KR101660294B1 - Novel bacterial vector system based on glmS - Google Patents

Abstract

The present invention provides attenuated Gram negative bacteria in which the glmS gene has been deleted for the in vivo delivery of an efficient therapeutic gene using bacteria.

Description

A glmS-based host vector system {Novel bacterial vector system based on glmS}

The present invention relates to a novel host vector system, and more particularly to a host vector system based on glmS .

Cancer is defined as the unregulated and invasive growth of cells. Conventional chemotherapy, including surgical resection, radiation therapy and chemotherapy, is effective in the treatment of many complaints, but about half of cancer patients do not benefit from these traditional therapies. Accordingly, alternative techniques have been developed as medical therapies which are claimed to be able to treat cancer by treating, ameliorating, supplementing, or replacing traditional methods. Among them, some bacterial species have been reported to prefer proliferation and accumulation in tumors, and also enable design to provide specific therapeutic proteins directly at the tumor site. Moreover, because bacteria have advantageous properties, including the ability to simultaneously retain and express multiple therapeutic proteins, and because they can be easily removed by antibiotics, (Nauts et al ., Acta Med. Scand. Suppl ., 276: 1-103, 1953). In addition, there is an attempt to directly use live attenuated or genetically modified non-pathogenic bacteria to kill tumor cells, or to use the bacteria as a cancer drug by transferring tumor-killing molecules to cancer tissues, (Forbes, Nat. Biotechnol ., 24: 1484-1485, 2006). Among them, Salmonella typhimurium ( S. typhimurium ) is an invasive gram-negative bacterium with mobility and can be clustered in solid tumors (Pawelek et al ., Cancer Res ., 57: 4537-4544, 1997). In vitro, chemotactic compounds produced by recurrent tumor cells have been shown to induce this effect (Kasinskas and Forbes, Biotechnol. Bioeng ., 94: 710-721, 2006). In vivo, salmonella has been shown to clusters primarily in nourishment-rich necrotic areas (Forbes et al ., Cancer Res ., 63: 5188-93, 2003). Salmonella has also been successfully used to deliver chemotherapeutic molecules such as prodrug-converting enzymes and antigens to tumors (King et al ., Hum. Gene Ther ., 13: 1225-1233, 2002).

However, since the conventional plasmid carrying the therapeutic gene is not essential for the survival of the bacteria, the conventional bacteria for gene therapy for anticancer therapy has a problem that when the plasmid-transformed bacteria is put into the living body, The plasmid is lost in the proliferation process, and thus the expression efficiency of the therapeutic gene is deteriorated over time.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems and it is an object of the present invention to provide a gene carrier capable of preventing loss of therapeutic gene in vivo. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the invention, glmS gene is deleted attenuated gram-negative selection markers (selection marker) to the glmS gene and a transformant introduced a plasmid comprising a foreign gene operably linked to a promoter transformed attenuated gram-negative bacteria in There is provided a composition for preventing the destruction of the plasmid in the transformed attenuated gram-negative bacterium which is administered into an animal body without using an antibiotic containing as an active ingredient.

In this composition, the attenuation is selected from the group consisting of pab, nadA, pncB, pmi, rpsL, ompR, htrA, hemA, rfc, poxA, galU, aro , galE , cya , cyp, crp , cdt , pur , phoP, , guaA, guaB, clpP, clpX fliD, flgK, flgL, relA or spoT gene.

In such a composition, the attenuated gram-negative bacteria are selected from the group consisting of Salmonella spp., Helicobacter spp., Shigella spp., Enterobacter spp., Serratia spp. , Stenotrophomonas spp., Bdellovibrio spp., Legionella spp., Pseudomonas spp., Moraxella spp., Proteus spp. It may belong to the genus Proteus spp., Escherichia spp. Or Yersinia spp.

According to another aspect of the present invention, there is provided a therapeutic or imaging gene delivery composition comprising an attenuated Gram-negative bacillus in which the glmS gene is deleted as an active ingredient.

In the above composition for therapeutic gene delivery, the above-mentioned attenuated gram-negative bacteria are as described above.

According to a further aspect of the invention, glmS gene is deleted attenuated selection markers (selection marker) in the transformed introducing a plasmid containing the gene coding for the anti-cancer protein operably linked to a glmS gene and the promoter transfected in gram-negative bacteria An anticancer agent containing an attenuated gram negative bacterium as an effective ingredient is provided.

In the anticancer agent, the attenuated gram negative bacteria are as described above.

