, arabinose 2%. The yeast involved mainly uses YPD full culture medium and SD / -Trp selection culture medium, and the relevant preparation method refers to the instructions of commercial reagents.

[0123] 1. LPS lysis solution: In every 80 mL of ddH2O, add 10 mL of 0.5 M Tris pH 6.8, 8 mL of propylene glycol, 16 mL of 10% SDS, and 4 mL of β-mercaptoethanol, mix well, and store at 4°C for later use.

[0124] 2.LPSSDS-PAGE loading solution: In every 80mL ddH2O, take 10mL 0.5 M Tris pH 6.8 and 8mL 0.05g bromophenol blue, mix well, and store at 4℃ for later use.

[0125] 3. LPS fixative: Measure 80 mL of anhydrous ethanol, 10 mL of glacial acetic acid, and 110 mL of ddH2O and mix well.

[0126] 4. LPS oxidation solution: Add 1.4g periodic acid to the LPS fixative solution and dissolve it.

[0127] 5.LPS silver staining solution: Measure 2.9mL 1M NaOH, 2mL NH4OH and 250mL ddH2O, dissolve 1g AgNO3 in 5mL ddH2O, add to the above solution, mix well, it will become turbid at first and then clear and transparent, and it is ready for use.

[0128] 6.LPS developer: 0.025 g citric acid, 0.25 mL formaldehyde, dissolved in 500 mL ddH2O, mix well.

[0129] 7.12.5% ​​SDS-PAGE gel, SDS-PAG electrophoresis buffer (10×), TAE electrophoresis buffer (50×), 1% agarose gel, 5% skimmed milk powder, TBS buffer (10×), TBST buffer (1×), transfer buffer (1×), PBS buffer (10×), 1× PBS-T washing solution: For related preparation methods, nucleic acid electrophoresis, SDS-PAGE electrophoresis and Western-bot experimental steps, please refer to the Molecular Cloning Experiment Guide (4th Edition).

[0130] (II) Strains and plasmids

[0131] Wild-type Salmonella Typhimurium (S.Typhimurium), wild-type Salmonella Enteritidis (S.Enteritidis), and wild-type Salmonella Paratyphi A (S.Paratyphi A) have been disclosed in the document “Li P, Zhang K, Lei T, Zhou Z, Luo H. Multiple immunodominant O-epitopes co-expression in live attenuated Salmonellaserovars induce cross-protective immune responses against S.Paratyphi A, S.Typhimurium and S.Enteritidis. PLoS Negl Trop Dis. 2022Oct 13; 16(10): e0010866.”

[0132] Wild-type Salmonella Typhi (S. Typhi), E. coliχ7232, and E. coliχ7213 strains have been disclosed in the literature "Li P, Liu Q, Luo H, Liang K, Han Y, Roland KL, Curtiss R 3rd, Kong Q. Bi-valent polysaccharides of Vi capsular and O9 O-antigen in attenuated Salmonella Typhimurium induce strong immune responses against these two antigens. NPJ Vaccines. 2018 Jan 9; 3: 1." The engineered bacteria E. coliχ7232 and E. coliχ7213 can normally express the pir gene, and the conjugative transfer machinery element is integrated into the genome of E. coliχ7213, which can mediate the conjugative transfer of transferable plasmids through sex pili.

[0133] E. coli W3110 and E. coli W3110ΔwecA strains have been published in the literature "Bian Xiaoping, Liu Qing, Kong Qingke, Luo Hongyan, Li Pei. Establishment and verification of Escherichia coli CRISPR / Cas9 gene editing system [J]. Chinese Journal of Veterinary Science, 2021, 41(1): 110-116".

[0134] pKD3 and pCP20 plasmids have been disclosed in the literature "Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci US A. 2000 Jun 6; 97(12): 6640-5." The pKD3 plasmid contains the characteristic sequence of FRT-Cm-FRT and chloramphenicol resistance; pCP20 is a temperature-sensitive plasmid that can be naturally lost when cultured at 42°C. It encodes a FRT site-specific recombinase and ampicillin resistance.

[0135] The pRE112 suicide plasmid was disclosed in the literature "Edwards RA, Keller LH, Schifferli DM. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene. 1998 Jan 30; 207(2): 149-57." The pRE112 plasmid contains the R6κori conditional replication origin (its self-replication ability depends on the host bacteria that can normally express the pir gene), transferable elements (traJ and oriT), chloramphenicol or kanamycin resistance positive selection markers and the sacB gene negative selection marker.

[0136] The wzy_ST, wzy_SE, wzy_wzzB_ST and wzy_wzzB_SE genes in the pET9a-wzy_ST, pET9a-wzy_SE, pET9a-wzy_wzzB_ST and pET9a-wzy_wzzB_SE plasmids were fully synthesized by Nanjing GenScript Biotech and cloned into the commercial plasmid vector pET9a. The DNA sequences of the wzy_ST, wzy_SE, wzzB_ST and wzzB_SE genes can be referred to GenBank Accession No. AEF07203.1, WP_000905565.1, AEF07997.1 and WP_000215251.1, respectively.

[0137] (II) Experimental methods

[0138] 1. Primer design

[0139] With reference to the sequences of the Escherichia coli W3110 genome (GenBank Accession No. NC_007779.1), Salmonella typhi viaB locus (GenBank Accession No. D14156.1), Salmonella typhimurium genome (GenBank Accession No. CP002614.1), and Salmonella enterica serovar Enteritidis genome (GenBank Accession No. CP007361.2) published in the NCBI (https: / / www.ncbi.nlm.nih.gov / ) public database, the deletion, insertion, and expression primers of the relevant gene clusters were designed using the software SnapGene 2.3.2 (Table 2). All primers were synthesized by BGI.

[0140] Table 1 All primers used in the present invention

[0141]

[0142]

[0143] 2. Extraction of bacterial genome: The bacterial genome was extracted using the phenol-chloroform-isoamyl alcohol method. The relevant experimental methods refer to the Molecular Cloning Experiment Guide (4th edition). The extracted genome can be used as a PCR template later.

