MXPA00011604A - Method of producing thy - Google Patents
Method of producing thyInfo
- Publication number
- MXPA00011604A MXPA00011604A MXPA/A/2000/011604A MXPA00011604A MXPA00011604A MX PA00011604 A MXPA00011604 A MX PA00011604A MX PA00011604 A MXPA00011604 A MX PA00011604A MX PA00011604 A MXPA00011604 A MX PA00011604A
- Authority
- MX
- Mexico
- Prior art keywords
- thya
- gene
- strain
- vibrio cholerae
- cholerae
- Prior art date
Links
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Abstract
A method of producing a thy A-strain of vibrio cholerae comprising the step of site-directed mutagenesis in the V. cholerae chromosome at the locus of the thy A gene SEQ ID NO:1 of FIG. 1, is described. Particularly, a&Dgr;thy A strain of Vibrio cholerae lacking the functionality of the thy A is disclosed. This strain may comprise one or several episomal autonomously replicating DNA elements, such as plasmids, having an optionally foreign, e.g. E. coli, functional thy A gene that enables the strain to grow in the absence of thymine in the growth medium, and optionally having a structural gene encoding a homologous or heterologous protein. Further, proteins encoded by a structural thy A gene and the 5'-flanking region are described as SEQ ID NO:4 of FIG. 4 and SEQ ID NO:5 of FIG. 5, respectively. Additionally, a vaccine comprising a Vibrio cholerae&Dgr;thy A strain of the invention or a thy A-strain of Vibrio cholerae produced by the method of the invention is disclosed.
Description
METHOD FOR PRODUCING STRAIN "STRAINS FROM Vibrio cholerae, SUCH STRAINS AND ITS USE
FIELD OF THE INVENTION The present invention relates to a method for producing thyA strains of Vibrio cholerae, to such strains and to their use The present invention particularly relates to a strain of Vijbrio cholerae that has been deprived of its thyK gene in the chromosome, ie, a strain? thyA lacking the functionality of the thyA gene.This strain may comprise one or more episode elements of autonomously replicating DNA, such as plasmids, which have a functional, optionally foreign thyA gene, eg coli, which makes it possible for the strain to grow in the absence of tintin in the culture medium and optionally have a structural gene coding for a homologous or heterologous protein.The present invention also relates to thyA nucleotide sequences and proteins encoded by they, and a vaccine that comprises as an immunizing component a strain of Vibrio cholerae A thyA of the present invention or a strain thyA "of Vibrio cholerae produced by the method of the present invention. BACKGROUND OF THE INVENTION The expression of recombinant genes in hosts
Ref: 125004 Bacteria is often achieved by introducing self-replicating episomal elements (e.g. plasmids) that encode the structural gene of the protein of interest, under the control of an appropriate promoter, in host bacteria. Such plasmids are commonly maintained by the inclusion of selective genetic markers that encode proteins that confer resistance to specific antibiotics (such as ampicillin, chloramphenicol, kanamycin, tetracycline, etc.). They are then maintained in the host by the addition of the appropriate antibiotic to the culture medium. Stable maintenance of plasmids in the host strains often requires the addition of the appropriate selected antibiotic without which they could segregate or could give rise to a significant number of cells in any culture that are devoid of the plasmid and, therefore, not They can express the desired product. However, the use of antibiotics in the production of recombinant proteins is undesirable for a number of reasons. Apart from the obvious increase in cost that arises from the need to add them as a supplement to the culture medium, the use of antibiotics is considered a problem in the production of any recombinant protein that has a human or veterinary purpose. This is mainly due to three reasons. First, residual antibiotics can, in sensitive individuals, cause severe allergic reactions. Second, there is the possibility of selecting bacteria resistant to antibiotics in the natural bacterial flora of those who use the product and, finally, the DNA that codes for the resistance to antibiotics can also be transferred to sensitive bacteria in the individuals that consume the product , thus spreading undesirable resistance to antibiotics in a group. There are already inventions dealing with this problem, one such is the even gene, which effectively kills all cells that do not retain a copy of the plasmid after each cell division [1]. Another patent application [2], which touches on the subject of the invention described herein, was based on knowledge of the thyA DNA sequence in E. coli. The authors introduced the thyA gene into a plasmid, but used host strains that were spontaneous thyA mutants selected based on the resistance to trimethoprim, such mutants were not well defined (point mutations or small deletions) and can revert to the wild type (ie , thyA +) at unacceptably high frequencies, this would lead to the host bacterium eliminating the plasmid and, thus, losing production or not giving a consistent and reliable production of the desired recombinant product.An additional problem with the selection with trimethoprim is 5 the possibility that thymine dependence may arise due to a mutation in the enzyme dihydrofolate reductase (folA) gene and, in this way, not be complemented by a thyA gene of plasmidic origin [3]. This patent application was discontinued at least in Europe. The use of V. cholerae for the expression of recombinant genes has proven to be advantageous over common prokaryotic expression systems in that specific recombinant products can be produced in large quantities and secreted into the culture medium, thus facilitating the subsequent purification procedures. This is in contrast to E. coli, where the product is often assembled in the periplasmic space [4]. An important factor that endows V. cholerae with this property are the eps genes in V. 20 cholerae [5]. Thymidine synthetase encoded by the thyA gene of Escherichia coli and other bacteria, catalyzes the methylation of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) and is an essential enzyme in the biosynthesis of deoxyribothymidine triphosphate (dTTP) to be
- ^^ a ^ ^ Sx á ^ ¡j? The bacterium becomes dependent on an external source of thymine, which is incorporated into the dTTP by a wild-type route encoded by the deo genes. 6] Spontaneous mutants that are t.hyA "can be easily isolated based on their resistance to trimethoprim. This antibiotic inhibits the regeneration of tetrahydrofolate from the dihydrofolate produced by the synthesis of dTMP catalyzed by the enzyme thymidylate synthetase. Thus, if the cells are thyA, they become thymine-dependent but no longer deplete tetrahydrofolate in the presence of trimethoprim DESCRIPTION OF THE INVENTION The present invention, in its different aspects, is based on the new nucleotide sequence of the thyA gene. V. cholerae A useful application of the thyA gene is, for example, in maintaining the recombinant plasmids used in the overproduction of recombinant proteins in V. cholerae, and in the use of the sequence for the insertion of foreign genes in a selectable manner and specifies site on the chromosome of V. cholerae One aspect of the present invention relates to a method for producing a thyA strain of Vibrio cholerae comprising the step of performing site-directed mutagenesis on the chromosome of V. cholerae for the deletion and / or
- ~ a »i A i ié-ag ^^? ^^^ insertion of gene nucleotides at the locus of ten thyA, essentially having the nucleotide sequence of SEQ ID NO. 1, of Figure 1. The term "having essentially the nucleotide sequence" as used herein and in the claims, is intended to comprise the nucleotide sequences having some extensions, truncated portions, deletions or additions, natural or unnatural , that do not interfere with the natural function of the nucleotide sequence in question. Another aspect of the present invention relates to a strain of V. cholerae thyA "which is a strain? ThyA that lacks the functionality of the thyA gene.In one embodiment of this aspect of the present invention, the strain? ThyA of V. cholerae comprises one or more episomal DNA elements of autonomous replication having a functional thyA gene, which make it possible for the strain to grow in the absence of thymine in the culture medium In a preferred embodiment, the episomal DNA element of autonomous replication is a plasmid In another preferred embodiment, the strain thyA according to the present invention comprises an episomal DNA element of autonomous replication, especially a plasmid, a foreign thyA gene, such as an E. coli gene.
