KR101898973B1 - Plasmid-based Vibrio cholerae CTX phage replication system - Google Patents

Abstract

The present invention relates to a plasmid-based Vibriol cholera CTX phage cloning system, which comprises cloning a CTX phage genomic sequence into a plasmid capable of replicating the plasmid, amplifying it in a host cell, and introducing it into a V. cholera strain, Can be reproduced. Also, the plasmid of the present invention can be used to produce a strain having a plurality of CTX phages in a single Vibrio cholera host cell.

Description

[0001] Plasmid-based Vibrio cholerae CTX phage replication system [0002]

The present invention relates to a plasmid-based Vibrio cholera CTX phage replication system.

Cholera is a diarrheal disease caused by a strain of V. cholerae that produces toxins. Serotype O1 strains and O139 serotype strains, which can be subdivided into two types of biotypes, produce toxins and thus produce epidemic cholera. The toxin-producing strains are produced by lysogenization by infecting the CTX phage, which contains the cholera toxin gene. Three major CTX phages are known, CTX-cla, CTX-1 and O139 serotype of CTX-cla and El Tor strains possessed by Classical strains. A model for an evolutionary mechanism in which each of the bio-type strains is infected with a bio-type-specific phage to produce a toxin-producing strain is presented, but infection and replication of the CTX phage is not limited by the host strain's biotype . In recent years, atypical El Tor strains with CTX phages that appear to be formed by the mosaics of CTX-cla and CTX-1 have been around the world.

Evidence that cloning of CTX-1 and CTX-O139 phage is possible under laboratory conditions and evidence of the mechanism by which the Wave 2 and 3 atypical El Tor strains are made suggest that the cloning of the CTX-cla phage and its analogous phage, CTX-2 It occurs in the natural world, but it is not proven experimentally.

On the other hand, cloning of the conventional CTX phage replicated the CTX phage using the El Tor bio-type cholera that has already been lysogenized by the CTX phage (Fig. 1, Brigid M Davis et al. Current Opinion in Microbiology , 2003 , 6: 35-42), and CTX ^{E1 Tor} (CTX-1) can be made into a replicative form in a prophage state and can also be transduced. However, CTX-CTX repeat and CTX-RS1 sequences are required, and it is known that only the classical biotypes can be transfected. The replicative form of CTX-1 is plasmid-like (pCTX-1), and the cloned CTX-1 transduced with the classical biotypes can replicate the CTX phage and transduce the CTX phage into other classical strains .

On the other hand, CTX ^cla (CTX-cla) has no CTX: CTX or CTX: RS1 sequence and is not replicable because there is no transgenic E1 Tor strain, and cloning of CTX ^cla in laboratory conditions has not been proven.

 Brigid M Davis et al. Current Opinion in Microbiology, 2003, 6: 35-42

It is an object of the present invention to provide a recombinant plasmid for Vibrio cholera CTX phage replication comprising a Vibrio cholera CTX phage genomic sequence and replicable in a host cell.

Another object of the present invention is to provide a microorganism transformed with said plasmid.

Yet another object of the present invention is to provide a method for producing Vibrio cholera CTX phage from a microorganism transformed with said plasmid.

In order to achieve the above object, the present invention provides a Vt. Cholera CTX phage genomic sequence having the full-length sequence of the ctxA gene according to SEQ ID NO: 1 and the full-length sequence of the ctxB gene according to SEQ ID NO: 2, or a fragment thereof, A recombinant plasmid for replication of Vibrio cholera CTX phage.

The present invention also provides a microorganism transformed with the recombinant plasmid for cloning the Vibrio cholera CTX phage.

The present invention also relates to a method for producing a recombinant plasmid comprising the steps of: culturing a microorganism transformed with the recombinant plasmid for cloning of Vibrio cholera CTX phage; And isolating and purifying the CTX phage in the microorganism or culture thereof.

According to the present invention, when CTX phage genomic sequences are cloned into a plasmid as a replicable unit, the CTX phage genome sequence is amplified in a host cell and then introduced into a V. cholera strain, whereby CTX phage replication is possible regardless of host biotype. It is also possible to clone CTX-cla, CTX-2, CTX-env, etc., which have not yet been proven to be cloned.

Since it is possible that replication CTX using the plasmid can be in a single host cell with all of the plurality of phage such as ^{E1 Tor} CTX and CTX ^cla, CTX-O139, CTX-env strain produced.