[0144] 3. PCR reaction

[0145] In the following examples, colony PCR (2×Taq PCR Mix), high fidelity PCR (HF-PCR, 2×PrimeSTAR Max Premix), fusion PCR (2×PrimeSTAR Max Premix), long-range PCR (PrimeSTAR GXL DNA Polymerase) and circular PCR (CPEC PCR, 2×PrimeSTAR Max Premix) are commonly used. For related experimental methods, refer to "Quan J, Tian J. Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One. 2009 Jul 30; 4 (7): e6441.", the standard operating procedures of commercial PCR enzymes or the Molecular Cloning Experiment Guide (4th Edition). Colony PCR is mainly used for the construction of recombinant plasmids, confirmation of strain deletion or insertion mutations, etc.; high-fidelity PCR is mainly used to ensure the accuracy of the base sequence of the DNA target fragment during amplification; fusion PCR is mainly used for the fusion between two linear homologous arms of the suicide plasmid structure; long-fragment PCR is mainly used for the amplification of ultra-long DNA target fragments and to ensure the accuracy of the base sequence during amplification; circular PCR is mainly used for the construction of recombinant plasmids.

[0146] Example 1 Construction of a scarless deletion of the E. coli W3110 O antigen gene cluster (E. coli W3110ΔOAg)

[0147] O antigen refers to the specific polysaccharide chain on the outer membrane of the cell wall of Gram-negative bacteria, also known as O-specific chain or O-polysaccharide. O antigen is a common protective antigen in bacteria and can induce the body to produce an immune response. However, the O antigen gene cluster of Escherichia coli K12 series strains, such as W3110, cannot normally synthesize its own O antigen polysaccharide chain structure. The main reason is that the wbbL gene in the O antigen gene cluster of this series of strains has a natural insertion mutation and cannot synthesize rhamnose glycosyltransferase, resulting in the obstruction of the synthesis of its own O antigen. The total length of the Escherichia coli W3110 O antigen gene cluster is about 13Kbp ( Figure 1 ), the main steps for its precise and traceless knockout are as follows:

[0148] 1. Preparation of upstream and downstream homology arm fusion fragments of O antigen gene cluster

[0149] Using the E. coli W3110 genome as a template, high-fidelity PCR amplification was performed using primers D_OAg(K12)-1F / 1R and D_OAg(K12)-2F / 2R, respectively. The upstream and downstream homology arm fragments of the O antigen gene cluster were purified and recovered using a DNA purification and recovery kit. Then, the upstream and downstream homology arms obtained were used as templates, and primers D_OAg-1F / 2R were used for fusion PCR amplification. After purification and recovery by the kit, the upstream and downstream homology arm fusion fragment D_OAg-1F2R of the O antigen gene cluster was obtained.

[0150] 2. Preparation of linearized pRE112-Cm suicide plasmid vector fragment

[0151] Using pRE112-Cm suicide plasmid as a template and primers Vec-112-F / R, high-fidelity PCR amplification was performed. After purification and recovery by a kit, a linearized pRE112 suicide plasmid vector fragment was obtained.

[0152] 3. Construction of pRE112-Cm-D_OAg recombinant suicide plasmid

[0153] Using the circular PCR method, the D_OAg-1F2R fragment obtained in step 1 was connected with the pRE112-Cm suicide plasmid vector fragment obtained in step 2; the connection product was electrotransformed into the χ7232 electrotransformation competent cell, coated with chloramphenicol resistance screening plate, and monoclonal bacteria were obtained the next day; the monoclonal strain was identified by colony PCR; the correct monoclonal strain was selected and inoculated into LB liquid culture medium for expansion, and the recombinant suicide plasmid was extracted using a bacterial plasmid extraction kit and sent to the company for sequencing, and finally it was confirmed that the pRE112-Cm-D_OAg recombinant suicide plasmid was successfully constructed ( Figure 2 Finally, the successfully constructed pRE112-Cm-D_OAg recombinant suicide plasmid was electroporated into the χ7213 electroporation competent cell, and the chloramphenicol + DAP screening plate was coated, and the χ7213 (pRE112-Cm-D_OAg) transformant was obtained the next day.

[0154] Among them, the circular PCR (circular PCR does not require additional primers, template 1 and template 2 are primers for each other, and finally form a circle, method reference: Quan J, Tian J. Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One. 2009 Jul 30; 4 (7): e6441.) reaction system is: 0.4 μL of template 1 and template 2, 5 μL of 2× PrimeSTAR Max Premix, add ddH2O to 10 μL. The amplification program of circular PCR is: preheating and denaturation at 98℃ for 2 minutes; denaturation at 98℃ for 10 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 1 to 3 minutes (extension time is adjusted according to the length of the amplified DNA fragment), 15 cycles; 72℃ for 5 minutes; storage at 16℃.

[0155] 4. Construction of E. coli W3110 O antigen-deficient strain (E. coli W3110ΔOAg)

[0156] The χ7213 (pRE112-Cm-D_OAg) transformant and the E. coli W3110 parent strain were combined and transferred; after positive screening on chloramphenicol resistance plates, negative screening on sucrose plates, and colony PCR identification (identification primers D_OAg-1F / 2R), the O antigen gene cluster precise and seamless deletion strain E. coli W3110ΔOAg ( Figure 3 ).

[0157] Example 2 Construction of a scarless deletion of the E. coli W3110 ECA gene cluster (E. coli W3110ΔECA)

[0158] ECA is a common sugar chain structure on the surface of Enterobacteriaceae bacteria, and its full name is Enterobacterial Common Antigen. ECA is an important virulence factor of Enterobacteriaceae bacteria, which can stimulate the body to produce an immune response and is a good protective antigen. The total length of the ECA gene cluster of Escherichia coli W3110 is about 13Kbp ( Figure 1 ), the main steps for its precise and traceless knockout are as follows:

[0159] 1. Preparation of upstream and downstream homology arm fusion fragments of ECA gene cluster

[0160] Using the W3110 genome as a template, high-fidelity PCR amplification was performed using primers D_ECA(K12)-1F / 1R and D_ECA(K12)-2F / 2R, respectively. The upstream and downstream homology arm fragments of the ECA gene cluster were purified and recovered using a DNA purification and recovery kit. Then, the upstream and downstream homology arms obtained were used as templates, and primers D_ECA-1F / 2R were used for fusion PCR amplification. After purification and recovery by the kit, the upstream and downstream homology arm fusion fragment D_ECA-1F2R of the ECA gene cluster was obtained.