In a particularly preferred embodiment of this aspect of the present invention, the strain? thyA according to the present invention comprises, in one or several elements of episomal DNA of autonomous replication, especially plasmids, in addition to a foreign thyA gene, such as an E. coli gene, also a structural gene coding for a homologous protein or heterologous, such as subunit B of the thermolabile enterotoxin of Escherichia coli (LTB) or the glutathione S-transferase protein of 26 kD of Schistosoma japonicum (GST 26 kD). A third aspect of the present invention relates to a nucleotide sequence of a 5 'flanking region of a structural thyA gene of Vibrio cholerae having essentially the nucleotide sequence of SEQ ID NO. 2, of Figure 2. A fourth aspect of the present invention relates to a nucleotide sequence of a 3 'flanking region of a structural thyA gene of Vibrio cholerae having essentially the nucleotide sequence of SEQ ID NO. 3, of Figure 3. The nucleotide sequence SEQ ID NO. 1 is useful for the insertion of foreign genes in a selectable and site-specific manner into the chromosome of V. cholerae, and for site-directed mutagenesis in the production of thyA strains of Vijbrio cholerae.
A fifth aspect of the present invention, refers to a protein encoded by a nucleotide sequence of a thyA gene of Vibrio cholerae according to the present invention, such as a protein having the amino acid sequence of SEQ ID NO. 4, of Figure 4. A sixth aspect of the present invention relates to a protein encoded by a nucleotide sequence of a 5 'flanking region of a structural thyA gene of Vijbrio cholerae according to the present invention, such as the protein having the amino acid sequence of SEQ ID NO. 5, of Figure 5. The proteins according to the fifth and 15th aspects of the present invention are useful for research purposes and are potential targets for antimicrobial therapy. A seventh aspect of the present invention relates to a vaccine comprising as an immunizing component, a strain of Vijbrio cholerae? thyA, in accordance with the present invention or a strain of Vibrio cholerae thyA 'produced by the method of the present invention. The vaccine will be used for the prophylactic and therapeutic treatment of cholera and, optionally, of 25 other infectious diseases, especially in cases of
. »- > 3- JMg - * »mt J, *,. i »¡" ». ..... .. ".. .."., ",, R.h > . "_ ^" JjjJ "where the strain used has been manipulated to express foreign proteins. The vaccine, in addition to the immunizing component or components, will comprise a vehicle such as physiological saline solution, and other components frequently used in vaccines such as regulatory solutions and adjuvants. Vehicles, regulatory solutions, adjuvants and other useful components are described, for example, in the European Pharmacopoeia and the United States Pharmacopoeia. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the nucleotide sequence SEQ ID NO. 1 of the thyA gene of Vijbrio cholerae. Figure 2 shows the nucleotide sequence SEQ ID NO. 2 of the 5 'flanking region of the structural thyA gene of Vibrio cholerae. • Figure 3 shows the nucleotide sequence SEQ ID NO. 3 of the 3 'flanking region of the structural thyA gene of Vijbrio cholerae. Figure 4 shows the amino acid sequence SEQ ID NO. 4 of the protein encoded by the structural thyA gene of Vibrio cholerae. Figure 5 shows the amino acid sequence SEQ ID NO. 5 of the protein encoded by the 5 'flanking region of the structural thyA gene of Vibrio cholerae. Figure 6 shows the cloning of a J-TcoRI / HindlII fragment containing the thyA gene of V. cholerae in pUC19. Figure 7 shows a composition of thyA gene products from E. coli [16], V. cholerae and H. influenzae [17] that shows the high degree of homology between V. cholerae and H. influenzae, compared to E. coli Figure 8 shows the insertion of a block of KanR resistance genes in the PstI site of the thyA gene of V. cholerae, in pUC19. Figure 9 shows a polymerase catalyzed chain reaction (PCR) to generate a thyA-Kan fragment with Xbal ends. Figure 10 shows the ligation of the thyA-Kan fragment with Xbal ends in the plasmid pNQ705. Figure 11 shows a partial deletion of the thyA gene and the start of the Kan gene in the plasmid pNEB193. Figure 12 shows the break with Xbal to cut the gene? thyA? kan of plasmid pNEB193, and ligation in pDMA4 restricted by Xbal. Figure 13 shows a schematic of a strategy to completely eliminate the thyA gene from V. cholerae. Figure 14 shows the insertion of the 5 'region upstream of thyA in pMT-SUICIDE 1; the generation of the pMT with 5 prim. Figure 15 shows the insertion of the 3 'region
* > - »- *.» ».» •. * & ..
chain down from thyA in the pMT with 5 prim; the generation of pMT? thyA V. cholerae. Figure 16 shows the expression of the vector pMT-eltB (thyA) used for the expression of LTB in V. 5 cholerae JS1569? thyA Figure 17 shows the expression vector pMT-GST (thyA) used for the expression of GST in V. cholerae JS1569? thyA DESCRIPTION OF EXPERIMENTS 10 Strategy used In order to produce defined thyA mutants of V. cholerae that could be used as suitable producing strains for recombinant proteins encoded in the plasmids maintained by thyA complementation, first it was necessary to clone and characterize the natural gene and its flanking regions 5 'and 3'. Our strategy was to first clone the thyA gene of V. cholerae in a plasmid, at the base of the complementation of the thyotrophic auxotrophy in a strain of E. coli K12. The restriction analysis and 20 subcloning experiments were performed in order to locate the thyA structure gene in the large DNA fragment initially obtained. Then, the appropriate region containing the thyA gene and its 5"and 3 'flanking regions was sequenced.