In the anticancer agent, the promoter may be a homeostatic promoter or an inducible promoter, and the inducible promoter may be a P _BAD promoter. The anti-cancer protein may be a protein toxin, an antibody specific for a cancer antigen, a fragment of the antibody, a tumor suppressor gene, or an antiangiogenic factor. At this time, the protein toxin is a botulinum toxin (Botulinum toxin), Te tanuseu toxin (Tetanus toxin), Shiga toxin (Shiga toxin), diphtheria toxin (Diphtheria toxin, DT), ricin (ricin), Pseudomonas exotoxin (Pseudomonas exotoxin, PE ), Cytolysin A (ClyA), and r-Gelonin. The tumor suppressor gene is a gene that inhibits the development of tumors. Examples of the tumor suppressor gene include VHL (von Hippel Lindau), APC (Adenomatous polyposis coli), CD95 (cluster of differentiation 95), ST5 (Suppression of tumorigenicity 5), YPEL3 ), p53, ST7 (Suppression of tumorigenicity 7) and ST14 (Suppression of tumorigenicity 14). The anti-angiogenic proteins include anti-VEGF antibody, angiostatin, endostatin, and the Kringle V domain of apolipoprotein.

According to a further aspect of the invention, glmS gene is deleted attenuated gram-negative selection markers (selection marker) to the glmS gene and a transformant introduced with a plasmid including the foreign gene operably linked to a promoter transformed attenuated gram-negative bacteria in A method for inhibiting the loss of said plasmid in said transformed attenuated Gram-negative bacteria administered to an animal comprising administering to said animal an effective amount of said plasmid,

In the above method, the attenuated gram-negative bacteria and the promoter are as described above.

In the above method, the foreign gene may be a gene encoding a therapeutic gene, a coding gene, or an antigen protein.

The composition of the present invention may be administered into a body by a method commonly used in the technical field of the present invention. Such administration methods may include oral administration, intravenous administration, intramuscular administration, intratumoral administration, transdermal administration, intranasal administration, gastrointestinal injection, inhalation by aerosol spray, eye drops administration and the like.

In addition, the effective dose of the composition of the present invention can be adjusted depending on the age, sex, severity of disease, etc. of the individual, and can be generally determined in the range of 0.0001 g / kg to 100 mg / kg.

According to another aspect of the invention, glmS transfected gene is attenuated selection marker for gram-negative bacteria (selection marker) is a plasmid containing the gene coding for the antigen protein operably linked to a glmS gene and the promoter transfected with the deletion introduced There is provided a vaccine composition containing the converted attenuated gram-negative bacteria as an effective ingredient.

In the vaccine composition, the antigen protein may be an immunogenic protein derived from bacteria, fungi, parasites or viruses. Thus, the vaccine composition may be effective in the treatment of infectious diseases caused by infection of bacteria, fungi, parasites or viruses, where local immunity is important and may be the primary line of defense. Examples of such vaccines include pneumonic plague caused by Yersinia pestis , gonorrhea caused by Neisseria gonorrhoeae , Treponema pallidum , Such as syphilis and other sexually transmitted diseases caused by Clamydia trachomatis , as well as ocular infections caused by Clamydia trachomatis . Streptococci belonging to Groups A and B may cause sore throat or heart disease and may be caused by Neisseria meningitidis , Mycoplasma pneumoniae , Hemophilus influenza , Bordetella pertussis , Mycobacterium tuberculosis , Mycobacterium leprae , Bordetella avium , Escherichia coli , and the like. , a bacterial genus Streptococcus ekuyi (Streptococcus equi), Streptococcus pneumoniae (Streptococcus pneumoniae), brucellosis Abo tooth (Brucella abortus), par stew Relais H. Emory Utica (Pasteurella hemolytica), Vibrio cholera (Vibrio cholera), Shigella ( also Shigella species), and Legionella pneumophila (Legionella pneumophila) included in the prevention target bacteria of the vaccine composition of the invention Can. In addition, in the case of an antiviral vaccine, the subject virus may be a DNA virus or an RNA virus and may be, for example, an influenza virus, papovirus, adenovirus, herpesvirus, Poxvirus, parvovirus, reovirus, picovirus, mixovirus, paramyxovirus or retrovirus. The term " virus "

In addition, the immunogenic component of the vaccine composition may be an allergen source of the subject that can be used in an exposure regimen designed to specifically desensitize an individual exhibiting allergic symptoms.

The vaccine composition may be administered to the animal by known or standard techniques. Such administration includes oral administration, gastric intubation, and bronchial-nasal spray. Of these methods, any of the vaccine compositions of the present invention that can reach gut-assocated lymphoid tissue (BALT) or bronchus-associated lymphoid tissue (BALT) cells to induce antibody formation are preferred in the present invention.