[0161] 2. Construction of pRE112-Cm-D_ECA recombinant suicide plasmid

[0162] The D_ECA-1F2R fragment obtained in step 1 was connected with the pRE112-Cm suicide plasmid vector fragment obtained in Example 1 by using the circular PCR method; the connection product was electrotransformed into the χ7232 electrotransformation competent state, and chloramphenicol resistance was applied for screening, and a monoclonal strain was obtained the next day; the monoclonal strain was identified by colony PCR; the correctly identified monoclonal strain was selected and inoculated into LB liquid culture medium for expansion, and the recombinant suicide plasmid was extracted using a bacterial plasmid extraction kit, and sent to the company for sequencing, and finally it was confirmed that the pRE112-Cm-D_ECA recombinant suicide plasmid was successfully constructed ( Figure 4 Finally, the successfully constructed recombinant suicide plasmid was electroporated into the χ7213 electroporation competent cell, and the chloramphenicol + DAP screening plate was coated, and the χ7213 (pRE112-Cm-D_ECA) transformant was obtained the next day.

[0163] 3. Construction of E. coli W3110 ECA deletion strain (E. coli W3110ΔECA)

[0164] The χ7213 (pRE112-Cm-D_ECA) was transformed and combined with the parent strain of E. coli W3110 for transfer; after positive screening on chloramphenicol resistance plates, negative screening on sucrose plates and colony PCR identification (identification primers D_ECA-1F / 2R), the ECA gene cluster precise and seamless deletion strain E. coli W3110ΔECA ( Figure 3 ).

[0165] Example 3 Construction of a scarless deletion of the E. coli W3110 CA gene cluster (E. coli W3110ΔCA)

[0166] Colanic Acid (CA) is a bacterial extracellular polysaccharide that can be produced by most Escherichia coli. CA has a large molecular weight and is loosely wrapped on the surface of the bacteria, making the bacteria appear slimy. When encountering unfavorable growth conditions, such as dryness, low pressure and low pH, CA can help the bacteria improve their chances of survival. The total length of the Escherichia coli W3110 CA gene cluster is about 23Kbp ( Figure 1 ), the main steps for its precise and traceless knockout are as follows:

[0167] 1. Preparation of upstream and downstream homology arm fusion fragments of CA gene cluster

[0168] Using the W3110 genome as a template, high-fidelity PCR amplification was performed using primers D_CA(K12)-1F / 1R and D_CA(K12)-2F / 2R, respectively. The upstream and downstream homology arm fragments of the CA gene cluster were purified and recovered using a DNA purification and recovery kit. Then, the upstream and downstream homology arms obtained were used as templates, and primers D_CA-1F / 2R were used for fusion PCR amplification. After purification and recovery by the kit, the upstream and downstream homology arm fusion fragment D_CA-1F2R of the CA gene cluster was obtained.

[0169] 2. Construction of pRE112-Cm-D_CA recombinant suicide plasmid

[0170] The D_CA-1F2R fragment obtained in step 1 was connected with the pRE112-Cm suicide plasmid vector fragment obtained in Example 1 by using the circular PCR method; the connection product was electrotransformed into the χ7232 electrotransformation competent state, and chloramphenicol resistance was applied for screening, and a monoclonal strain was obtained the next day; the monoclonal strain was identified by colony PCR; the correctly identified monoclonal strain was selected and inoculated into LB liquid culture medium for expansion, and the recombinant suicide plasmid was extracted using a bacterial plasmid extraction kit, and sent to the company for sequencing, and finally it was confirmed that the pRE112-Cm-D_CA recombinant suicide plasmid was successfully constructed ( Figure 5 Finally, the successfully constructed recombinant suicide plasmid was electroporated into the χ7213 electroporation competent cell, and the chloramphenicol + DAP screening plate was coated, and the χ7213 (pRE112-Cm-D_CA) transformant was obtained the next day.

[0171] 3. Construction of E. coli W3110 CA deletion strain (E. coli W3110ΔCA)

[0172] The χ7213 (pRE112-Cm-D_CA) was transformed and combined with the parent strain of E. coli W3110; after positive screening on chloramphenicol resistance plates, negative screening on sucrose plates and colony PCR identification (identification primers D_CA-1F / 2R), the CA gene cluster precise and seamless deletion strain E. coli W3110ΔCA ( Figure 3 ).

[0173] Example 4: Precise and seamless insertion of the Escherichia coli W3110 genome into the Salmonella typhi Vi capsular polysaccharide gene cluster

[0174] Vi capsular polysaccharide is mainly present on the outer membrane surface of Salmonella typhi and is an important virulence factor of the bacterium. In addition, Vi capsular polysaccharide is widely used to prepare Vi capsular polysaccharide or polysaccharide protein vaccines that can prevent Salmonella typhi infection. The Vi capsular polysaccharide gene cluster is located in the ViaB locus (viaB_locus), and the total length of viaB_locus is about 14Kbp ( Figure 1 ), using E. coli W3110ΔOAg as the background bacteria, the main steps of accurately inserting the viaB locus at the deletion site of the original W3110 strain OAg polysaccharide gene cluster are as follows:

[0175] 1. Preparation of large DNA fragments of viaB locus

[0176] High-fidelity PCR amplification was performed using the Salmonella typhi genome as a template and primer pairs viaB_F1 / R1 and viaB_F2 / R2. The upper half viaB_up and the lower half viaB_down DNA fragments of the viaB locus were purified and recovered using a DNA purification and recovery kit.

[0177] 2. Preparation of linearized pRE112-Cm-D_OAg suicide plasmid vector fragment

[0178] Since the pRE112-Cm-D_OAg suicide plasmid constructed in Example 1 already carries the upstream and downstream exchange arms for homologous recombination with the background strain, it is only necessary to connect the target fragment to be inserted in the middle of the exchange arm.

[0179] Therefore, high-fidelity PCR amplification was performed using the pRE112-Cm-D_OAg suicide plasmid as a template and primer pair 112-D_OAg_viaB-F / R. After purification and recovery by a kit, the linearized pRE112-Cm-D_OAg suicide plasmid vector fragment was obtained.