In order to verify that one of the sequenced genes was in fact the thyA gene of V. cholerae, homology comparisons were made with thyA sequences from other organisms. The cloned gene could also be complemented with the thyA phenotype of a mutant strain of V. cholerae that had been selected based on its resistance to trimethoprim. The sequence analysis of this mutant showed that in fact it had a change of a single base in the gene that had been identified as the thyA, which resulted in a stop codon giving rise to a non-functional truncated gene product. The knowledge of the thyA sequence and that of the surrounding region allowed us to use appropriate suicide vectors to perform site-directed mutagenesis. The strategies taken into account were (a) insertional inactivation (b) a combination of insertional inactivation and gene deletion and (c) complete gene removal: (a) Insertional inactivation of the thyA gene was achieved by inserting a gene block KanR (with suicide vector pNQ705 [14]). (b) A deletion of approximately 400 bp was performed on the carrier strain of the KanR gene block that removed 200 bp of the thyA gene upstream of the insertion site and the kanamycin resistance gene, which was inactivated in this manner. Then a gene was obtained
^ jgj¡ ^ thyA deleted, where the deletion or deletion was in the central part of the gene and was followed by an insertion of a non-coding DNA region. This construct was inserted into the chromosome of V. cholerae using the suicide vector pDM4 and resulted in a strain called JS1569? ThyA? Kan. (c) The complete removal of the thyA gene was performed by ligating the flanking regions of the structural gene, taking care not to interrupt other open reading frames (the interruption of the adjacent igt gene is also lethal). The DNA carrying the deletion was cloned into a new suicide vector (PMT-SUICIDE-1) used for the insertion of the sequence into the chromosome of V. cholerae. The resulting strain was designated JS1569? ThyA. For the expression of recombinant genes in these strains? ThyA of V. cholerae, two expression vectors were constructed. Each consisted of the thyA gene of E. coli, the origin of replication of the high-copy, general-purpose vector pUC19, the tac promoter, and the rho-independent trpA transcription terminator. In one of the two vectors, the lacl9 gene was inserted in order to regulate the expression of the tac promoter, which also contained the iac operator sequence. Two genes were cloned into these plasmids and expressed in the newly generated V. cholerae strain with thyA deletion, JS1569 thyA. The first one coded for subunit B of the human heat labile enterotoxin of E. coli (LTB) (Figure 16), the second coded for the sj26 glutathione-S-transferase (GST) of Schistosoma japonicum (Figure 17). The LTB is similar in structure to the subunit B of the cholera toxin produced naturally by the host cell and was secreted into the culture medium. The other protein is of eukaryotic origin, coming from the asian hepatic trematode. It is known that sj26 GST is expressed in high levels in E. coli and accumulates in the cytoplasm. The expression of the two recombinant proteins was evaluated based on a GMl ELISA test of the culture supernatant in the case of LTB and a commercially available assay in the case of GST. Both proteins were also analyzed based on a polyacrylamide gel electrophoresis with sodium dodecylsulfate (SDS-PAGE) and by western blotting. Origin of the fchyA gene The thyA gene was cloned from the V. cholerae strain JS1569. This strain originates from the strain of V. cholerae Inaba 569B of the classical biotype (ATCC No. 25870). The strain suffered a deletion in the ctxA gene ([7] and became resistant to rifampicin [8].) Cloning of a 1.14 kb HindIII / EcoRI fragment spanning the thyA gene of V. cholerae Chromosomal DNA was prepared by the method C [9] and was digested completely with the restriction enzyme HindlII.The digested DNA was ligated into the vector plasmid for general purposes pBR322 (New England Biolabs Inc. Beverly, MA USA), which had been digested with HindlII and treated with alkaline phosphatase The ligation mixture was subjected to electroporation [10] in a strain of E. coli HB101 that was phenotypically thyA "(selected based on resistance to trimethoprim) and the culture was inoculated onto modified Syncase (MS) agar plates [11] supplemented with 50 μg / ml ampicillin, but without thymine, then transformants were selected based on the acquisition of the plasmid as well as the presence of a functional thyA gene. were inoculated to obtain isolated colonies on the same type of agar plates and then grown in MS broth supplemented with ampicillin. The plasmid DNA was prepared by the "Wizard miniprepps" method (ProMega Corp. Madison Wis., USA) and was digested with finc / III. A fragment of
^ fcjáj approximately 10-12 kb, and this clone was called thyA B2. To reduce the size of the fragment, the plasmid was cut with the restriction enzyme £ coRI and ligated again using T4 ligase. The ligated DNA was again subjected to electroporation in the E. coli strain described above, using the same selective conditions to grow the transformants. The colonies resulting from this experiment
isolated in the manner previously described and the plasmid DNA was purified and analyzed by double digestion with 2ScoRI and ífindlll. The remnant was a DNA fragment of approximately 1.4 kb that retained the ability to complement the thyA mutation in the E. coli host cell.
This fragment was cloned into the plasmid pUC19 (New England Biolabs) which had been digested with the same two enzymes and treated with alkaline phosphatase. After electroporation, the transformants of the experiment were isolated and characterized in the manner above
described. This clone was called thyA 1: 2 (Figure 6). Verification that the H ndlII / EcdRI fragment of 1.4 kb contains the thyA gene. Southern blot immunoblot analysis (Southern blot). To verify that the cloned fragment
in fact it was of chromosomal origin of V. cholerae, the DNA
of strain JS1569 was digested completely with i? indIII and EcóRI and HindlII. The DNA fragments were separated by agarose electrophoresis together with the clone digested with ífindlll thyA B2 and the clone digested with EcóRI and HindIII thyA 1: 2. After electrophoresis, the DNA was transferred to a nylon membrane, immobilized by UV irradiation and hybridized (under stringent conditions) with the 1.5 kb fragment cut from the thyA 1: 2 clone that had been labeled with 32P dCTP using the Amersham Multiprime package. Results In both the chromosomal DNA digested with HindlII and in the clone thyA B2 digested with ífindlll, a band of approximately 10 kb was evident. Similarly, in the chromosomal DNA digested with H-'coRI / J-indlII and in the plasmid DNA of the clone thyA 1: 2, a band of 1.4 kb was evident (data not shown). These data demonstrated that the cloned fragment was derived from the DNA of V. cholerae JS1569. Transformation of thyA JS1569 with plasmid thyA 1: 2 To verify that the cloned JJcoRI / i? IndlII fragment of 1.4 kb could support the growth of V. cholerae phenotypically thyA ", a thymine-dependent mutant of strain JS1569 (V. cholerae JS1569 4.4) was subjected to electroporation with the plasmid thyA 1: 2. The
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Electroporation and the selective medium were as previously described. Strain JS1569 4.4 did not grow in MS medium without the addition of thymine. Results The colonies of the strain were isolated
JS1569 4.4 that grew in the absence of thymine. All were shown to be carriers of the thy A 1: 2 plasmid, thus supporting the assumption that the cloned fragment contained the thyA gene of V. cholerae. DNA sequencing of plasmid thyA 1: 2. He
Plasmid DNA was sequenced by the dideoxy chain termination method [12] using the ABI PRISM ™ Dye cyclic terminator sequencing kit (Perkin Elmer). Both are commercially available, as well as tailor-made primers. The DNA sequences were
analyzed in an ABI PRISM 373 automatic sequencer (Perkin Elmer). The data was analyzed using the AutoAssembler software package (Perkin Elmer). The homology search with the DNA sequence found was performed with the GCG program [13]. 20 Results. The best homologies were with the thymidylate synthetases from several species. Note that homology with E. coli thymidylate synthetase was low (Figure 7). Strategy for the deletion of the thyA gene in V. cholerae
JS1569.