Other methods of administration, such as intravenous injection, may also be used to deliver the delivery microorganisms into the blood vessels of an individual. Intravenous, intramuscular or intramammary injections may also be used in other embodiments of the present invention.

According to one embodiment of the present invention as described above, it is possible to realize a novel host vector system which reduces the disappearance of the fibrilloid containing the therapeutic gene introduced into the bacteria to thereby increase the therapeutic effect. Of course, the scope of the present invention is not limited by these effects.

1 is a graph showing the growth of glmS ^- mutant E. coli at various conditions of culture, according to one embodiment of the present invention:
○: 0.2% N- acetyl -D- glucosamine (N-acetyl-D-glucosamine , GlcNAc) supplemented LB overnight and then cultured in a culture medium, glmS for 24 hours was diluted 50-fold in minimal medium cultures ^- mutant E. coli (CKS1001) ;
●: glmS ^- mutant Escherichia coli harboring the E. coli glmS ⁺ plasmid cultured overnight in LB medium supplemented with 0.2% GlcNAc and diluted 50 times with minimal medium and incubated for 24 hours;
?: GlmS ^- mutant Escherichia coli cultured overnight in 0.2% GlcNAc supplemented LB medium and cultured in 0.2% GlcNAc supplemented LB medium for 24 hours; And
▲: Wild-type parent strain ( E. coli , CH1436) cultured in a minimal medium for 24 hours.
2 is φhdeABp overnight incubation in 0.2% GlcNAc supplemented LB medium in accordance with an embodiment of the invention: the wild-type E. coli (CH1436, left panel) and held by the glmS lacZYA ^- The mutant E. coli (right panel) was diluted 50 times with minimal medium and incubated for 5 hours. After centrifugation, β-galactosidase activity was measured for the supernatant (white rod) and pellet (black rod) The result is a graph.
Figure 3 shows that glmS ^- mutant (CKS1001) carrying an E. coli glmS ⁺ pGFP plasmid or a pGFP plasmid, respectively, is cultured overnight in 0.2% GlcNAc supplemented LB medium, diluted 50 times with minimal medium, (S) and pellet (P) obtained by centrifuging the supernatant after incubation for 24 hours.
Figure 4 is a graph showing plasmid retention in E. coli using the glmS system according to one embodiment of the present invention:
○: glmS ^- mutant bacteria (CKS1001); and
●: parent strain (CH1018) held by the ^Ec GlmS ⁺ p.
Figure 5 is a graph showing targeting and spread of various mutant E. coli strains in CT26 mouse colon cancer cells according to one embodiment of the present invention:
□: glmS ^- mutant bacteria (CKS1001);
▒: ^A glmS ^- mutant bacterium ^{carrying an} EcGlmS ⁺ p vector;
▨: asd ^- mutant bacteria (HJ1019); and
■: Wild type Escherichia coli strain (CH1436).
FIG. 6 is a photograph of a bioluminescence signal obtained by inducing the expression of a lux gene according to an embodiment of the present invention and superimposing the bioluminescence signal on an optical image of the mouse in a virtual color image.
Figure 7 is a graph showing photon intensity in a tumor area according to one embodiment of the present invention:
FIG. 6 is a photograph of a bioluminescence signal obtained by inducing the expression of a lux gene according to an embodiment of the present invention and superimposing the bioluminescence signal on an optical image of the mouse in a virtual color image.
Figure 7 is a graph showing photon intensity in a tumor area according to one embodiment of the present invention:
?: GlmS ^- mutation; And
●: Wild type Escherichia coli.
Figure 8 is a graph showing the total number of bacteria in accordance with one embodiment of the present invention:
□: total number of bacteria; And
■: the number of bacteria have the ^Ec GlmS ⁺ pLux.
9 is a glmS in various medium conditions according to the date of the present ^invention; A graph showing the growth of a mutant S. typhimurium :
○: glmS ^- mutant Salmonella (SKS1001) cultured in minimal medium;
●: glmS ^- mutant Salmonella strains with ^St GlmS ⁺ p cultured in minimal medium;
△: glmS have a 0.2% GlcNAc The p ^{+ St} GlmS cultured in supplemented medium ^- Mutant Escherichia coli; And
▲: Wild-type Salmonella parent strain (SCH2005) cultured on a minimal medium.
10 is a graph showing the intracellular growth of glmS ^- mutant S. typhimurium according to one embodiment of the present invention:
?: Wild-type Salmonella (SCH2005); And
●: glmS ^- mutant salmonella (SKS1001).
FIG. 11 is a graph showing Salmonella counted according to each intracellular condition for 0 hours to 24 hours in HeLa cells (A) and peritoneal macrophages (B) according to an embodiment of the present invention :
■: glmS ^- mutant strain;
□: glmS ^- mutant strains with ^St GlmS ⁺ p; And
▨: Wild type strain.
Figure 12 shows the maintenance of ^St GlmS ⁺ p plasmid in culture in a minimal medium (l) of a glmS ^- mutant Salmonella strain () or in a 0.2% GlcNAc supplemented medium (o) using an in vitro conditional glmS system according to one embodiment of the present invention. when cultured in the graph does not contain (a) and ampicillin containing medium () or wild-type Salmonella strains representing the degree of ampicillin medium is a graph (B) showing the degree of maintenance of the plasmid p ^{+ St} GlmS.
13 is held by the glmS ^st GlmS ⁺ p being proliferation in the tumor tissue, according to one embodiment of the invention ^- mutant strain (SKS1002, A) and Of bacteria (Kan ^R Cat ^R) of ^St GlmS ⁺ p wild-type Salmonella strains (SHJ2036, B) held by the total number (■) and ampicillin number of held by the resistance plasmid (Amp ^R) bacteria (□) measured by the illustrated graph, the .