[0180] 3. Construction of pRE112-Cm-D_OAg-viaB recombinant suicide plasmid

[0181] The viaB_up, viaB_down obtained in step 1 and the linearized pRE112-Cm-D_OAg vector fragment obtained in step 2 were sequentially connected using the NEB high-fidelity DNA large fragment assembly kit; the connection product was electroporated into the χ7232 electroporation competent cell, coated with chloramphenicol resistance screening plates, and a monoclonal strain was obtained the next day; the monoclonal strain was identified using colony PCR; the correctly identified monoclonal strain was selected and inoculated into LB liquid culture medium for expansion, and the recombinant suicide plasmid was extracted using a bacterial plasmid extraction kit and sent to the company for sequencing, and finally it was confirmed that the pRE112-Cm-D_OAg-viaB recombinant suicide plasmid was successfully constructed ( Figure 6 Finally, the successfully constructed recombinant suicide plasmid was electroporated into the χ7213 electroporation competent cell, and the chloramphenicol + DAP screening plate was coated, and the χ7213 (pRE112-Cm-D_OAg-viaB) transformant was obtained the next day.

[0182] 4. Construction of a scarless insertion mutant of the viaB locus at the genomic level in E. coli W3110

[0183] The χ7213 (pRE112-Cm-D_OAg) was transformed and combined with the E. coli W3110ΔOAg mutant strain; positive screening on chloramphenicol resistance plates, negative screening on sucrose plates and colony PCR identification (identification primers Check-middle-F / Check-middle-R) ( Figure 7 ) and finally obtained the insertion mutant strain E. coli W3110ΔOAg::viaB_locus, in which the viaB locus was precisely and seamlessly inserted at the missing site of the O antigen gene cluster of the original W311 strain.

[0184] Example 5 Precise insertion of the Escherichia coli W3110 genome band into the Salmonella Typhimurium O antigen gene cluster

[0185] The total length of the O antigen gene cluster of Salmonella typhimurium is about 18Kbp ( Figure 1 ), the main steps for accurate insertion of the band mark are as follows:

[0186] 1. Construction of pYES1L-D_OAg recombinant plasmid

[0187] Using the W3110 genome as a template, high-fidelity PCR amplification was performed using primer pairs ye-galF-F / R and ye-gnd-F / R, respectively. After the PCR product was purified and recovered by the kit, the upstream and downstream homology arm fragments of the O antigen gene cluster were obtained. Then, using the pYES1L vector as a template, high-fidelity PCR amplification was performed using primer pairs ye-gnd-VF / ye-galF-VR. After the PCR product was purified, a linearized pYES1L vector fragment was obtained. Using the circular PCR method, the upper homology arm, lower homology arm and linearized pYES1L vector fragment obtained above were sequentially connected, and finally the YES1L-D_OAg recombinant plasmid ( Figure 8 ).

[0188] 2. Construction of pYES1L-D_OAg-Cm_FRT2 recombinant plasmid

[0189] High-fidelity PCR amplification was performed using the pKD3 plasmid as a template and the primer pair FRT2-CM-F / R. After the PCR product was purified and recovered, a chloramphenicol resistance gene fragment Cm-FRT2 with FRT-specific sequences at both ends was obtained;

[0190] Using the YES1L-D_OAg recombinant plasmid as a template and primer pair V-yes-D_OAg-F / R, high-fidelity PCR amplification was performed. After the product was purified and recovered by reagents, the linearized YES1L-D_OAg vector fragment was obtained.

[0191] The Cm-FRT2 fragment was connected to the linearized YES1L-D_OAg vector fragment using the circular PCR method; the connection product was electrotransformed into the electrocompetent Escherichia coli ER2566, and chloramphenicol resistance was applied for screening, and a monoclonal strain was obtained the next day; the monoclonal strain was identified using colony PCR; the correct monoclonal strain was selected and inoculated into LB+chloramphenicol liquid culture medium for expansion, and the recombinant plasmid was extracted using a bacterial plasmid extraction kit and sent to the company for sequencing, and finally it was confirmed that the pYES1L-D_OAg-Cm_FRT2 recombinant plasmid was successfully constructed ( Fig. 9 ).

[0192] 3. Construction of pYES1L-D_OAg-ST_OAg-Cm_FRT2 recombinant plasmid

[0193] Using the pYES1L-D_OAg-Cm_FRT2 recombinant plasmid constructed in step 2 as a template, use the primer pair V-yes-FRT-F / V-yes-galF-R to amplify the entire plasmid backbone with high fidelity PCR. After purification and recovery by the kit, the linearized pYES1L-D_OAg-Cm_FRT2 vector fragment can be obtained. This fragment will be used for seamless connection of the O antigen polysaccharide gene cluster of Salmonella typhimurium.

[0194] Using the genome of Salmonella typhimurium as a template, long-range PCR amplification was performed using primer pair Sal_OAg-F / R. After the product was purified and recovered by a kit, the complete DNA fragment ST_OAg of the entire O antigen polysaccharide gene cluster of Salmonella typhimurium was obtained.

[0195] The linearized pYES1L-D_OAg-Cm_FRT2 vector fragment was connected to the ST_OAg large fragment using the GeneArt advanced genetic assembly system, and the ligation product was transferred into the Mav203 yeast competent cell and coated on the SD / -Trp solid selective plate. After a few days, a monoclonal yeast strain was obtained. The recombinant plasmid in the monoclonal yeast strain was extracted using a yeast plasmid kit. The recombinant plasmid was identified using the primer pair Who_3110galF-F / Who-3110gnd-R, and finally the pYES1L-D_OAg-ST_OAg-Cm_FRT2 recombinant plasmid was successfully constructed ( Fig.10 ).

[0196] 4. Construction of pRE112-Kan-D_OAg-ST_OAg-Cm_FRT2 recombinant suicide plasmid

[0197] Using suicide plasmid pRE112-Kan as a template and primer pair 112-kan-STAE-F / R, high-fidelity PCR amplification was performed. After purification and recovery by a kit, a linearized pRE112-Kan vector fragment was obtained.

[0198] Using the pYES1L-D_OAg-ST_OAg-Cm_FRT2 recombinant plasmid constructed in step 3 as a template, long-range PCR amplification was performed using the primer pair ye-galF-F / ye-gnd-R. After the PCR product was purified and recovered using a kit, the D_OAg-ST_OAg-Cm_FRT2 large fragment was obtained.

