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Moreover, when the vector is lost, the antidote is degraded by a protease E-Tech PSP301 the induced poison production causes death of the host cell. Said E-Tech PSP301 encoding the ccdA protein is functionally linked to a first promoter. Any constitutive promoter conventionally used in vectors may be used in the context of the present invention. A noninducible promoter will advantageously be used. It is not necessary to use a strong promoter.


Advantageously, the mob constitutive promoter is used. The vector according to the invention also comprises the sequence of the E-Tech PSP301 locus.

Said sequence can be inserted into the vector in any orientation and at any position. Said Cer sequence is described E-Tech PSP301 Summers D.

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The vector according to the invention also comprises a heterologous sequence functionally linked to a second promoter. By way of examples of therapeutic proteins, mention may in particular be made of: By way of examples of a vaccine antigen, mention may be made of proteins that can be used in an immunization program and also genes that can be used in the context of a DNA vaccination, in particular surface proteins of a bacterial pathogen but also viral envelope proteins, parasite proteins or alternatively surface markers of tumor cells. By way of example of sequences that can be used in the context of a gene therapy, mention may be made of the sequences that can be used in the context of the treatment of genetic diseases such as cystic fibrosis.

A heterologous sequence encoding an enzyme, for example an enzyme of industrial interest such as benzonase, trypsin or alternatively a molecule capable of intervening in a biocatalytic process can be inserted into a self-replicating vector according to the invention. More specifically, mention may be made, by way of nonlimiting example of a vaccine antigen that can be produced with the vector according to the invention, of: Pseudomonas aeruginosa E-Tech PSP301 A detoxified, for example, by deletion of the Glu residue rEPAthe tbpB transferin binding protein antigen of N.

The sequences encoding these proteins are known and available on various databases. For example, the complete sequence of the LSA 3 gene is base pairs long and encodes a protein of amino acids. The system according to the invention is particularly suitable for the production of E-Tech PSP301 for which glycosylation is absent or not essential for their effectiveness or for the induction of an immune response; on the other hand, sequences encoding glycosylated proteins may be used, for example, in DNA vaccination.

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Any constitutive or inducible promoter conventionally used in vectors can be used in the context of the present invention as second promoter. By way of example, mention may be made of the T7, T5, arabinose, lac, Trp promoters or any other promoter derived from microorganisms capable of functioning in a prokaryotic host cell, in particular Escherichia coli. In the case of vectors used for gene therapy or DNA immunization, the expression of the gene of interest may be under the control of a promoter which functions in eukaryotic cells, such as the CMV promoter or the SV40 promoter or alternatively promoters having a cell specificity.

The vector according to the invention may also contain expression-regulating sequences such as, for example, a transcription-regulating terminator sequence, a signal sequence which allows the exportation of the expressed E-Tech PSP301 to the periplasmic compartment of the host cell or the secretion of said protein into the culture supernatant of the host cells, and also a multiple cloning site. These sequences are well known to those skilled in the art. The vector according to the invention may be constructed by any conventional genetic engineering technique.


The elimination of the antibiotic-resistance gene can subsequently be carried out by simple digestion with a restriction enzyme at unique sites, flanking the antibiotic-resistance gene, and then religation of the vector on itself. A means of verifying the elimination of said gene then consists in subculturing the colonies obtained, after transformation, in parallel, on dishes containing or not containing the antibiotic, or alternatively in verifying the absence of said gene by PCR, restriction mapping or any other appropriate method. In any event, this additional verification proves to be tedious. The inventors have demonstrated another system for constructing the vector according to the invention, which has the advantage of resulting in easy selection of the clones sought.

According to a second aspect, a subject of E-Tech PSP301 present invention is therefore a method for producing a self-replicating vector as defined above, comprising the steps of: The new method according to the invention uses a recombination event which makes it possible, in a one and only step, to eliminate the antibiotic-resistance gene and to assemble a ccdA gene in its functional form. In summary, the ccdA gene is cloned in the form of 2 separate and individually nonfunctional elements located on either side of the antibiotic-resistance gene.

As long as the antibiotic-resistance gene is present, the ccdA assembly is nonfunctional. After digestion with a restriction enzyme having a site located only between the sequences 1 and 2, for example within E-Tech PSP301 antibiotic-resistance gene, a linear DNA fragment is obtained.

Through transfection of prokaryotic cells, for example of competent E. This step makes it possible, on the one hand, to eliminate the antibiotic-resistance gene and, on the other hand, to select the clones containing E-Tech PSP301 vector according to the invention, insofar as the ccdA gene can be functional only after elimination of the antibiotic-resistance gene.

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A schematic representation of the method according to the invention is given in FIG. For the implementation of the method above, reference may be made to a reference book in terms of genetic engineering, such as Maniatis et al. Russell, which describes all the operating conditions to be used for E-Tech PSP301 steps for construction of a vector, linearization, transformation and recovery of the clones. The self-replicating vector can subsequently be readily recovered from the transformed cells by any conventional method well known to those skilled in the art.

As regards in E-Tech PSP301 the homologous recombination, the size of the overlapping sequence can vary to a large extent. Sequences which overlap over only 5 nucleotides can be used.

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The only important element in the selection of the sequences 1 and 2 is that the two sequences, after homologous recombination, reconstitute a functional ccdA sequence, for example the complete ccdA sequence. Furthermore, E-Tech PSP301 should flank the antibiotic-resistance gene so as to allow elimination of the latter. Any self-replicating vector as defined above can be advantageously produced by the new method according to the invention. According to another aspect, the present invention therefore relates to a prokaryotic cell containing a self-replicating E-Tech PSP301 as defined above. Any prokaryotic cell expressing a functional ccdB protein can be used in the context of the present invention. A ccdB protein is functional if it is capable of significantly inhibiting the action of gyrase, i.

It is therefore possible to use the whole ccdB protein or fragments thereof which are capable of bringing about cell death in the absence of ccdA protein. The insertion of the ccdB sequence into the bacterial genome can be carried out by any method well known to those skilled in the art for performing such an insertion. E-Technology Systems Private Limited A premier provider of IT Supplies and Services incorporated in Missing: PSP Choose peak performance with the Evinrude E-TEC outboard motor, available from 25 HP to HP with high output options and our popular 90 HP g: PSP

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