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. It should be understood, however, that the present invention is not limited to the embodiments and examples described below, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. To fully inform the category of the invention to those who possess it. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Example 1: glmS ^-  Host cell production

1-1: glmS ^- Preparation of E. coli

The bacterial strains used in this study are summarized in Table 1. All E. coli strains were derived from the MG1655 background and the glmS ^- mutant strain (IBPC750) was from J. Plumbridge (France). glmS: tet ^R was transferred to the test strain by P1 phage (Garrett et al ., J. Bacteriol. , 162: 840-844, 1985). A streptomycin resistant strain (CH1436) was obtained from a colony of LB medium containing 10 mu g / ml of streptomycin.

The bacterial strain used in the present invention Strain Explanation References or sources E. coli MG1655 The wild type ( E. coli K-12) ATCC CH1018 arg / lac Kim et al., J. Microbiol., 42: 103-110, 2004 HJ1020 asd Gallan et al., Gene , 94: 29-35, 1990 IBPC750 GlmS :: tet J. Plumbridge (FR) CH1436 Streptomycin resistant CH1018 Invention CKS1001 CH1436, glmS: Tet ^r Invention ^E.c GlmS ⁺ p Coli glmS ⁺ include ^{(Ec +} GlmS) pUC19 Invention pGFP Including GFP gene pUC19 Clontech ^E.c GlmS ⁺ pGFP Including GFP gene ^Ec GlmS ⁺ p Invention pLux LacP :: including luxCDABE pUC19 Invention ^E.c GlmS ⁺ pLux LacP :: including luxCDABE ^Ec GlmS ⁺ p Invention S.typhimurium SCH2005 S. typhimurium 14028s strain ATCC SHJ2037 SCH2005, rela :: blood, spoT :: cat, Kan ^r , Cam ^r Song et al., J. Biol . Chem., 279: 34183-34190, 2004 SMR2130 SHJ2037, Drela, DspoT Invention SKS1001 SCH2005, glmS :: kan, kan ^r Invention SKS1002 SMR2130, glmS :: kan, Kan ^r Invention ^S.t GlmS ⁺ p Salmonella containing glmS ^{+ (St +} GlmS) pBAD24 Invention ^S.t GlmS ⁺ pLux LacP :: luxCDABE including ^St GlmS ⁺ p Invention

1-2: glmS ^-  Production of attenuated Salmonella

The glmS ^- mutant S. typhimurium (SKS1001) was constructed from SCH2005 (14028s) by the method developed by Datsenko and Wanner (Kaiser et al ., Microbiology , 146 Pt 12: 3217-3226, 2000). DNA fragment held by the kan cassette into the ^R position of the glmS open reading frame (glmS open reading frame) was generated using PCR amplification. PCR was performed using the following primer pairs. The primer pair was pKD ^St glmS 5 '(SEQ ID NO: 1: 5'-TTA CTC AAC CGT AAC CGA TTT TGC CAG GTT ACG CGG CTG GTC AAC GTC GGT GCC TTG ATT GTG TAG GCT GGA GCT GCT TCG AA-3') and pKD ^St glmS 3 '(SEQ ID NO: 2: 5'-ATG TGT GGA ATT GTT GGC GCG ATC GCG CTT CGT GAT GTA GCT GAA TCC TTC TTG AAG GTC ATA TGA ATA TCC TCC TTC GTT CC-3').

ppGpp / glmS ^- mutant Salmonella strains (ppGpp / glmS ^- mutant Salmonella , SKS1002) were transfected using p22 phage.