[0199] The linearized pRE112-Kan vector fragment and the D_OAg-ST_OAg-Cm_FRT2 large fragment were connected using the NEB high-fidelity DNA large fragment assembly kit, and the connection product was electrotransformed into the χ7232 electrotransformation competent medium, coated with kanamycin resistance screening plates, and monoclonal strains were obtained the next day; colony PCR was used to identify the monoclonal strains; the correctly identified monoclonal strains were selected and inoculated into LB+kanamycin liquid culture medium for expansion, and the recombinant suicide plasmid was extracted using a bacterial plasmid extraction kit and sent to the company for sequencing, and finally it was confirmed that the pRE112-Kan-D_OAg-ST_OAg-Cm_FRT2 recombinant suicide plasmid was successfully constructed ( Fig.11Finally, the successfully constructed recombinant suicide plasmid was electroporated into the χ7213 electroporation competent cell, and the kanamycin + DAP screening plate was coated, and the χ7213 (pRE112-Kan-D_OAg-ST_OAg-Cm_FRT2) transformant was obtained the next day.

[0200] 5. Construction of E. coli W3110 genome-level ST_OAg gene cluster with scar insertion mutant

[0201] The χ7213 (pRE112-Kan-D_OAg-ST_OAg-Cm_FRT2) transformant was combined and transferred with the Escherichia coli W3110 parent strain; after kanamycin resistance plate screening, sucrose-chloramphenicol plate screening and PCR colony identification (identification primer Check-wbaP-F / R), the insertion mutant strain W3110ΔOAg::ST_OAg-Cm_FRT2 was obtained, in which the original Escherichia coli W3110 OAg polysaccharide gene cluster was precisely replaced by ST_OAg_Cm-FRT2.

[0202] The pCP20 plasmid was electroporated into the W3110ΔOAg::ST_OAg-Cm_FRT2 mutant strain, and the ampicillin resistance plate was coated. The chloramphenicol gene was eliminated by expressing the FRT site-specific recombinase, and finally a large fragment of the gene cluster with no resistance selection marker but a FRT characteristic sequence was obtained. The mutant strain W3110ΔOAg::ST_OAg ( Fig.12 ).

[0203] Example 6 Precise insertion of Escherichia coli W3110 genome band into the Salmonella enteritidis O antigen gene cluster

[0204] The total length of the O antigen gene cluster of Salmonella enteritidis is about 22Kbp ( Figure 1 ), the main steps for accurate insertion of the band mark are as follows:

[0205] 1. Construction of pYES1L-D_OAg-SE_OAg-Cm_FRT2 recombinant plasmid

[0206] Using the genome of Salmonella enteritidis as a template, long-range PCR amplification was performed using primer pair Sal_OAg-F / R. After the product was purified and recovered by a kit, the complete DNA large fragment SE_OAg of the entire O antigen polysaccharide gene cluster of Salmonella enteritidis was obtained.

[0207] The linearized pYES1L-D_OAg-Cm_FRT2 vector fragment in Example 5 was connected to the SE_OAg large fragment using the GeneArt high-order genetic assembly system, and the connection product was transferred to the Mav203 yeast competent medium, coated with SD / -Trp solid selective plates, and a monoclonal yeast strain was obtained after a few days; the recombinant plasmid in the monoclonal yeast strain was extracted using a yeast plasmid kit; the recombinant plasmid was identified using the primer pair Who_3110galF-F / Who-3110gnd-R and the Long-range PCR method, and finally it was confirmed that the pYES1L-D_OAg-SE_OAg-Cm_FRT2 recombinant plasmid was successfully constructed ( Fig.13 ).

[0208] 2. Construction of pRE112-Kan-D_OAg-SE_OAg-Cm_FRT2 recombinant suicide plasmid

[0209] Using the pYES1L-D_OAg-SE_OAg-Cm_FRT2 recombinant plasmid constructed in step 1 as a template, long-range PCR amplification was performed using the primer pair ye-galF-F / ye-gnd-R. After the PCR product was purified and recovered using a kit, the D_OAg-SE_OAg-Cm_FRT2 large fragment was obtained.

[0210] The pRE112-Kan vector fragment obtained in Example 5 and the D_OAg-SE_OAg-Cm_FRT2 large fragment were connected using the NEB high-fidelity DNA large fragment assembly kit, and the connection product was electrotransformed into the χ7232 electrotransformation competent medium, coated with kanamycin resistance screening plates, and monoclonal strains were obtained the next day; colony PCR was used to identify the monoclonal strains; the correctly identified monoclonal strains were selected and inoculated into LB+kanamycin liquid culture medium for expansion, and the recombinant suicide plasmid was extracted using a bacterial plasmid extraction kit and sent to the company for sequencing, and finally it was confirmed that the pRE112-Kan-D_OAg-SE_OAg-Cm_FRT2 recombinant suicide plasmid was successfully constructed ( Fig.14 Finally, the successfully constructed recombinant suicide plasmid was electroporated into the χ7213 electroporation competent cell, and the kanamycin + DAP screening plate was coated, and the χ7213 (pRE112-Kan-D_OAg-SE_OAg-Cm_FRT2) transformant was obtained the next day.

[0211] 3. Construction of the SE_OAg gene cluster insertion mutant at the genome level of Escherichia coli W3110

[0212] The χ7213 (pRE112-Kan-D_OAg-SE_OAg-Cm_FRT2) transformant was combined and transferred with the Escherichia coli W3110 parent strain; after kanamycin resistance plate screening, sucrose-chloramphenicol plate screening and PCR colony identification (identification primer Check-wbaP-F / R), the insertion mutant strain W3110ΔOAg::SE_OAg-Cm_FRT2 in which the original Escherichia coli W3110 OAg polysaccharide gene cluster was precisely replaced by SE_OAg-Cm_FRT2 was obtained.

[0213] The pCP20 plasmid was electroporated into the W3110ΔOAg::SE_OAg-Cm_FRT2 mutant strain, and the ampicillin resistance plate was coated. The chloramphenicol gene was eliminated by expressing the FRT site-specific recombinase, and finally the mutant strain W3110ΔOAg::SE_OAg ( Fig.15 ).