Figure 1 shows a conventional Vibrio cholera CTX phage cloning technique.
Figure 2 shows a plasmid-based Vibrio cholera CTX phage cloning system of the invention.
FIG. 3A illustrates the replication mechanism of the CTX phage. The cloned (plasmid-form) pCTX can not be cloned in one CTX, which can be completely cloned and inserted into the chromosome of cholera as a lysogen, CTX: This shows that CTX phage can be cloned if CTX profile is consecutive like CTX or if CTX: RS1 array exists. Figure 3B depicts the genomic genome of the CTX-1: RS1 sequence of the E1 Tor CTX phage introduced into the plasmid, and the CTX phage genome and the 5 ', 3 ' -encrypted And then cloning the plasmid into the plasmid.
Figure 4 shows the CTX-1: mapping of the gene (A) of the RS1 sequence and the construction (B) of the pUC-CTX plasmid. In Figure 4A, strain PM8 comprises the CTX-1: RS1 sequence of Figure 4A except that some sequences of ctxA and ctxB are replaced by a kanamycin resistant cassette. The block arrows indicate the transcription direction of each gene, and the black triangle indicates the att sequence. The ig-1 (intergenic sequence one) between the Att sequence and the stop codon of rstR is indicated. The recombinant pUC-CTX plasmid of Fig. 4B contains three DNA fragments, i.e., a replication origin fragment of pUC18, a DNA fragment containing 245 nucleotides of zot amplified from PM8 to the rstR termination codon of RS1, and an att sequence of CTX phage Or an upstream-119th nucleotide of the truncation codon of rstR to 244 nucleotides of zot. The third DNA fragment is amplified from the other CTX phages (CTX-1, CTX-cla, CTX-2, CTX-O139, CTX-env, etc.) and linked to the other two fragments. Thus, the zot-3'UTR fragment is included in all constructs, and the 5'UTR-zot fragment is phage type specific.
Fig. 5 is an electron microscope photograph of the CTX phage, (A) CTX-1kan phage produced in O395 transfected with pCTX-1kan, and (B) CTX-2kan phage cloned in MG116025 transgenic pCTX- , And (C) CTX-O139kan phage produced in the O395 transgenic form of pCTX-O139kan.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention provides a Vibrio cholera CTX phage comprising the full-length sequence of the ctxA gene described in SEQ ID NO: 1 and the full-length sequence of the ctxB gene according to SEQ ID NO: 2, or a fragment thereof having the Vibrio cholera CTX phage genome sequence substituted with a selectable marker gene To a recombinant plasmid for replication.

In addition, the present invention provides a microorganism transformed with the recombinant plasmid for cloning of Vibrio cholera CTX phage.

The present invention also relates to a method for producing a recombinant plasmid comprising the steps of: culturing a microorganism transformed with the recombinant plasmid for cloning of Vibrio cholera CTX phage; And isolating and purifying the CTX phage in the microorganism or culture thereof.

Generally, CTX ^{El Tor} can be replicated in the protease state and transduction is possible, but CTX: CTX repeat or CTX: RS1 sequence is required and only strains of Classical bio-type can be transduced . However, CTX ^cla has not been found to be cloned because there is no CTX: CTX sequence and there is no El Tor strain capable of receiving CTX phage by transduction.

Thus, the present inventors have demonstrated the replication process of CTX-cla in a laboratory condition by producing plasmid-based CTX phage cloning system and El Tor strain capable of being transduced by CTX phage. Reproduction of various CTX phages was demonstrated through this plasmid-based CTX phage cloning system.

According to one embodiment of the present invention, a plasmid can be constructed by cloning a CTX phage into a unit capable of replicating, amplified in E. coli , and then replicated in V. cholerae . The CTX phage generated in the plasmid is the same as the phage generated in the natural state. The genome of the CTX phage put into the plasmid can also be artificially synthesized. Because CTX replication is possible in plasmids, both CTX ^{El Tor} and CTX ^cla are available. This shows that other CTX phages (CTX-2, CTX-O139, CTX-env) are also producible. El Tor strain, which is capable of receiving CTX ^cla , can also be newly discovered, and CTX ^cla can be transfected.

It has already been shown that El Tor strains are transduced by CTX-1 in the body (specifically in the intestines of mice), but the transduction under laboratory conditions has not been proven. Thus, if only appropriate laboratory conditions are provided, El Tor strains can be expected to be transduced by CTX-1 as well as CTX-cla or CTX-2. Wave 2 atypical El Tor strains include but is expected to be in the CTX-2 having a chromosome 2 rstR ^cla example in tandem repeat (tandem repeat) is CTX-2 could be replicated has been proved experimentally. This can also be attributed to the absence of the El Tor strain, which can be transduced by CTX-2 or CTX-cla.

The recombinant plasmid for cloning of Vibrio cholera CTX phage of the present invention will be described in detail as follows.

The recombinant plasmid for cloning of Vibrio cholera CTX phage includes a full-length sequence of the ctxA gene and a full-length sequence of the ctxB gene or a fragment of the ctxB gene of the Vibrio cholera CTX phage genomic sequence substituted with a selectable marker gene.