The recombinant Escherichia coli strain and recombinant Salmonella strain prepared above were cultured in LB medium (Luria Bertani medium, Difco Laboratories, USA) or 0.2% glucose, 1 μg / ml thiamine, 1 L sodium chloride Ml < / RTI > of calcium pantothenate. To the solid support medium, 1.5% bacto agar was added. All the medium was supplemented with 100 μg / ml ampicillin, 15 μg / ml tetracycline and 10 μg / ml streptomycin and 100 μg / ml GlcNAc as needed.

Example 2: glmS ⁺  Plasmid vector production with gene

2-1: Escherichia coli glmS ⁺  Plasmid production

The glmS gene was amplified from Escherichia coli genomic DNA by PCR using the following primer pairs:

Forward primer: 5'-GGAAGCTTATGTGTGGAATTGTTGGCG-3 '(SEQ ID NO: 3); And

Reverse primer: 5'-GGGTCGACTTACTCAACCGTAACAGATTTG-3 '(SEQ ID NO: 4).

Purified the amplified PCR product then was cut with Hind and XbaI restriction enzymes, by ligation of a fragment of 1.8 kb in size with the same restriction enzyme sites of the pUC19 vector was prepared, E. coli glmS ⁺ plasmid named them as ^Ec GlmS ⁺ p Respectively.

2-2: Salmonella glmS ⁺  Plasmid production

GlmS gene of S. typhimurium was amplified by a PCR reaction using the following primers from genomic DNA extracted from the strain S. typhimurium 14028s:

Forward primer: 5'-GGGCTAGAATGTGTGGAATTGTTGGC3 '(SEQ ID NO: 5); And

Reverse primer: 5'-GGGAAGCTTTTACTCTACGGTAACCGATTTC-3 '(SEQ ID NO: 6).

The amplified PCR product was purified and then digested with Xba I and Hin dIII restriction enzymes and ligated into pBAD24 vector (Guzman et al ., J. Bacteriol. , 177 (14): 4121-4130, 1995) to construct a Salmonella glmS ⁺ plasmid, which was named ^St GlmS ⁺ p .

2-3: glmS ⁺ pGFP  making

GFP gene was amplified by PCR using pEGFP plasmid (Clontech, USA) as a template and using the following primer pairs:

Forward primer: 5'-GGCCCGGGGTGAGTTAGCTCACTCATTAG-3 '(SEQ ID NO: 7); And

Reverse primer: 5'-AAACCCGGGGAATTCTAGAGTCGCCGC-3 '(SEQ ID NO: 8).

After the PCR amplification product was purified, the 1.1 kb DNA fragment was digested with Sma I restriction enzyme, ligated to ^Ec GlmS ⁺ p digested with the same restriction enzyme, and the E. coli glmS ⁺ plasmid vector expressing GFP was ligated And named ^Ec GlmS ⁺ pGFP .

2-4: glmS ⁺ pLux  making

Briefly , a luminescence-expressing plasmid ( pLux ) was constructed according to the prior art (Min JJ, et al. , Mol. Imaging Biol. , 10: 54-61, 2008) pear pLux containing the lux operon (lux operon, luxCDABE) of (Photobacterium leiognathi) using the Xba I restriction enzyme site was inserted into the pUC19 plasmid backbone (pUC19 plasmid backbone) (Min et al., Nat. Protoc., 3 : 629-636, 2008). The original pLux contains a lux operon of 9.5 kb and an unknown sequence upstream of approximately 700 bp. As the upstream sequence and the following sequence was replaced by the lac promoter (lac promoter sequence). The lux operon from which the upstream sequence was removed was amplified by PCR using the following primer pairs:

lux1 primer: 5'-GGGAATTCTATACCGAAACTACATAC-3 '(SEQ ID NO: 9); And

lux2 primer: 5'-GGGTCTAGAGCACTTAATGCCGCTACT-3 '(SEQ ID NO: 10).

After the PCR reaction product was purified, a DNA fragment of 8.8 kb was digested with restriction enzymes Xba I and Eco RI, inserted into a pUC19 vector digested with the same restriction enzyme, and ligated to produce a plasmid expressing the lux gene , And named it pNlux construct.

The E. coli lac promoter sequence was obtained by performing a PCR reaction using the following primer pairs:

Forward primer: 5'-GGGAATTCCATGGTCATAGCTGTTTC-3 '(SEQ ID NO: 11); And

Reverse primer: 5'-GTGAGCTCGGGATCCTCTAGAGTCGA-3 '(SEQ ID NO: 12).

PCR products of 100 bp fragment was then inserted into a pGEMT Easy vector (Promega, Madison, WI, USA) was cut with Eco RI restriction enzymes, by ligation to insert a pΔNlux vector digested with the same restriction enzymes, under the lac promoter lux was constructed and named as pLacP :: Lux ( pLux ).