[0214] Example 7 Precise insertion of Escherichia coli W3110 genome band into the Salmonella paratyphi O antigen gene cluster

[0215] The O antigen polysaccharide gene clusters of Enteritidis Salmonella and Paratyphoid Salmonella are very similar, with only one difference in the function of the tyv gene. The tyv gene of the former is normal, while the tyv gene of the latter has a natural frameshift mutation and loses the normal tyv gene function. Therefore, the O antigen polysaccharide of Salmonella Paratyphoid can be synthesized by simply removing the tyv gene in the O antigen polysaccharide gene cluster of Enteritidis Salmonella. The construction method based on this strategy includes the following steps:

[0216] 1. Construction of pRE112-Kan-D_OAg-Cm_FRT2 recombinant suicide plasmid

[0217] Using the pYES1L-D_OAg-Cm_FRT2 recombinant plasmid constructed in Example 5 as a template, high-fidelity PCR amplification was performed using the primer pair ye-galF-F / ye-gnd-R. After purification and recovery by the kit, the D_OAg-Cm_FRT2 fragment was obtained.

[0218] The pRE112-Kan vector fragment obtained in Example 5 and the D_OAg-Cm_FRT2 fragment were connected by circular PCR method; the connection product was electrotransformed into χ7232 electrotransformation competent cells, and kanamycin resistance was applied for screening, and a monoclonal strain was obtained the next day; the monoclonal strain was identified by colony PCR; the correctly identified monoclonal strain was selected and inoculated into LB+kanamycin liquid culture medium for expansion, and the recombinant suicide plasmid was extracted using a bacterial plasmid extraction kit, and sent to the company for sequencing, and finally it was confirmed that the pRE112-Kan-D_OAg-Cm_FRT2 recombinant plasmid was successfully constructed ( Fig.16 ).

[0219] 2. Construction of pRE112-Kan-D_OAg-SA_OAg-Cm_FRT2 recombinant suicide plasmid

[0220] Using the constructed pRE112-Kan-D_OAg-Cm_FRT2 recombinant plasmid as a template, high-fidelity PCR amplification was performed using the primer pair V-yes-FRT-F / V-yes-galF-R. After purification and recovery with the kit, the linearized pRE112-Kan-D_OAg-Cm_FRT2 vector fragment was obtained.

[0221] Using the genome of Salmonella enteritidis as a template, high-fidelity PCR amplification was performed using primer pairs Sal_OAg-F / SA-Prt-R and SA-NPrt-F / Sal_OAg-R. After the PCR products were purified and recovered by a kit, the upper half SE_OAg-up and lower half SE_OAg-down DNA fragments of the O antigen polysaccharide gene cluster of Salmonella enteritidis were obtained.

[0222] Using the NEB high-fidelity DNA large fragment assembly kit, SE_OAg-up, SE_OAg-down and linearized pRE112-Kan-D_OAg-Cm_FRT2 vector fragments were sequentially connected, and the connection products were electroporated into the χ7232 electroporation competent plate, coated with kanamycin resistance screening plates, and monoclonal strains were obtained the next day; colony PCR was used to identify the monoclonal strains; the correct monoclonal strains were selected and inoculated into LB+kanamycin liquid culture medium for expansion, and the recombinant suicide plasmid was extracted using a bacterial plasmid extraction kit and sent to the company for sequencing, and finally it was confirmed that the pRE112-Kan-D_OAg-SA_OAg-Cm_FRT2 recombinant suicide plasmid was successfully constructed ( Fig.17 Finally, the successfully constructed recombinant suicide plasmid was electroporated into the χ7213 electroporation competent cell, and the kanamycin + DAP screening plate was coated, and the χ7213 (pRE112-Kan-D_OAg-SA_OAg-Cm_FRT2) transformant was obtained the next day.

[0223] 3. Construction of the SA_OAg gene cluster insertion mutant at the genome level of Escherichia coli W3110

[0224] The χ7213 (pRE112-Kan-D_OAg-SA_OAg-Cm_FRT2) transformant was combined and transferred with the Escherichia coli W3110 parent strain; after kanamycin resistance plate screening, sucrose-chloramphenicol plate screening and PCR colony identification (identification primer Check-wbaP-F / R), the insertion mutant strain W3110ΔOAg::SA_OAg-Cm_FRT2 in which the original Escherichia coli W3110 OAg polysaccharide gene cluster was precisely replaced by SA_OAg-Cm_FRT2 was obtained.

[0225] The pCP20 plasmid was electroporated into the W3110ΔOAg::SA_OAg-Cm_FRT2 mutant strain, and the ampicillin resistance plate was coated. The chloramphenicol gene was eliminated by expressing the FRT site-specific recombinase, and finally the mutant strain W3110ΔOAg::SA_OAg ( Fig.18 ).

[0226] Example 8 Heterologous expression of pathogenic bacteria outer membrane polysaccharide structure by Escherichia coli engineered bacteria

[0227] Bacterial outer membrane polysaccharides, such as capsular polysaccharides and O-antigen polysaccharides, are important protective antigens of the body and are also frequently selected targets for bacterial vaccine development. The conventional method for obtaining bacterial outer membrane polysaccharides is to culture pathogens in large quantities in vitro, such as Streptococcus pneumoniae, Haemophilus parasuis and Salmonella typhi, and then extract the outer membrane polysaccharide antigens from the bacteria. Due to the special culture methods and special culture components of many pathogens, conventional methods face high acquisition costs. In addition, in vitro culture of pathogens also faces potential biological risks and other issues.

[0228] The above problems can be circumvented by heterologously expressing pathogenic outer membrane polysaccharides in Escherichia coli engineered bacteria, providing a low-cost method for obtaining bacterial outer membrane polysaccharide antigens without biosafety risks. Based on the above large-scale genetic manipulation method of bacterial gene clusters, the present invention successfully constructed E. coli W3110ΔwecA ΔOAg::ST_OAg, E. coli W3110ΔwecAΔOAg::SE_OAg, E. coli W3110ΔwecAΔOAg::SA_OAg and E. coli W3110ΔOAg::viaB_locus mutants. The mutants were transformed into pET9a-wzy_ST( Fig.19 )、pET9a-wzy_SE( Fig. 20 )、pET9a-wzy_wzzB_ST( Fig.21 ) and pET9a-wzy_wzzB_SE recombinant plasmid ( Fig. 22 ) as well as extraction, silver staining and immunoblotting experiments of bacterial outer membrane LPS can verify the successful expression of pathogenic bacteria outer membrane O antigen polysaccharide in Escherichia coli.