Two different strategies were used to obtain defined thyA mutants of V. cholerae JS1569, the first one included the inactivation of the thyA gene by the insertion of a block of KanR genes, detected by a partial deletion of the thyA gene and the KanR gene block. . The second strategy was to completely eliminate the thyA gene from the chromosome by means of a new suicide vector pMT SUICIDE-1. This vector contains the 5 'and 3"flanking regions of the thyA gene, as well as the R6K origin of replication and the mojb RP4 genes, to replace the thyA gene of strain JS1569, it was decided to use the already thymine-dependent strain JS1569 4.4, since preliminary experiments indicated that there is a strong selective disadvantage to go from the natural type to thymine dependence, even in the presence of high levels of exogenous thymine. Inactivation of the thyA gene by inserting a block of KanR genes The strategy involved the inactivation of the thyA gene by inserting a kanamycin resistance gene into a unique pstl site in the thyA gene, in the form of a block of KanR genes ( Pharmacia) (Ficnira 8). This construct was amplified by PCR (Expand ™ High Fidelity PCR system, Boehringer Mannheim) with primers incorporating the Xbal ends, in such a way that it could be transferred to the suicide plasmid pNQ705 [14], which is a carrier of a unique Xbal site and of the chloramphenicol resistance gene. The following primers were used for the PCR amplification of the insertion-inactivated gene: thyA-lp: b'GCT CTA GAG CCT TAG AAG GCG TGG TTCJ 'corresponds to bases 557 to 575 of SEQ ID NO. 2 (figure 2) with an aggregate site Xbal (in bold type) and thyA-11: 5'GCT CTA GAG CTA CGG TCT TGA TTT ACG GTA T3 'corresponding to the complementary sequence of bases 235 to 257 of SEQ ID NO . 2 (figure 3), with an added site Xbal (in bold type) (Figure 9 10).Subsequently, the resulting plasmid was transferred to the strain of E. coli S-17 that was used in the conjugation experiments. Since the recipient strain JS1569 4.4 is resistant to rifampin and sensitive to chloramphenicol, and the donor strain E. coli S-17 is resistant to both chloramphenicol and kanamycin, the transconjugants were selected based on their resistance to both rifampicin like to kanamycin. However, the resulting V. cholerae strains would be resistant to chloramphenicol, since the plasmid
initially it would be inserted into the chromosome. Exconjugants that had incorporated the inactivated thyA gene carrying the block of KanR genes into the chromosome and that had lost the plasmid pNQ705, could then be selected from those that were sensitive to chloramphenicol but remained resistant to kanamycin. To verify insertion of the kanamycin resistance gene into the thyA gene, the full thyA gene was amplified by PCR with primers thyA-10 and thyA-11, and the size of the resulting fragment was compared to that of the native thyA gene. The expected thyA fragment of 2.6 kb was found, compared to the 1.4 kb native thyA gene fragment. Results The exconjugantes showed to be resistant to kanamycin, sensitive to chloramphenicol and when they were amplified by PCR they did not show to have incorporated the gene block of resistance to kanamycin in their chromosome. Sequencing of the amplified fragment showed that only the defect in the gene was due to the insertion of the kanamycin gene. This indicated that the recombination event that had incorporated the gene inactivated by insertion into the chromosome had also eliminated the mutation point that had made the recipient strain (JS1569 4.4) dependent on thymine. The growth of the resulting strain was only observed if the culture medium was supplemented with thymine (200 μg / ml). Partial deletion of the thyA gene and the KanR gene block To further ensure the non-reversible thyA mutation, the insert inactivated thyA was subcloned as an Xbal fragment in the plasmid pNEB 193 (New England Biolabs). PCR primers were designed to eliminate 209 base pairs of the thyA gene and remove 261 base pairs
of the KanR gene block. Thus, the thyA gene was further excised and resistance to kanamycin was also inactivated (by removing the start of the coding region). The overall result of this procedure,
was a carrier strain of a thyA gene that underwent deletion that also contained a non-coding DNA insert. thyA - 14: b'GGG GGC TCG AGG GGC ACA TCA CAT GAA3 'thyA - 15: 5'CCC CCC TCG AGC GCC AGA GTT GTT TCT GAA3' Bold letters indicate the cleavage sites of the Xhol enzyme (Figure eleven).
After PCR amplification, a DNA fragment spanning the entire plasmid was obtained with
exception of the region subject to deletion. The amplified DNA was digested with XhoI, it was ligated and
transformed into E. coli HB101. Colonies were selected on plates containing ampicillin. Individual colonies were selected and replanted. Small-scale plasmid preparations from individual colonies yielded the expected restriction patterns when analyzed with the restriction enzymes Xbal, Xhol, flindlll and rsal. The incomplete thyA gene carrying an inactivated kanamycin resistance gene was cut from the vector
by digestion with Xbal, it was purified and ligated into the pDM4 vector [15] (Figure 12). PDM4 is a suicide vector derived from pNQ705 that contains the sacBR gene of
Bacillus subtilis and a modified multicloning site. After the transfer of plasmid pDM4
(? ThyA? Kan) to the E. coli strain S-17, a transconjugation experiment was performed. This time, the V. cholerae strain JS1569 thyAKan obtained above was used as the recipient strain. The coupling was done in the way
previously described with selection by rifampicin and chloramphenicol. After growing in this medium, the colonies were selected in a medium containing 10% sucrose in the absence of chloramphenicol. Sucrose induces the sacBR gene, which encodes the levansucrase that
transforms sucrose into levan. This compound is
It is toxic to many gram-negative microorganisms. In this way, the clones that were still carriers of the suicide plasmid died leaving only the exconjugants that had lost the plasmid. 5 Results A colony that was sensitive to chloramphenicol and kanamycin was selected. PCR amplification of the thyA region with primers thyA-10 and thyA-11 confirmed that the thyAKan fragment (2.6 kb) in the chromosome had been replaced by the fragment
? ThyA? Kan (2.1 kb). The growth of the resulting strain was also observed if the culture medium was supplemented with thymine 820 μg / ml). This strain was named V. cholerae JS1569? ThyA? Kan. 15 Direct deletion of the thyA gene in V. cholerae. For this procedure, the flanking sequences 5 'and 3' of the thyA gene were used. A new suicide vector pMT SUICIDE-1 was constructed (Figure 14) that contains the origin of replication R6K, the mob genes of
RP4, a chloramphenicol resistance gene and a multichannel site of Litmus 28 (New England Biolabs). In effect, a modified fragment was constructed in which the coding region of thyA was replaced by a multiclonation site (derived from Litmus 28), leaving
Only the 5 'and 3' region of the V. cholerae thyA locus. The resulting plasmid was used to generate a strain of V. cholerae in which the entire thyA gene had been eliminated. The plasmid pMT SUICIDE-1 (M. Lebens, unpublished data) was used as raw material for this construction. From the 5 'and 3' regions of the thyA locus, the following PCR primers were designed:
thyA-31: 'CGG GGT ACC TGG CTT GAT GGG TTT TAT 3' corresponding to bases 22 to 39 of SEQ ID NO. 3 (figure 3) (3 'region of the thyA region) with a Kpnl site
(indicated in bold letters) and thyA-32: 5'GAA GGC CTT CGC CTC TGC TTG CGA CT3 'corresponding to the complementary sequence of the bases
731 to 749 of SEQ ID NO. 3, with a Stul site (indicated in bold type). This pair of primers produces a 746-base PCR fragment corresponding to the 3 'flanking region of the thyA gene.