The CTX phage genomic sequence may be a genomic sequence of CTX-1, CTX-cla, CTX-2, CTX-env or CTX-O139.

According to one embodiment of the present invention, the Vibrio cholera CTX-1 phage genome is shown in Fig. According to Fig. 3, the gene is composed of genes of rstR, rstA, rstB, cep, orfU, ace, zot, ctxA and ctxB.

In the recombinant plasmid for cloning of Vibrio cholera CTX phage of the present invention, the full-length sequence of the ctxA gene and the full-length sequence of the ctxB gene or a fragment thereof are substituted with a selectable marker gene cassette.

The full-length sequence of the ctxA gene is shown in SEQ ID NO. 1. ≪ / RTI > In addition, the full-length sequence of the ctxB gene may be the nucleotide sequence of SEQ ID NO: 2. The fragment of the ctxB gene may comprise the nucleotide sequence of SEQ ID NO: 3.

In addition, a selectable marker gene such as a drug resistance gene can easily detect the transformant due to the phenotype of the selectable marker gene in the transformant. As such selectable marker genes, an ampicillin resistance gene, a kanamycin resistance gene, a straptomain resistance gene, a tetracycline resistance gene, an erythromycin resistance gene or a chloramphenicol acetyltransferase gene can be used. More specifically, the present invention replaces the kanamycin resistance gene in place of the full-length sequence of the ctxA gene and the full-length sequence of the ctxB gene or a fragment thereof. Most specifically, the substituted kanamycin resistance gene may comprise the nucleotide sequence set forth in SEQ ID NO: 4.

According to one embodiment of the present invention, the part in which the full-length sequence of the ctxA gene and the full-length sequence of the ctxB gene, or a fragment thereof, is substituted with the kanamycin resistance gene further includes 5 'and 3'- Can be described by the sequence of SEQ ID NO: 5.

The recombinant plasmid for cloning Vibrio cholera CTX phage of the present invention may contain a region (copy regulatory region, or gene expression cassette) necessary for autonomous replication of the plasmid. Even in the case where a region other than the replication control region, that is, a region other than the region including the replication origin and the genes necessary for replication, is deleted, replication can also be performed in the host cell.

The copy control region includes a promoter and is a nucleic acid sequence having an expression activity for regulating expression of the gene, that is, transcription and translation, after the gene is operatively linked to the gene to be expressed.

The recombinant plasmid for cloning of Vibrio cholera CTX phage may be derived from a plasmid for transforming Escherichia coli derived from the group consisting of pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219 and pMW218 .

The recombinant plasmid for cloning Vibrio cholera CTX phage can be constructed in the same manner as known cloning vectors, expression vectors and the like.

For plasmid DNA preparation, DNA cleavage and binding, transformation, and the like, methods known to those skilled in the art can be used. These methods are described in Sambrook, J., Fritsch, E. F., and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

According to one embodiment of the present invention, there is provided a Vibrio cholera CTX phage genome sequence in which the full-length sequence of the ctxA gene and the full-length sequence of the ctxB gene or fragments thereof are removed, and a selectable marker gene cassette operatively linked to the removed sequence region DNA fragments can be prepared by insertion into the replication origin of the plasmid.

More specifically, a recombinant plasmid for cloning Vibrio cholera CTX phage

A replication origin fragment of the plasmid;

A portion of the zot gene and a selectable marker gene cassette and a portion of the ctxB gene from the 245th nucleotide of the zot of the front Vibrio cholera CTX phage genome to the rear rstR codon in the cholera strain having the CTX: RS1 or CTX: CTX sequence, and A DNA fragment comprising a sequence up to 3'UTR; And

A chorea strain having an array with CTX at the frontmost position may contain DNA fragments containing the 5'UTR of the CTX phage and neureotide to rstR, rstA, rstB, cep, orfU and the 244th sequence of zot. DNA fragments were amplified from different CTX fragments (CTX-1, CTX-cla, CTX-2, CTX-O139 and CTX-env) and ligated to the other two fragments, Can be included.

An example of such a recombinant plasmid for cloning Vibrio cholera CTX phage of the present invention is shown in the cleavage map of Fig.

V. cholera in a recombinant plasmid for CTX phage clone of the invention can be replicated in Escherichia coli (E. coli), Salmonella (Samonella), Shigella (Shigella), keulrep when Ella (Klebsiella), Pseudomonas (Psuedomonas), and Vibrio (Vibrio) Do. Than may be specifically, Escherichia coli (E. coli) or Vibrio (Vibrio).