[0229] Extraction of LPS: Take 1mL of overnight cultured bacterial liquid, discard the supernatant after centrifugation, resuspend with ddH2O and centrifuge again, discard the supernatant, add 200μL of LPS lysis buffer, suspend and lyse evenly, boil at 100℃ for 10 minutes, cool, centrifuge at 12,000rpm for 10min, take 10μL of supernatant, add to 90μL of LPS SDS-PAGE loading solution, add 1μL of proteinase K to the mixture, digest at 37℃ for 1h, and the LPS extract after proteinase K digestion can be directly used in SDS-PAGE electrophoresis experiment.

[0230] LPS silver staining: LPS separated by 12.5% ​​SDS-PAGE gel can be developed by silver staining. Place the gel in a clean glass container, rinse once with ultrapure water, then pour in LPS fixative, fix for 4 hours, rinse 3 times with ultrapure water, 10 minutes each time, add LPS oxidizing solution, incubate for 10 minutes, rinse 3 times with ultrapure water, 10 minutes each time, add LPS silver staining solution, incubate for 10 minutes, rinse 3 times with ultrapure water, 10 minutes each time, finally add LPS developer until color appears and the bands are clear, rinse 3 times with ultrapure water, take pictures and save.

[0231] Immunoblotting of LPS: LPS separated by SDS-PAGE gel can also be transferred to a PVDF membrane, and then blocked with 5% skim milk powder blocking solution, incubated with the corresponding Salmonella O antigen single factor (anti-O4 / O9 / O2) antiserum (rabbit source), eluted with TBS-T buffer after 2 hours, and then incubated with HRP-labeled goat anti-rabbit antibody for 1.5 hours. After elution with TBS-T buffer, it was developed with an ECL chemiluminescence kit (Bio-Rad), and finally photographed and saved.

[0232] The results are as follows Figures 23 to 25 As shown, it is proved that the above mutant strains can heterologously express the corresponding target polysaccharide antigens well, laying a foundation for subsequent extended research based on polysaccharide antigens (preparation of antiserum, preparation of polysaccharide antigen monoclonal antibodies and biosynthesis of polysaccharide protein conjugate vaccines, etc.).

Claims

1. A vector suitable for genetic manipulation of large-scale DNA fragments in bacterial genomes, Features: A suicide plasmid vector is used as a backbone vector, carrying the following structures: a conditional replication origin, a homologous recombination element, a positive screening marker, a negative screening marker and a transferable element; the homologous recombination element is a deletion homologous recombination element or an insertion homologous recombination element; Further, the structure of the deletion homologous recombination element is an upstream homologous arm of the target segment to be deleted-a downstream homologous arm element of the target segment to be deleted, and the target segment is a gene or a gene cluster; Furthermore, the structure of the inserted homologous recombination element is an element of upstream homologous arm of the operating site-target fragment to be inserted-downstream homologous arm of the operating site, the operating site is a predetermined insertion site for the target fragment to be inserted in the background strain, and the target fragment is a gene or a gene cluster.

2. The carrier according to claim 1, Features: The gene cluster is an Escherichia coli O antigen polysaccharide gene cluster, a Salmonella typhimurium (S. Typhimurium) O antigen polysaccharide gene cluster, a Salmonella enteritidis (S. Enteritidis) O antigen polysaccharide gene cluster, a Salmonella paratyphi (S. Paratyphi A) O antigen polysaccharide gene cluster, an Escherichia coli ECA gene cluster, an Escherichia coli CA gene cluster or a Salmonella typhi (S. Typhi) Vi capsular polysaccharide gene cluster.

3. The carrier according to claim 1, Features: The positive selection marker is an antibiotic marker; Preferably, the negative selection marker is sacB, lacY or pheS; Preferably, the antibiotic is labeled Cm, Kan, Spec, Gm or Amp.

4. The carrier according to claim 1, Features: The backbone vector is pRE112.

5. The carrier according to claim 1, Features: In the case of scar insertion, the structure of the inserted homologous recombination element is: homologous arm upstream of the operation site-target fragment to be inserted-FRT-resistance gene-FRT-homologous arm downstream of the operation site; and the resistance gene is different from the positive selection marker.

6. An expression system comprising the vector according to any one of claims 1 to 5.

7. The expression system according to claim 6, It is characterized in that Also included is a vector expressing a FRT site-specific recombinase; Preferably, the vector expressing the FRT site-specific recombinase is a pCP20 plasmid.

8. A genetic manipulation method for seamless deletion of large-scale DNA fragments in bacterial genomes. Features: The steps include: S1, constructing a deletion homologous recombination element; the structure of the deletion homologous recombination element is the upstream homologous arm of the target fragment to be deleted-the downstream homologous arm of the target fragment to be deleted; S2, connecting the missing homologous recombination element obtained in step S1 to a linearized suicide plasmid backbone vector, transforming the engineering bacteria, and screening out positive strains; the suicide plasmid backbone vector carries a conditional replication origin, a positive screening marker, a negative screening marker and a transferable element; S3, combining and transferring the positive strain obtained in step S2 with the background strain of the target fragment to be deleted, and screening out the positive deletion mutant strain, thus completing the genetic operation of the target fragment.

9. The method according to claim 8, Features: In step S1, the construction process of the deletion homologous recombination element is as follows: amplify the upstream homologous arm of the target fragment to be deleted and the downstream homologous arm of the target fragment to be deleted, and fuse the two by fusion PCR to obtain; The operation of step S2 is: using the circular PCR method, connecting the deleted homologous recombination element with the linearized suicide plasmid vector, transforming Escherichia coli χ7232, screening out positive strains, extracting the recombinant suicide plasmid, transforming Escherichia coli χ7213 again, and screening out positive deletion mutants.