As template for the PCR reactions, a chromosomal DNA preparation of V. cholerae JS1569 was used (Figure 13). The amplified DNA was digested with the appropriate restriction enzymes and cloned into the vector pMT-SUICIDE-1 (FIGS. 14 and 15), obtaining the plasmid pMT? ThyA V. cholerae containing approximately 700 base pairs of the 5 * chain region above the thyA gene and the same number of base pairs of the 3 'region downstream of the thyA gene. This plasmid was transferred to E. coli strain S17-1 and used in conjugation experiments such as those described above. The strain of V. cholerae JS1569 4.4 was used as the receptor. Couplings were performed on LB agar supplemented with rifampin, chloramphenicol and thymine. The exconjugantes that had lost the suicide plasmid of the chromosome, were selected based on their sensitivity to chloramphenicol. Results A colony sensitive to chloramphenicol and resistant to rifampicin was selected. PCR amplification with thyA-10 and thyA-11 primers from the thyA region resulted in a 1.4 kb fragment of the native thyA gene and a 0.6 kb fragment of the? ThyA gene. This confirmed that the thyA structural gene in the chromosome had been eliminated. In addition, the bacteria could only grow in a culture medium supplemented with thymine. This strain was named V. cholerae JS1569? ThyA. Expression of subunit B of the thermolabile exotoxin of B. coli (LTB) and the sj26 glutathione-S-transferase (GST) of Schistossma japonicvm, in V. cholerae JS1569? thyA. Two expression vectors were constructed, each consisting of the thyA gene of E. coli, the origin of replication of the high copy vector pUC19, the tac promoter and the rho-independent trpA transcription terminator. In one of the two vectors, the laclq gene had been inserted in order to regulate the expression of the tac promoter, which also contained the iac operator sequence (Figures 16 and 17). Expression of the LTB protein in V. cholerae strain JS1569? ThyA.
The expression vector shown in Figure 10 was electroporated in V. cholerae JS1569? ThyA. Transformants were selected on MS agar. Individual colonies were grown to produce 5 minipreparations of plasmids that were verified by restriction enzyme analysis. For expression, a transformant was grown in MS medium at 37 ° C in a shaking culture. The culture medium was harvested and analyzed for the presence of LTB,
using the GMI-ELISA method. Results The culture was found to produce approximately 300 μg / ml of LTB, as measured by GMl ELISA. An SDS-PAGE and a Western blot analysis using specific monoclonal antibodies against LTB
further verified that the secreted protein was LTB. Expression of the GST protein in the V. cholerae strain JS1569? ThyA The sj26 glutathione-S-transferase (GST) of Schistosoma japonicum was cloned into the expression vector shown in Figure 17. This vector is identical to the first, except by the sequence of the gene l¿? clq. The iaclq allows the controlled expression of recombinant proteins. The vector was electroporated in V. cholerae JS1569? ThyA.
Transformants were selected on MS agar. Individual colonies were grown to produce plasmid miniprep, which were analyzed with restriction enzymes. For expression, a transformant was grown in MS medium at 37 ° C in a shaking culture, with the addition of IPTG. Results The recombinant protein was found in the cytoplasm of V. cholerae bacteria. Analysis of SDS-PAGE and Western blot with a monoclonal antibody specific against GST (Pharmacia Biotech, Uppala) confirmed that GST was expressed. The expression level of GST was more difficult to determine than the LTB, since the protein was expressed intracellularly, but it was judged to be in the same range as the LTB. References 1. Molin, S., K. A. Gerdes. 1984. Stabilized plasmids. US Patent 4,760,022. 2. Morona, R., and S. R. Attridge. 1987. Non-antibiotic marker system. EPC-A-0251579. 3. Green, J. M., B.P. Nichols, and R. G. Matthews. nineteen ninety six.
Folate biosynthesis, reduction and polyglutamylation.
In: F. C. Neidhardt, R. Curtiss III, J. L. Ingraha, E.
C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M.
Riley, M. Schaechter and H. E. Umbarger (Eds.) Escherichia coli and Salmonella cellular and molecular biology. ASM Press Washington D. C. pp 665-673. Neill, R. J., B. E. Ivins, and R. K. Holmes. 1983. Synthesis and secretions of the plasmid-coded heat-labile enterotoxin of Escherichia coli in Vibrio cholerae. Science. 221: 289-290. Sandkvist, M., M. Bagdasarian, S. P. Howard, and V. J. DiRita, 1995. Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is reguired for extracellular secretion in Vibrio cholerae. EMBO J. 14: 1664-1673. Newuhard, J. and R. A. Kelln. 1996. Biosynthesis and conversions of pyrimidines. Jn: F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter and H. E. Umbarger (Eds.) Escherichia coli and Salmonella cellular and molecular biology. ASM Press Washington D.C. pp. 580-599. Kaper, J. B., H. Lockman, M. M. Baldini, and M. M. Levine. 1984. A recombinant live oral cholera vaccine. Biotechnology 2: 345-349. Sánchez, J., and J. Holmgren. 1989. Reco binant system for overexpression of cholera toxin B subunit in Vibrio cholerae as a basis for vaccine development. Proc. Nati Acad. Sci. USA 86: 481-485. Wilson, K. 1994. Preparation of genomic DNA from Bacteria. In Current protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J. G. Seidman, J.A. Smith, and K. Struhl, eds.) Pp. 2.4.1-2.4.2 John Wiley & Sons, New York. 10. Sheen, J. 1994. High-efficency transformation by electroporation. In Current protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J. G. Seidman, J.A. Smith, and K. Struhl, eds.) Pp. 1.8.4-1.8.5. John Wiley & Sons, New York. 11. Lebens, M., S. Johansson, J. Osek., M. Lindblad and J. Holmgren. 1993. Large-scale production of Vibrio cholerae toxin B subunits for use in oral vaccines. Biotechnology. 11: 1574-1578. 12. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA seguencing with chain-terminating inhibitors. Proc.