The Vibrio (Vibrio) can be a classical bio-type E1 Tor bio type, atypical E1 Tor, O139 bio type, or V. cholera (Vibrio cholerae) for any serotype. More specifically, the CTX-1 genome-cloned plasmid can be transfected into a classical biotypic strain and CTX phage replication is possible. Plasmid cloning of the CTX-cla or CTX-2 genome can be introduced into any El Tor strain and CTX-cla and CTX-2 phages can be cloned, and the cloned CTX phage can be mutated into the toxT gene by the Tyr139Phe mutation And CTX phage replication is possible by transfection into Vibrio cholera strain. Such mutant strains include, but are not limited to, the A213-toxT ^Y139F strain or the IB4122-toxT ^Y139F strain.

Accordingly, the present invention provides a microorganism transformed with a recombinant plasmid for vibrio cholera CTX phage replication.

The method of transforming the host cell with the recombinant plasmid for cloning of Vibrio cholera CTX phage can be selected without any particular limitation in all transformation methods known in the art. For example, fusion of bacterial protoplasts, electroporation, And the like.

The microorganism may be selected from the group consisting of Escherichia coli (E. coli), Salmonella (Samonella), Shigella (Shigella), keulrep when Ella (Klebsiella), Pseudomonas (Psuedomonas), and Vibrio (Vibrio).

The cultivation of the transformed microorganism can be carried out in a suitable culture medium by various culture methods known in the art. Examples of the culture method include batch, continuous, and fed-batch cultivation. The fed-batch cultivation includes, but is not limited to, an implantation and a repeated implantation culture.

The media which can be used according to the present invention generally comprise at least one carbon source, a nitrogen source, inorganic salts, vitamins and / or trace elements. Preferred carbon sources are sugars such as monosaccharides, disaccharides or polysaccharides. The nitrogen source is generally an organic or inorganic nitrogen compound, or a material comprising these compounds. Examples of nitrogen sources include ammonia gas or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids, or complex nitrogen sources such as corn steep liquor, Yeast extract, and meat extract. The nitrogen source may be used alone or as a mixture. Inorganic salt compounds which may be present in the medium include chloride, phosphate or sulfate of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. As the phosphorus source, phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate, or the corresponding sodium-containing salts may be used. A chelating agent may be added to the medium to maintain metal ions in solution. More specifically, to promote replication of the CTX phage, a nanomolar concentration of mitomycin C can be added. All components of the medium are sterilized by heating (1.5 bar and 121 ° C for 20 minutes) or by sterile filtration. These ingredients can be sterilized together, or individually as needed. All components of the medium may be present at the start of the culture, or may optionally be added continuously or batchwise.

The CTX phage can be separated and purified from the transformed microorganism cells, culture of the microorganism, lysate of the microorganism or lysate of the culture to produce CTX phage.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1 Construction of Plasmid-Based CTX-1 Phage Replication System

The CTX phage can also be replicated in the plasmid-cloned CTX phage genome. The origin of replication of pUC18 and the entire ctxA and CTX-1 phage genome in which a portion of ctxB was replaced with a kanamycin cassette and the upstream and downstream non-coding sequences of the CTX phage were linked to E. coli and V. cholera ( V. cholerae ) (Fig. 3 and 4). The PCR primers used to amplify the replication origin of pUC18 were pUC18-ori-MluIF (5'-CCG CGC ACG CGT ATG TGA GCA AAA GGC-3 ') and pUC18-ori-KpnIR (5'- CGC GCC GGT ACC CCC GTA GAA AAG ATC-3 '). The DNA fragments from the 245th base to the 3'UTR of the zot gene were amplified from the PM8 strain by using Zot-MluIF (5'-CGC GCG ACG CGT TTC TCT TTA TCG ATG-3 ') primer and 3'UTR-KpnIR GCC GGT ACC CAA GAC TCG CTA GCG-3 ') primer and ligated with the cloning origin DNA fragment of pUC18. This ligated plasmid is a pUC18-zot-3'UTR plasmid and is commonly used in pUC-CTX-1kan, pUC-CTXclakan, pUC-CTX-2kan and pUC-CTX-O139kan. The DNA fragment from the 5 'UTR of the CTX phage to the 244 th base of the zot gene was amplified by PCR using primers MluIR (5'-CCG GCG ACG CGT CCT TTC TCG CCC AGT GCC-3') and 5'- UTR MluIF The plasmid pUC18-zot-3'UTR was amplified from the El Tor strain N16961 using an ACG CGTAG AGA CAA AAT GTT CCT-3 'primer and inserted into the plasmid pUC18-zot-3'UTR to which the pUC18 clone origin fragment and the zot- -CTX-1kan was prepared. The strain PM8 is described in a paper published by the present inventors in 2014 (Kim, EJ , et al. (2014) Molecular insights into the evolutionary pathway of Vibrio cholerae O1 atypical El Tor variants. PLoS Pathog . 10, e1004384). V. cholerae The plasmid-generated CTX-1 was constructed by using the PM14 strain, the classical strain O395, and the US Gulf Coast strain A213 as the host, in which the virulent phage CTX-1 was removed from the El Tor bio-type strain N16961. -1kan were transfected (Table 1).