Forward primer: 5'-AAGTCGACATGTGTGGAATTGTTGGCG-3 '(SEQ ID NO: 13); And

Reverse primer: 5'-GGGTCGACTTACTCAACCGTAACAGATTTG-3 '(SEQ ID NO: 14).

A 1.8 kb DNA fragment, which was amplified, was digested with Sal I restriction enzyme, ligated into a pLux vector digested with the same restriction enzyme, and ligated to produce a plasmid expressing the lux operon cassette and glmS ⁺ It was named ^ec GlmS ⁺ pLux.

On the other hand, Salmonella glmS gene was amplified from the genomic DNA of S. typhimurium strain 14028s through PCR using the following primer pairs:

Forward primer: 5'-AAAGTCGACATGTGTGGAATTGTTGGC -3 '(SEQ ID NO: 15)

Reverse primer: 5'-GGGGTCGACTTACTCTACGGTAACCGATTTC -3 '(SEQ ID NO: 16)

After digesting the amplified PCR product, the DNA fragment of 1.8 kb was digested with Sal I restriction enzyme and ligated into the pLux vector digested with the same restriction enzyme to ligate the lux cassette with Salmonella glmS ⁺ It was produced a plasmid expressing the gene, and named it as ^St GlmS ⁺ pLux.

Experimental Example 1: glmS ⁺  In vitro characteristics of plasmids

glmS ^- E. coli requires exogenously supplied GlcN / GlcNAc for survival. The present inventors examined the traits by cultivating the mutant strain (CKS1001) in a minimal medium (M9) medium after producing the glmS ^- mutant strain according to the above-mentioned embodiment (Fig. 1). The bacteria grown in the GlcNAc supplemented medium were transferred to the minimal medium and subcultured. The samples collected at the predetermined time were plated on LB agar medium supplemented with GlcNAc to count viable cells. On the LB plate medium, the glmS ^- mutant strain was replicated several times but dissolved within, indicating that sufficient nutrients were not present in the LB medium for the survival of these glmS ^- mutant strains. From the above analysis, it was confirmed that the viability of the glmS ^- mutant strain after 24 hours was rapidly decreased from 10 ⁶ CFU to 10 ² CFU. These traits were similar to the conventional results observed in glmM ^- mutant strains (Mengin-Lecreulx et al. , J. Bacteriol., 175: 6150-6157, 1993). The fate of the glmS mutant strains was then further investigated in the absence of GlcNAc supplementation by confirming whether bacterial killing was due to cell lysis. The glmS ^- mutant carrying a plasmid in which the DNA fragment of hdeABp :: lacZYA (Kim et al., J. Microbiol., 42: 103-110, 2004) was cloned was tested for the degree of bacterial dissolution in the absence of GlcNAc (Fig. 2). Specifically, after 5 hours of passage culture in the minimal medium, the bacteria were centrifuged and β-galactosidase assays were performed on pellets and supernatants as reported by Miller et al . (Zubay et al ., Proc. Natl. Acad Sci. USA , 69: 1100-1103, 1972). The pellet was dissolved in Koch's lysis solution and used for analysis. Due to the significant reduction in CFU compared to the period of five hours glmS ^- because there was an A ₆₀₀ value of the mutant strain (cell weight) it is significant _{A 420 / min / ㎖ / A} 600 instead of the A ₄₂₀ / min / ㎖ to ONP . As a result, as shown in Fig. 2, the sum of A ₄₂₀ / min / ml of the supernatant (white rod) and pellet (black rod) was 0.85 in the wild type strain and 0.35 in the glmS ^- mutant strain. However glmS ^- an A ₄₂₀ / min / ㎖ measured in the mutant strain in most (> 80%), while the value in the supernatant, was the value of entirely in the pellet in the wild type strain, which glmS ^- that is soluble under these culture conditions, the mutant strain .

To further confirm lysis in the glmS ^- mutant strain at the time of absence of supplement GlcNAc, the wild-type strain and the glmS ^- strain were transformed with pGFP and cultured in the minimal medium as shown in Fig. Western blot analysis for GFP was performed using a GFP-specific antibody after obtaining the supernatant and pellet samples at set times (FIG. 3). First, the protein samples were boiled for 5 minutes, and the total protein (10 μg) was developed by electrophoresis on a 12% SDS-polyacrylamide gel. The proteins were electrophoresed on nitrocellulose membrane (Bio-Rad, USA) Lt; / RTI > The protein-transferred membrane was blocked with 5% skim milk and treated with mouse anti-GFP antibody monoclonal antibody (Sigma-Aldrich, UK, 1: 5000 dilution) The reaction was allowed to proceed overnight at 4 ° C. After incubation for 1 h with wasp peroxidase-conjugated anti-mouse secondary antibody (anti-mouse IgG antibody linked to horseradish peroxidase, Sigma-Aldrich, UK, 1: 5000 or 1: 10000 dilution) (ECL, Amersham Bioscience).