Nati Acad. Sci. USA 74: 5463-5467. 13. Program Manual for the Wisconsin Package. Version 8. September 1994. Genetics Computer Group, 575 Science Drive, Madison Wisconsin. 14. Milton, D.L., R. OsToole, P. Hógstedt, and H. Wolf-Watz. 1996. Flagellin A is essential for the virulence of Vibrio anguillarum J. Bacteriol. 176: 1310-1319. 16. Belfort, J., G. Maley, J. Pedersen-Lane and F. Maley. 1983. Primary tructure of the Escherichia coli thyA gene and its thymidylate synthase product. Proc. Nati Acad. Sci. USA 80: 4914-4918. 17. Fleischmann, RD, Adams, MD, White, 0., Clayton, RA, Kirkness, EF, Kerlavage, AR, Bult, CJ, Tomb, JF, Dougherty, BA, Merrick, JM, McKenney, K., Sutton, G., FitzHugh, W., Fileds, CA, Gocayne, JD, Scott, JD, Shirely, R., Liu, LI., Glodek, A., Kelley, JM Weidman, JF, Phillips, CA, Spriggs, T., Hedblom, E., Cotton, MD, Utterback, TR, Hanna, MC, Nguyen, DT, Saudek, DM, Brandon, RC, Fine, LD, Fritchman, JL, Fuhrmann, JL, Geoghagen NSM, Gnehm, CL, McDonald, LA, Small, KV, Fraser, CM, Smith, HO, and JC Venter. 1995. Whole-genome random sequencing and assembly of Hae ophilus influenzae RD. Science 269: 496-512. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.
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< 110 > SBL Vaccine AB < 120 > METHOD TO PRODUCE STRAA "STRAINS FROM Vibrio cholerae,
SUCH STRAINS AND YOUR USE < 130 > 29772 < 150 > SE 9801852-6 < 151 > 1998-05-26 < 160 > 5 < 170 > PatentIn Ver. 2.1 < 210 > 1 < 211 > 2909 < 212 > DNA < 213 > Vibrio cholerae < 400 > 1 gagaaggttt gttatgcctc agggttatct gcagtttccc aatatcgacc ccgtattgtt 60 ttcgatcggc cctctagcgg tgcgctggta tggcttgatg tatttggtgg gtttectttt 120 tgctatgtgg ttggccaatc gcegagcgga tcgegcgggc agtggttgga cgcgtgagca 180 AGTC etgae ttg tattcg ccggettttt aggtgtagtg atcggtggcc gagttggtta 240 tgtgatcttc tacaattttg atctgttcct tgctgaccct ctttatttat tcaaag gtg 300 gactggcggc atgtccttcc acggcggctt attgggegtg atcaccgcca tgttctggta 360 aaccaacgca egcgegtaaa ccttctttgg tgtggccgat TTTG tgccc ctttag gcc "20 attcggtttg gggatgggac gtatcggtaa ctttatgaat aggaacttt gggga gagt 480 aacggatgtg ccttgggctt ttgtattccc taatggtggc ccactgccgc GCCA ccttc 540 acagctttat gaattcgcct tagaaggcgt gg tctgttc tttattctta attggtttat 600 tggtaaacct cgtccgctag gcagcgtatc cggactgttt ttagctggat acggtacatt 660 ccgcttcctt gtggaatacg tccgtgagce agatgc cag ttgggtctgt ttggtggctt 720 catttcaatg gggcaaatcc tctccttacc tatggtgatc ateggtattt tgatgatggt 7B0 ttggtcttac aagcgcgg t tg atcaaga ccggtagca gcaaaatagg g agttaggt 840 gaaacagtat ttagatet tt gtcagegcat cgtcgatcaa ggtgtttggg ttgaaaatga 900 acgaacgggc aagcgttgtt tgactgtgat taatgccgat ttgacctacg atgtgggcaa 960 cctctagtga caatcagttt ctacacgcaa gagtttttgg aaagctgccg tagccgagtt 1020 gctcggctat attcgtggtt acgataatgc ggcggatttt cgccaattag gtaccaaaac 1080 ctgggatgct aatgccaatt taaaccaagc atggctcaac aatccttacc gtaaaggtga 1140 ggatgacatg ggacgcgtgt atggtgttca gggtagagct tgggctaagc ctgatggtgg 1200 cagttgaaaa tcatattgac agattgttga tgatttgage cgtggcgttg atgaccgagg 1260 aacttctaca tgaaattctt atccgggtga atttcacatg gggtgt tgc gcccttgcat 1320 gtacagccat catttttcat tgctggggga taccttgtat ctcaacagta ctcagcgttc 1380 atgtgatgtg cccttggggt tgaatttcaa catggtgcag gtttatgtgt tccttgcgct 1440 gatggcacag atcacaggga aaaagccggg cttggcgtat cacaagatcg tcaatgcgca 1500 catttaccaa gatcaactcg aattgatgcg cgatgtgcag ctaaaacgtg agccattccc 1560 agcgcctcag ttccatatca atccaaagat taaaacactg caggatttgg aaacttgggt 1620 cactttggat gattttgacg tcaccggata tcagttccac gatcctattc aatacccgtt 1680 ttcagtctaa tcccgtattc aggcg gtatg gcttgatggg ttttatataa aaaaagctcc 1740 cgaaggtcgg gagctttttt tatacagatg atgctttaac gcttaagcgg ttagggcaag 1800 aatgctgccg gggatgacga caaacacacc caataagtaa ctcaccacca ccattttgct 1860 caagttgaga cttacaagcc tgagctcagc acctttaata ggcagttcgc gtaagaaagg 1920 aataccgtaa atcaagaccg tagccatcaa gttaaagctt aagtgcacca gcgcaatttg 1980 cagagcaaac acggcaaact caccagagac agcggttgcg gcgagcagag cagtaa aca 2040 agtgccaatg ttcgcaccta aggtaaatgg cgcactttca gtagatttca gcacgccaga 2100 ggaaccatta gcccacgaga ggtcgatgaa ggctggttgt gattgaacta ataccgtaac 2160 cactgtacct gaagcaatac cgtgtagtgg gcctcggcca atcgcatttt gtagaatttc 2220 ccaaccatca acgtgcgcgg aactcttcat cagtttgccc tggcgacgaa atcaccgtaa 2280 atacccaata aatggtcgca cgataagtgc gacaccaccg aaagtattac ccaataccga 2340 aagctgggtt tcaagccctg tgatgacagg tttggtaatc ggtttgataa aatcaaaacc 2400 tttcatgctc atatcgccag tcgcaagcag aggcgaaacg agccagtgtg agactttctc 2460 taaaatgcca aacatcattt ctagaggtag gaagatcagc accgcgagaa gattgaaaaa 2520 atcgtggatg gtggcactgg egaaagcacg gcgaaactct tctttacagc gcatatggcc 2580 aaggctgacg agagtattgg tcacagtagt accaatattg gcacccatca ccataggaat 2640 cgcggtttca accggtaacc caccggcaac gagac caaca ataatagaag tcaccgtgct 2700 tgaggattga atcagtgccg ttgccactaa accaatcatc aatcctgcaa ttgggtggga 2760 agcaaattca aatagaactt tggcttgatc gccggttgcc catttaaaac cgctgccgac 2820 catcgcgact gcaagaagta gtaaatacag catgaaagcc aagtttgccc aacgtaggcc 2880 tttcgtggtc agcgaaatcg gcgctgcag 2909