Transduction efficiency of CTX phage-based replication-a Vibrio cholerae (V. cholerae) Strain list and plasmids used in the experiments Strain name Explanation Gene structure Produced CTX phage A transformed receptor strain Transfection efficiency Chromosome 1 Chromosome 2 PM20 N16961 derivative TLC: CTX-1: RS1 No element CTX-1kan O395 10 ⁵ MG116025 10 ² PM14 N16961 derivative TLC: RS1 No element no PM14-U1 PM14 transformed with pUC-CTX-1can TLC: RS1 No element CTX-1kan O395 10 ⁵ PM14-U2 PM14 transformed with pUC-CTX-2can TLC: RS1 No element CTX-2kan MG116025 2 x 10 PM14-U3 PM14 transformed with pUC-CTX-clakan TLC: RS1 No element CTX-clakan MG116025 10 ² PM14-U4 PM14 transformed with pUC-CTX-O139kan TLC: RS1 No element CTX-O139kan O395 2 x 10 ² MG116025 10 O395 Classical strain TLC: ^Trun CTX- ^cla : CTX-cla CTX-cla no O395-U1 O395 transformed with pUC-CTX-1can TLC: ^Trun CTX- ^cla : CTX-cla CTX-cla CTX-1kan O395 5 × 10 ⁴ A213 US Gulf Coast strain TLC No element no A213-U1 A213 transformed with pUC-CTX-1can TLC No element CTX-1kan O395 3 x 10 ³

The Vibrio cholerae (V. cholerae) is that it was confirmed that the CTX phage made from the plasmid in the presence of transition to mitomycin transfected with the plasmid in the strain (FIG. 5). The CTX phage actually delivered is a complete CTX phage without a pUC18 origin of replication.

As shown in Table 1, PM14 is a strain in which CTX-1 prophage is removed from N16961, which is a Wave 1 El Tor strain, O395 is a classical strain, A213 is a US Gulf Coast strain, and CTX phage is not contained. The pUC-CTX-1kan plasmid is transformed into these strains. When mitomycin is present, up to 10 ⁵ of CTX-1kan phage are transferred to O395, which is used as a transduction receptor strain. In fact, similar CTX-1 kans are produced in N16961 in rheumatoid CTX-1, and in previous reports of the present inventors, it is similar to that in CT2-1 in the case of V212-1, about 10 ⁵ CTX-1 phages are produced. Cloning of the CTX phage is not determined by the host biotype. This result shows that phage replication can occur in complete form in the plasmid-cloned CTX phage genome.

Example 2 Construction of plasmid-based CTX-cla or CTX-2 and CTX-O139 phage cloning system

After confirming that the CTX-1 phage was generated from the CTX-1 genome cloned into the plasmid, the CTX-2 clone, which was not proven experimentally, and its genetic ortholog, CTX-2, Cloning into the plasmid could be expected to produce a phage of the offspring unless these phage were indeed cloning deficient.

For this, pUC-CTX-clakan and pUC-CTX-2kan were constructed in a manner similar to the construction of pUC-CTX-1kan. The origin of replication was amplified from the pUC18 plasmid by PCR. The PCR primers used were pUC18-ori-MluIF (5'-CCG CGC ACG CGT ATG TGA GCA AAA GGC-3 ') and pUC18-ori-KpnIR (5'-CGC GCC GGT ACC CCC GTA GAA AAG ATC- ) to be. The DNA fragments from the 245th base to the 3'UTR of the zot gene were amplified from the PM8 strain by using Zot-MluIF (5'-CGC GCG ACG CGT TTC TCT TTA TCG ATG-3 ') primer and 3'UTR-KpnIR GCC GGT ACC CAA GAC TCG CTA GCG-3 ') primer and ligated with the cloning origin DNA fragment of pUC18. This ligated plasmid is a pUC18-zot-3'UTR plasmid and is commonly used in pUC-CTX-1kan, pUC-CTXclakan, pUC-CTX-2kan and pUC-CTX-O139kan. The DNA fragment from the 5 'UTR of the Classical CTX phage to the 244th base of the zot gene was amplified by PCR using primers MluIR (5'-CCG GCG ACG CGT CCT TTC TCG CCC AGT GCC-3') and 5'- UTR MluIF The plasmid pUC18-zot-3'UTR was amplified from the classical strain Cairo48 using the primer CCG ACG CGT TAG AGA CAA AAT GTT CCT-3 'and inserted into the plasmid pUC18-zot-3'UTR to which the pUC18 clone origin fragment and the zot- -CTX-clakan was prepared. Primer B33Ch2MluF (5'-CGC CCG ACG CGT ATG ATT TTT TTA TTC TTC CAC-3 ') and primer MluIR (5'-CCG GCG ACG CGT CCT TTC TCG CCC AGT GCC-3') were used to construct pCTX-2kan The DNA fragment from the 5'UTR of the CTX-2 phage to the 244th base of the zot gene was amplified from the wave 2 strain B33 and inserted into the plasmid pUC18-zot-3'UTR to construct pUC18-CTX-2kan.