Bacterial spent media and bacterial pellets were prepared as follows. The pellet was sonicated in phosphate-buffered saline (PBS) with lysis buffer containing 10 mM lysozyme and 10% SDS for dissolution. The medium used was filtered with 0.22 μm filter paper, and the protein was precipitated with 10% trichloroacetic acid (1 hour, 4 ° C).

As a result of the Western blot analysis, as shown in FIG. 3, the sample contained GFP in the supernatant after 1 hour from the culture of pGFP- bearing glmS ^- mutant strain (CKS1001 / pGFP ) , GFP in the pellet was measured only up to 2 hours. glmS held by the wild type strain have the pGFP (CH1018 / pGFP) and ^Ec GlmS pGFP ^{+ -} samples from the mutant strain (CKS1001 / GlmS pGFP ⁺⁾ could have most of the GFP throughout the experimental period check appears in the pellet.

Based on the results, a balanced-lethal host-vector system was constructed in which the glmS gene inserted into the plasmid can complement the glmS ^- mutation on the chromosome.

Then, the present inventors with the wild type glmS ^- it was confirmed that the transformed result, the plasmid is maintained without the need for antibiotic in the mutant strain ^Ec GlmS ⁺ p (Fig. 4). Specifically, the present inventors subcultured the overnight culture overnight in fresh medium (1/1000) without ampicillin for a total of 4 days. Samples were taken every 24 hours and serially diluted in sterile 0.9% sodium chloride (sterile 0.9% NaCl). Were plated in appropriate volumes on triple LB agar plates containing GlcNAc with or without ampicillin. The bacteria were plated on a plate containing ampicillin, and the samples were collected on a designated date to confirm the maintenance of the plasmid. The number of colonies was used to calculate the ratio of total viable cell concentration to plasmid-bearing bacteria. In the case of the wild type strain, the plasmid was lost more than 92% on the 2nd day and 99% on the third day. glmS ^- In the case of the mutant strain, no loss of plasmid was observed within 4 days of the experiment. Clearly, the above results demonstrate the possibility of using glmS ^- mutant bacteria in a balanced-lethal system to maintain the expression of the plasmid in the absence of antibiotics.

Experimental Example 2: glmS ⁺  Characteristics of plasmids in mouse models

2-1: Survival of bacteria in vivo

The prerequisite for a balanced - lethal host - vector system based on glmS is that glmS ^- There is no sufficient supply of nutrients required for the growth of mutant bacteria. Some bacterial species, including Escherichia coli, have been shown to target tumor tissues (Forbes, Nat. Rev. Cancer , 10: 785-642, 2010) in tumor tissues (Jiang et al ., Mol. Ther. , 18: 794, 2010), we have determined viability of glmS ^- mutant bacteria in tumor tissues (Min et al ., Nat. Protoc. , 3: 629-636, 2008). Tumor mouse models were prepared by subcutaneous transplantation of CT26 cells on the right thigh side of BALB / c mice to obtain xenograft model mice. CT26 cells were cultured in high glucose Dulbecco's modified Eagle medium containing 10% FBS and 1% penicillin-streptomycin. 14 days after wild type or glmS it does not hold or retain the ^Ec GlmS ⁺ p ^- Mutant bacteria (1 × ^{10 8} CFU) were injected into each mouse via the tail vein. To quantify bacteria in tumor mouse models, streptomycin-resistant mutants were prepared and alleles were transferred to test strains. This is because the presence of bacteria already present in the mouse or the need to correct for contamination. Resistance to chromosomally acquired streptomycin is primarily due to mutations in the gene encoding ribosomal protein S12, rpsL (Wittmann et al. , Mol. Gen. Genet. , 141: 331-341, 1975).