< 210 > 2 < 211 > 838 < 212 > DNA < 213 > Vibrio cholerae < 400 > 2 gagaaggttt gttatgcctc agggttatct gcagtttccc aatattgacc ccgtattgtt 60 ttcgatcggc cctctagcgg tgcgctggta tggcttgatg tatttggtgg gtttcctttt 120 tgctatgtgg ttggccaatc gccgagcgga tcgcgcgggc agtggttgga cgcgtgagca 180 agtctctgac ttgttattcg ccggcttttt aggtgtagtg atcggtggcc gagttggtta 240 tgtgatcttc tacaattttg atctgttcct tgctgaccct ctttatttat tcaaagtgtg 300 gactggcggc atgtccttcc acggcggctt attgggtgtg atcaccgcca tgttctggta 360 aaccaacgca tgcgcgtaaa ccttctttgg tgtggccgat tttgttgccc ctttagtgcc 420 attcggtttg gggatgggac gtatcggtaa ctttatgaat agtgaacttt ggggacgagt 480 aacggatgtg ccttgggctt ttgtattccc taatggtggc ccactgccgc gccatccttc 540 acagctttat gaattcgcct tagaaggcgt ggttctgttc tttattctta attggtttat 600 tggtaaacct cgtccgctag gcagcgtatc cggactgttt ttagctggat acggtacatt 660 ccgcttcctt gtggaatacg tccgtgagcc agatgctcag ttgggtctgt ttggtggctt 720 catttcaatg gggcaaatcc tctccttacc tatggtgatc atcggtattt tgatgatggt 780 ttggtcttac aagcgcggtt tgtatcaaga ccgtgtagca gcaaaatagg gtagttag 838
< 210 > 3 < 211 > 1222 < 212 > DNA < 213 > Vibrio cholerae < 400 > 3 taatcccgta ttcaggcggt atggcttgat gggttttata taaaaaaagc tcccgaaggt 60 cgggagcttt ttttatacag atgatgcttt aacgcttaag cggttagggc aagaatgctg 120 ccggggatga cgacaaacac acccaataag taactcacca ccaccatttt gctcttacaa 180 gcccaagttg agatgagctc agcaccttta ataggcagtt cgcgtaagaa aggaataccg 240 ccgtagccat taaatcaaga cttaagtgca caagttaaag ttgcagagca ccagcgcaat 300 aetcaccaga aacacggcaa gacagcggtt gcggcgagca gagcagtaat acaagtgcca 360 atgttcgcac ctaaggtaaa tgggtagatt tcaegcactt tcagcacgcc agagcccacg 420 agaggaacca ttaggctggt tgtggtcgat gaagattgaa ctaataccgt aaccactgta 480 cctgaagcaa taccgtgtag tgggcctcgg ccaatcgcat tttgtagaat ttcacgtgcg 540 cggccaacca tcaaactctt eatcagtttg eccatcaccg taatggcgac gaaaatggte 600 gcaataccca atacgataag tgcgacacca ccgaaagtat tacccaatac cgaaagctgg 660 gtttcaagcc ctgtgatgac aggtttggta atcggtttga taaaatcaaa acctttcatg 720 ctcatatcgc cagtcgcaag cagaggcgaa acgagccagt gtgagacttt ctctaaaatg 780 ccaaacatcá tttctagagg taggaagatc agcaccgcga gaagattgaa aaaatcgtgg 840 atggtggcac tggcgaa agc acggcgaaac tcttctttac agcgcatatg gccaaggctg 900 acgagagtat tggtcacagt agtaccaata ttggcaccca tcaccatagg aatcgcggtt 960 tcaaccggta acccaccggc aacgagacca acaataatag aagtcaccgt gcttgaggat 1020 tgaatcagtg ccgttgccac taaaccaatc atcaatcctg caattgggtg ggaagcaaat 1080 tcaaatagaa ctttggcttg atcgccggtt gcccatttaa aaccgctgcc gaccatcgcg 1140 gtagtaaata actgcaagaa cagcatgaaa gccaagtttg eccaacgtag gcctttcgtg 1200 gtcagcgaaa tcggcgctgc ag 1222 < 210 > 4 < 211 > 283 < 212 > PRT < 213 > Vibrio cholerae < 400 > 4 Val Lys Oln Tyr Leu Asp Leu Cys Gln Arg He Val? Sp Gn Gly Val 1 5 10 15 Trp Val Olu? Sn Olu? Rg Thr Oly Lys Arg Cys Leu Thr Val lie Asn 20 25 30 Wing Aßp Leu Thr Tyr? Sp Val Gly? Sn? Sn Gln Fhe Pro Leu val Thr 35 40 45 Thr? Rg Lys Ser Phe Trp Lys Ala Ala Val Ala ßlu Leu Leu (ily Tyr 50 55 60 He? Rg Gly Tyr Aßp Asa Ala Ala? Sp Phe? Rg Gln Leu Gly Thr Lys 65 70 75 80
Tbr Trp? ßp? A? Sn Wing? Sn Leu Aan ßln Wing Trp Leu? Sn? Sn Pro 85 '90 95 Tyr? Rg Lys Gly Olu? ßp? Sp Met Gly? Rg Val Tyr Gly Val ßln Gly 100 105 110? rg? the Trp? the Lys Pro? ßp ß and ßly Bis He? ßp Gln Leu Lys Lyß 115 120 125 He Val? ßp? ßp Leu Ser? rg Gly Val? ßp? ßp? rg Gly Glu He Leu 130 135 140? ßn Phe Tyr Aßn Pro Gly Glu Phe Hiß Met Gly Cyß Leu? Rg Pro Cyß 145 150 155 160
Met Tyr Ser Hiß Hiß Phe Ser Leu Leu Gly? ßp Thr Leu Tyr Leu? ßn 165 170 175 Ser Thr ßln? Rg Ser Cys Aßp Val Pro Leu ßly Leu Aßn Phe Aßn Met 180 185 190 Val ßln Val Tyr Val Phe Leu Ala Leu Met Ala Oln He Thr ßly Lyß 195 200 205 Lyß Pro ßly Leu? The Tyr Hiß Lyß He Val? ßn? The Hiß He Tyr ßln 210 215 220 Aßp ßln Leu ßlu Leu Met? Rg? Sp Val Oln Leu Lys Arg ßlu Pro Phe 225 230 235 240
Pro Ala Pro ßln Phe His He? Sn Pro Lys He Lys Thr Leu Oln? Sp 245 250 255 Leu aiu Thr Trp Val Thr Leu? Sp? ßp Phe? Sp Val Thr ßly Tyr ßln 260 265 270 Phe Hiß? ßp Pro He ßln Tyr Pro Phe Ser Val 275 280