O395 was not suitable as a host for the plasmid containing CTX-2 and CTX-cla, and only PM14, an El Tor strain, was used as a host. Even if CTX-cla and CTX-2 are produced from these, classical strains can not act as transduction receptor strains because of phage immunity. Therefore, several El Tor strains were prepared to prepare classical strains as transduction receptor strains and tested to see if CTX-2 or CTX-cla could be transduced. Other strains of El Tor strains were not transduced by CTX-cla or CTX-2 phage cloned in the plasmid, whereas MG116025 strains were obtained by CTX-2kan and CTX-clakan phage, It can be confirmed that a solid body is generated (Tables 2 and 3).

The replication efficiency of CTX-2 phage from pUC-CTX-2kan and the secondary transfection efficiency of the cloned CTX-2 phage Primary transduction Secondary transduction donor PCTX produced in donor Transduction
Receptor strain Transduction
efficiency Transgenic
(Transduced pCTX) Transduction
Receptor strain Transduction
efficiency PM14-U2 pCTX-2kan MG116025 2 x 10 MG116025 (pCTX-2kan) MG116025 2 x 10 ⁴ MG116025- toxT ^F139Y 0 A213- toxT ^Y139F 3 x 10 ³ IB4122 toxT ^Y139F 0 O395 0

The replication efficiency of CTX-cla phage from pUC-CTX-clakan and the secondary transfection efficiency of cloned CTX-cla phage Primary transduction Secondary transduction donor PCTX produced in donor Transduction
Receptor strain Transduction
efficiency Transgenic
(Transduced pCTX) Transduction
Receptor strain Transduction
efficiency PM14-U3 pCTX-clakan MG116025 1 x 10 ² MG116025 (pCTX-clakan) MG116025 3 x 10 ⁴ MG116025- toxT ^F139Y 0 A213- toxT ^Y139F 2 x 10 ⁴ IB4122- toxT ^Y139F 0 O395 0

As shown in Tables 2 and 3, since the CTX phage clones are more likely to replicate in the CTX phage clone, pCTX-2kan or pCTX-clakan, since the clone CTX phage is known to be better produced, Secondary transfection using the introduced material as the donor strain (receptor strain MG116025) revealed that about 10 ⁴ transformants were produced in pCTX-clakan and pCTX-2kan. Although cloning of CTX-2 and CTX-cla cloned in the plasmid resulted in fewer numbers of 20 and 100 transgenic lines in primary transduction, the number of secondary transcripts generated from pCTX-2 and CTX- Transduction is often made at 10 ⁴ levels (Tables 2 and 3).

(replication of CTX-O139 phage from pUC-CTX-O139kan)

It has already been reported that a progeny phage is produced in CTX-O139 that has already been inserted into the chromosome of cholerae in a protease state. No replication of CTX-O139 cloned into the plasmid has been reported. (5'-CGG CCG ACG CGT CCT TTC TCG CCC AGT GCC-3 ') primer and 5'-UTR MluIF (5'-CGC CCG ACG CGT TAG AGA CAA AAT GTT CCT- PUC-CTX-O139kan was prepared by amplifying the DNA fragment from the 5 'UTR of the O139 phage to the 244th base of the zot gene from the strain strain AR1961537 and inserting it into the plasmid pUC18-zot-3'UTR. When CTX-O139 was cloned into the plasmid in this manner, it was confirmed that CTX virus was produced (Tables 1 and 4). The O139 phage can be transfected into O395 and MG116025 at about 200, 10 (Table 4), and from each of these, the secondary O139 can be made at a level of 10 ⁶ . That is, the above results show that the appropriate use of the transformed receptor strain can produce various CTX phages from the plasmid-based CTX phage cloning system.