10. A genetic manipulation method for the seamless insertion of large-scale DNA fragments into bacterial genomes. Features: The steps include: S4, obtaining the target fragment to be inserted; S5, constructing a suicide plasmid vector containing an upstream homology arm of the operation site and a downstream homology arm of the operation site, and inserting the target fragment to be inserted between the upstream homology arm of the operation site and the downstream homology arm of the operation site; Obtaining a suicide plasmid vector containing an inserted homologous recombination element; Transforming the engineered bacteria and screening out the positive strains; the suicide plasmid vector carries a conditional replication origin, a positive screening marker, a negative screening marker and a transfer element; The structure of the inserted homologous recombination element is the homology arm upstream of the operation site-the target fragment to be inserted-the homology arm downstream of the operation site, and the operation site is the predetermined insertion site of the target fragment to be inserted in the background strain; S6, combining and transferring the positive strain obtained in step S5 with the background strain to screen out the positive insertion mutant strain, thus completing the genetic operation on the target fragment. Preferably, in step S5, the suicide plasmid vector containing the upstream homology arm of the operation site and the downstream homology arm of the operation site is obtained as follows: amplify the upstream homology arm of the operation site and the downstream homology arm of the operation site, fuse the two by fusion PCR to obtain a fusion fragment, and use a circular PCR method to connect the fusion fragment to the linearized suicide plasmid backbone vector.

11. The method according to claim 10, Features: In step S4, the target segment to be inserted is obtained in one of the following ways: a. The target fragment to be inserted is divided into an upstream fragment to be inserted and a downstream fragment to be inserted, and the fragments are amplified separately; or the target fragment to be inserted can be divided into multiple fragments, and each fragment is amplified separately; b. Long-range PCR amplification was used to obtain the full length of the target fragment; c. Total chemical synthesis.

12. The method according to claim 10, Features: The operation of step S5 is: linearizing the suicide plasmid vector containing the homology arm upstream of the operation site and the homology arm downstream of the operation site; The upstream fragment of the target fragment to be inserted, the downstream fragment of the target fragment to be inserted and the linearized suicide plasmid vector containing the upstream homology arm of the operation site and the downstream homology arm of the operation site were connected using DNA seamless cloning technology; Escherichia coli χ7232 was transformed, positive strains were screened, the recombinant suicide plasmid was extracted, and Escherichia coli χ7213 was transformed again to screen positive strains.

13. A genetic manipulation method for inserting large-scale DNA fragments into bacterial genomes. Features: The steps include: S7, obtaining the upstream homology arm and the downstream homology arm of the background strain to be inserted into the site, and constructing them into the linearized E. coli-yeast shuttle vector by circular PCR to obtain the E. coli-yeast shuttle vector carrying the upstream homology arm and the downstream homology arm; S8, obtaining a resistance gene fragment FRT-resistance gene-FRT with FRT sites at both ends; using a circular PCR method, connecting FRT-resistance gene-FRT with a linearized E. coli-yeast shuttle vector with upstream homology arm-downstream homology arm; obtaining an E. coli-yeast shuttle vector carrying upstream homology arm-FRT-resistance gene-FRT-downstream homology arm; S9, linearizing the E. coli-yeast shuttle vector carrying upstream homology arm-FRT-resistance gene-FRT-downstream homology arm; obtaining the target fragment to be inserted; connecting the first two using yeast transformation-mediated homologous recombination technology (TAR); obtaining the E. coli-yeast shuttle vector carrying upstream homology arm-target fragment-FRT-resistance gene-FRT-downstream homology arm; S10, using long-range PCR to amplify the upstream homology arm-target fragment-FRT-resistance gene-FRT-downstream homology arm fragment, and connecting it to the linearized suicide plasmid backbone vector; transforming the engineering bacteria to obtain a positive strain; the suicide plasmid vector carries a conditional replication origin, a positive screening marker, a negative screening marker and a transferable element; and the positive screening marker is different from the resistance gene; S11, combining and transferring the positive strain obtained in step S10 with the background strain, screening out the positive insertion mutant strain, transferring the vector expressing the FRT site-specific recombinase into the positive insertion mutant strain, eliminating the resistance gene in the middle of the FRT, and completing the genetic operation of the target fragment.

14. The method according to claim 13, Features: Possess at least one of the following characteristics: a. In step S7, the E. coli-yeast shuttle vector is pYES1L; b. In step S8, FRT-resistance gene-FRT is connected to the linearized E. coli-yeast shuttle vector with upstream homology arm-downstream homology arm, and the connection product is transferred into E. coli ER2566 to screen the positive clones with successful connection; c. In step S9, the E. coli-yeast shuttle vector carrying upstream homology arm-FRT-resistance gene-FRT-downstream homology arm is linearized; Obtain the target fragment to be inserted; connect the first two using yeast transformation-mediated homologous recombination technology (TAR); transfer the connection product into yeast mav203, and screen the positive clones with successful connection; d. In step S10, the linearized suicide plasmid backbone vector and the upstream homology arm-target fragment-FRT-resistance gene-FRT-downstream homology arm fragment are connected by using DNA seamless cloning technology, and the connection product is electrotransformed into Escherichia coli χ7232 to screen out positive clones; the plasmid of the positive clone is provided and transferred into Escherichia coli χ7213 to screen out positive strains; e. In step S11, the vector expressing the FRT site-specific recombinase is a pCP20 plasmid; f. The resistance gene is a gene resistant to Cm, Kan, Spec, Gm or Amp.

15. The method according to any one of claims 8 to 14, Features: Possess at least one of the following characteristics: g. The engineering bacteria is Escherichia coli; h. The positive screening marker is an antibiotic marker; i. The negative selection marker on the suicide plasmid vector is sacB, lacY or pheS; j. The suicide plasmid backbone vector is pRE112; k. The target fragment length is 10 to 30 Kbp; 1. The target fragment is a gene or a gene cluster.

16. The method according to claim 15, Features: The gene cluster is an Escherichia coli O antigen polysaccharide gene cluster, a Salmonella typhimurium (S. Typhimurium) O antigen polysaccharide gene cluster, a Salmonella enteritidis (S. Enteritidis) O antigen polysaccharide gene cluster, a Salmonella paratyphi (S. Paratyphi A) O antigen polysaccharide gene cluster, an Escherichia coli ECA gene cluster, an Escherichia coli CA gene cluster or a Salmonella typhi (S. Typhi) Vi capsular polysaccharide gene cluster; Alternatively, the antibiotic is labeled Cm, Kan, Spec, Gm or Amp.

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