^^^^^^^^ faith? ^^^^ < 210 > 5 < 211 > 271 < 212 > PRT < 213 > Vibrio cholerae < 400 > 5 Met Pro Oln Oly Tyr Leu Oln Phe Pro? An He? ßp Pro Val Leu Phe 1 5 10 15 Ser He Oly Pro Leu Ala Val? Rg Trp Tyr-Oly Leu Met Tyr Leu Val 20 25 30 Gly Pbe Leu Fhe? La Met Trp Leu? La? Sn? Rg? Rg? La? Sp Arg? La 35 40 45 Gly Ser Gly Trp Thr? Rg Glu ßln Val Ser? ßp Leu Leu Phe Ala ßly 50 55 60 Phe Leu Gly Val Val He Gly Gly ? rg val Gly Tyr val He l? he Tyr 65 70 75 80? sn Pbe? ßp Leu Phe Leu? la? ßp Pro Leu Tyr Leu Phe Lyss Val Trp 85 90 95 Thr ßly ßly Het Ser Phe Hiß ßly ßly Leu Leu ßly Val He Thr? La 100 105 110 Met Phe Trp Tyr? La? Rg Lyß? ßn ßln? Rg Thr Phe Phe ßly Val? 115 120 125? ßp Phe Val? Pro Leu Val Pro Phe ßly Leu ßly Met ßly Arg He 130 135 140 ßly? ßn Phe Met? ßn Ser ßlu Leu Trp ßly? Rg Val Thr? Sp Val Pro 145 150 155 160 Trp Wing Phe Val Phe Pro Aßn ßly ßly Pro Leu Pro Arg His Pro Ser 165 170 175 ßln Leu Tyr ßlu Phe? The Leu Olu Oly Val Val Leu Phe Phe He Leu 180 185 190 Asn Trp Phe He Oly Lyß Pro Arg Pro Leu Gly Ser- Va l Ser ßly Leu 195 200 '205 Phe Leu? the ßly Tyr ßly Thr Phe? rg Phe Leu Val Glu Tyr Val? rg 210 215 220 ßlu Pro? ßp? the ßln Leu ßly Leu Phe ßly ßly and Phe He 3er Met Gly 225 230 235 240 ßln He Leu 8er Leu Pro Met Val He He ßly He Leu Met Met Val 245 250 255 Trp Ser Tyr Lyß? Rg ßly Leu Tyr ßln? ßp? Rg Val? The? Lyß 260 265 270
* O *
Claims (16)
- - 33 - CLAIMS Having described the invention as an antecedent, the content of the following claims is claimed as property: 1. A method to produce a strain thyA "of Vibrio cholerae, characterized in that it comprises the step of performing site-directed mutagenesis on the chromosome of V. cholerae for the deletion and / or insertion of gene nucleotides at the thyA gene locus having essentially the nucleotide sequence of SEQ ID NO : 1 of figure 1.
- 2. A strain thyA "of Vibrio cholerae characterized because it is a strain? ThyA lacking the functionality of the thyA gene
- 3. A strain? ThyA of Vibrio cholerae according to claim 2, characterized in that comprises one or more autonomously replicating episomal DNA elements having a functional thyA gene, which makes it possible for the strain to grow in the absence of thymine in the culture medium
- 4. A strain of Vibrio cholerae thyA according to claim 3 , characterized in that the episomal DNA element of autonomous replication is a plasmid. - 3. 4 -
- 5. A strain thyA of Vibrio cholerae according to any of claims 3 or 4, characterized in that it comprises a foreign thyA gene.
- 6. A strain? ThyA of Vibrio cholerae according to claim 5, characterized in that the foreign thyA gene is an E. coli gene.
- 7. A strain? ThyA of Vibrio cholerae according to any of claims 3 to 6, characterized in that the one or more episomal DNA elements of autonomous replication can also comprise a structural gene coding for an homologous or heterologous protein.
- 8. A strain? ThyA of Vibrio cholerae according to claim 7, characterized in that the encoded protein is selected from the group consisting of the subunit B of the thermolabile enterotoxin of Escherichia coli (LTB) and the glutathione-S-transferase protein of 26 kD (GST 26 kD) of Schistosoma japonicum.
- 9. A nucleotide sequence of a thyA gene of Vibrio cholerae characterized in that it has essentially the nucleotide sequence of SEQ ID NO: 1 of Figure 1.
- 10. A nucleotide sequence of a 5 'flanking region of a thyA gene structure of Vibrio cholerae characterized in that it essentially has the sequence of -35-nucleotides of SEQ ID NO: 2 of Figure 2.
- 11. A nucleotide sequence of a 3 'flanking region of a structural thyA gene of Vibrio cholerae characterized by has essentially the nucleotide sequence of SEQ ID NO: 3 of Figure 3-
- 12. A protein characterized in that it is encoded by a nucleotide sequence of a thyA gene of Vibrio cholerae according to claim 9.
- 13. A protein of according to claim 12, characterized in that it has the amino acid sequence of SEQ ID NO: 4 of Figure 4.
- 14. A protein characterized in that it is encoded by a sequence of nucleotides of a 5 'flanking region of a structural thyA gene of Vibrio cholerae according to claim 10.
- 15. A protein according to claim 14, characterized in that the protein has the amino acid sequence of SEQ ID NO: 5. of Figure 5.
- 16. A vaccine characterized in that it comprises as an immunizing component a strain? thyA of Vibrio cholerae according to any of claims 2 to 8 or a strain thyA "of Vibrio cholerae produced by the method according to the claim 1.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE9801852-6 | 1998-05-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA00011604A true MXPA00011604A (en) | 2002-07-25 |
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