The replication efficiency of CTX-O139 phage from pUC-CTX-O139kan and the secondary transfection efficiency of the cloned CTX-O139 phage Primary transduction Secondary transduction donor PCTX produced in donor Transduction
Receptor strain Transduction
efficiency Transgenic
(Transduced pCTX) Transduction
Receptor strain Transduction
efficiency PM14-U4 pCTX-O139kan O395 2 x 10 ² O395 (pCTX-O139kan) O395 10 ⁶ MG116025 5 x 10 ³ MG116025- toxT ^F139Y 0 A213- toxT ^Y139F 10 ⁶ IB4122- toxT ^Y139F 10 ² MG116025 10 MG116025 (pCTX-O139kan) O395 5 × 10 ⁶ MG116025 3 x 10 ⁴ MG116025- toxT ^F139Y 0 A213- toxT ^Y139F 3 x 10 ³ IB4122- toxT ^Y139F 4 × 10 ²

Example 3: Replication of various CTX phages

When the El Tor strain can be transduced, the degree of transduction of various CTX phages can be examined for classical strains, MG116025, and transgenic El Tor strains. The genome of CTX-env, a new type of rstR gene among CTX phage, which has not been experimentally produced, can also be cloned by cloning in a plasmid-like manner.

It was also confirmed that various transduced CTX phages were introduced into the receptor strains through transduction and then lysogenized on chromosomes 1 and 2 (not shown).

In conclusion, the present invention can 1) produce progeny phage from various CTX phage genomes cloned into plasmid, and 2) enable El Tor strain to act as a transduction receptor strain. 3) In the receptor strains obtained by transfection of CTX phage produced by plasmid, the CTX phages can be converted into two chromosomes of the receptor strains, respectively, or both, and repeating this process, CTX phage can be used as a strain.

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Plasmid-based Vibrio cholerae CTX phage replication system <130> P16U22C1951 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 777 <212> DNA <213> Artificial Sequence <220> <223> ctxA <400> 1 atggtaaaga taatatttgt gttttttatt ttcttatcat cattttcata tgcaaatgat 60 gataagttat atcgggcaga ttctagacct cctgatgaaa taaagcagtc aggtggtctt 120 atgccaagag gacagagtga gtactttgac cgaggtactc aaatgaatat caacctttat 180 gatcatgcaa gaggaactca gacgggattt gttaggcacg atgatggata tgtttccacc 240 tcaattagtt tgagaagtgc ccacttagtg ggtcaaacta tattgtctgg tcattctact 300 tattatatat atgttatagc cactgcaccc aacatgttta acgttaatga tgtattaggg 360 gcatacagtc ctcatccaga tgaacaagaa gtttctgctt taggtgggat tccatactcc 420 caaatatatg gatggtatcg agttcatttt ggggtgcttg atgaacaatt acatcgtaat 480 aggggctaca gagatagata ttacagtaac ttagatattg ctccagcagc agatggttat 540 ggattggcag gtttccctcc ggagcataga gcttggaggg aagagccgtg gattcatcat 600 gcaccgccgg gttgtgggaa tgctccaaga tcatcgatga gtaatacttg cgatgaaaaa 660 acccaaagtc taggtgtaaa attccttgac gaataccaat ctaaagttaa aagacaaata 720 ttttcaggct atcaatctga tattgataca cataatagaa ttaaggatga attatga 777 <210> 2 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ctxB <400> 2 atgattaaat taaaatttgg tgtttttttt acagttttac tatcttcagc atatgcacat 60 ggaacacctc aaaatattac tgatttgtgt gcagaatacc acaacacaca aatatatacg 120 ctaaatgata agatattttc gtatacagaa tctctagctg gaaaaagaga gatggctatc 180 attactttta agaatggtgc aatttttcaa gtagaagtac caggtagtca acatatagat 240 tcacaaaaaa aagcgattga aaggatgaag gataccctga ggattgcata tcttactgaa 300 gctaaagtcg aaaagttatg tgtatggaat aataaaacgc ctcatgcgat tgccgcaatt 360 agtatggcaa attaa 375 <210> 3 <211> 166 <212> DNA <213> Artificial Sequence <220> <223> ctxB fragment <400> 3 atgattaaat taaaatttgg tgtttttttt acagttttac tatcttcagc atatgcacat 60 ggaacacctc aaaatattac tgatttgtgt gcagaatacc acaacacaca aatatatacg 120 ctaaatgata agatattttc gtatacagaa tctctagctg gaaaaa 166 <210> 4 <211> 816 <212> DNA <213> Artificial Sequence <220> <223> kanamycin gene <400> 4 atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat 60 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc 120 tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc 180 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct 240 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg 300 atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt 360 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct 420 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg 480 gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa 540 gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca 600 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc 660 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct 720 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa 780 ttgcagtttc atttgatgct cgatgagttt ttctaa 816 <210> 5 <211> 1542 <212> DNA <213> Artificial Sequence <220> <223> CTX prophage genome substituted ctxA and ctxB fragment with          kanamycin resistant gene <220> <221> gene (420). (1251) <223> kanamycin resistant gene <220> <221> gene <222> (1264). (1472) <223> ctxB <400> 5 tgcatcgagc taccgctaca aggtgctacc gttaccggat ttcaatcact ttgtggtgtt 60 cgataccttt gcagcgcaag cgctgtgggt agaagtgaaa cggggtttac cgataaaaac 120 agaaaatgat aaaaaaggac taaatagtat attttgattt ttgatttttg acttttgatt 180 tcaaataata caaatttatt tacttattta attgttttga tcaattattt ttctgttaaa 240 gatctcaaga 300 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 360 gattttggtc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta 420 tgagccatat tcaacgggaa acgtcttgct ctaggccgcg attaaattcc aacatggatg 480 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct 540 atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg 600 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc 660 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga 720 tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg 780 ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt 840 ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg 900 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag 960 aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac 1020 ttgataacct tatttttgg gaggggaaat taataggttg tattgatgtt ggacgagtcg 1080 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc 1140 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat 1200 tgcagtttca tttgatgctc gatgagtttt tctaagccct gcagggcgag agatggctat 1260 cattactttt aagaatggtg caatttttca agtagaagta ccaggtagtc aacatataga 1320 ttcacaaaaa aaagcgattg aaaggatgaa ggatatcctg aggattgcat atcttactga 1380 agctaaagtc gaaaagttat gtgtatggaa taataaaacg cctcatgcga ttgccgcaat 1440 tagtatggca aattaagata taaaaaagcc cacctcagtg ggcttttttg tggttcgatg 1500 atgagaagca accgttttgc ccaaacatgt attactgcaa gt 1542

Claims (13)

The Vt cholera CTX phage genome sequence in which the full-length sequence of the ctxA gene described in SEQ ID NO: 1 and the full-length sequence of the ctxB gene described in SEQ ID NO: 2 or a fragment thereof is substituted with a selectable marker gene is inserted into the replication origin of the plasmid A recombinant plasmid for cloning of Vibrio cholera CTX phage.
The method according to claim 1,
Wherein the fragment of the ctxB gene comprises the nucleotide sequence as set forth in SEQ ID NO: 3. 3. The recombinant plasmid for the Vibrio cholera CTX phage cloning according to claim 1,
The method according to claim 1,
Wherein the Vibrio cholera CTX phage genomic sequence is a genomic sequence of CTX-1, CTX-cla, CTX-2, CTX-env or CTX-O139.
The method according to claim 1,
Wherein the selectable marker gene is selected from the group consisting of an ampicillin resistance gene, a kanamycin resistance gene, a straptomain resistance gene, a tetracycline resistance gene, an erythromycin resistance gene, and a chloramphenicol acetyltransferase gene.
The method according to claim 1,
The recombinant plasmid for Vibrio cholera CTX phage cloning, wherein the selectable marker gene comprises the nucleotide sequence of SEQ ID NO: 4.
The method according to claim 1,
Wherein the recombinant plasmid for cloning of Vibrio cholera CTX phage is selected from the group consisting of Vibrio cholera CTX derived from a plasmid for transformation derived from Escherichia coli selected from the group consisting of pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219 and pMW218 Recombinant plasmid for phage cloning.
delete The method according to claim 1,
The recombinant plasmid for Vibrio cholera CTX phage cloning has a cleavage map of pUC-CTX-kan of Fig. 4B.
The method according to claim 1,
Wherein the Vibrio cholerae phage CTX recombinant plasmid for replication of E. coli (E. coli), Salmonella (Samonella), Shigella (Shigella), keulrep when Ella (Klebsiella), Pseudomonas that can be reproduced in (Psuedomonas) and Vibrio (Vibrio), Recombinant plasmid for cloning Vibrio cholera CTX phage.
The method according to claim 1,
The Vibrio cholerae phage CTX recombinant plasmids for cloning are classical bio type, E1 Tor bio type, atypical E1 Tor bio type or types of bio-O139 V. cholera (Vibrio cholerae ). &lt; / RTI &gt; A recombinant plasmid for cloning Vibrio cholera CTX phage.
A microorganism transformed with the recombinant plasmid for cloning of Vibrio cholera CTX phage of claim 1.
12. The method of claim 11,
Wherein the microorganism is Escherichia coli (E. coli), Salmonella (Samonella), Shigella (Shigella), keulrep when Ella (Klebsiella), Pseudomonas (Psuedomonas), and Vibrio (Vibrio), the microorganism is selected from the group consisting of.
Culturing the transformed microorganism of claim 11; And
And isolating and purifying the Vibrio cholera CTX phage in the culture medium of the transformed microorganism.

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