Tumor tissues were harvested at the indicated date, homogenized and counted at designated time (Figure 4) by plating on LB medium supplemented with GlcNAc containing streptomycin. The result, in the wild type strain at 1 day (point bars in) and glmS As shown in Figure 4 ^was observed in mutant strains (white bar) is about the same number (~ 1x10 ⁶⁾ the number of bacteria in. The number of bacteria in the wild-type strain increased to approximately 10 ⁹ CFU at 5 days, while that of the glmS ^- mutant strain decreased to approximately 5 × 10 ³ CFU at 7 days. While ^Ec GlmS ⁺ p transformed glmS ^to-mutant strain (black bars) was maintained for more than 10 ⁹ CFU similar to the wild-type by day 7. As a control experiment the inventors in the asd ^- Mutant strains (diagonal bars) were also counted. As a result, day 1 asd ^- The number of bacteria in the mutant strain was similar to the number of wild-type bacteria, but on the 7th day the value decreased to approximately 5 × 10 ⁴ CFU. From the above results, it was confirmed that the animal system lacks sufficient nutrients required for proliferation of glmS ^- mutant bacteria similarly to asd ^- mutant bacteria. Held by the proliferation, as well as the wild-type strain ^Ec GlmS ⁺ glmS ^- The mutant strain proliferated in vivo, indicating that the glmS gene in the plasmid can compensate for chromosomal mutations in animal models in vivo.

2-2: In vivo imaging of bacteria

The present inventors have previously reported quantitative and noninvasive techniques for monitoring the migration of bacteria in living subjects (Min et al ., Mol. Imaging. Biol., 10: 54-61, 2008). This technique , The bioluminescent bacteria are produced by bacteria transformed with an expression plasmid ( pLux ) containing the luxCDABE operon (Min et al., Mol. Imaging Biol., 10: 54-61, 2008; Min et al. Nat. Protoc. 3: 629-636, 2008). The present inventors using the above methods glmS holds the ^Ec GlmS ⁺ pLux tumor mouse models of the CT26 mouse colon cancer cells implanted ^- after floating the mutant E. coli strain with the wild-type E. coli strain, respectively 1x10 ⁸ reviews of 100 ㎕ PBS the After injection into the tumor mouse model via the tail vein, the expression pattern of the lux gene was monitored using a cooled CCD (charged couple detector) camera (FIG. 6). The mouse tumor models of colon cancer cell line CT26 cells 1x10 ⁶ dogs suspended to 20-30 g of a 5 to 6-week-old male weight BALB / c and BALB / c athymic (athymic) in 100 PBS nu ^- / nu ^- Mice were subcutaneously transplanted into the right lateral flank of the mice (Sam Taco, Korea). On the other hand, the imaging of the bioluminescence of the bacteria was carried out by placing a live animal anesthetized in a light intercepting chamber of an IVIS 100 imaging system (Caliper, Hopkinton, Mass., USA) equipped with a cooled CCD camera and measuring the fluorescence emitted from the luciferase- Photons were counted. The counted photons were integrated for one minute and virtual color images representing the counted photons were overlaid on the photographs of the mice using Living Image software V. 2.25 (Caliper, Hopkinton, MA, USA).

As a result, the bioluminescent signal was detected in the spleen and liver of the mouse within 30 minutes after the injection of the bioluminescent bacteria, as shown in Fig. On day 1, two types of bacterial signals were reduced in the liver and exclusively detected at the tumor site. GlmS have the ^Ec GlmS ⁺ pLux ^- signal of the mutated bacteria was found significantly stronger than the signal of wild-type bacteria.

In order to quantify the degree of bioluminescence, the present inventors selected ROIs based on the intensity of the signal and then measured the fluorescence intensity of the designated 1, 3, 5, 7, 9, On day 12, the maximum number of photons in each ROI (photons S ^-1 cm ^-2 sr ^-1 ) was recorded (FIG. 7). As a result, the glmS held GlmS ^{Ec +} pLux As shown in Figure ^7-photon outlet (photon flux) of the mutant bacteria was river about 10 to 100 times compared to the wild-type bacteria photon leakage.

2-3: Check whether the plasmid is maintained

After the inventors is the result was held to CFU coefficient, ^{Ec +} GlmS pLux to check whether according to whether maintenance of the plasmid (Fig. 8). To count the bacteria, tumor tissues were harvested at the indicated dates, homogenized and plated on LB agar medium containing streptomycin and supplemented with GlcNAc. On the other hand, bacteria were plated on LB agar medium containing ampicillin and GlcNAc to measure plasmids resistant to ampicillin (plasmid (Amp ^R )). The mutant strain (B in FIG. 8) both the total number of bacteria ^- a result, the wild type strain (A in FIG. 8) and glmS holds the ^Ec GlmS ⁺ pLux on streptomycin containing culture medium (white bars), as shown in FIG. 8 On day 1, it increased from 10 ⁷ to day 5 to 10 ⁹ and gradually decreased thereafter. Wild-type strain in ampicillin containing medium (black bars) (Fig. 8 A) is approximately the other hand, decreased by about 50 fold and decreased by 1000-fold in 5 days at 1 il, glmS holds the ^Ec GlmS ⁺ pLux ^- number of mutant bacteria Did not decrease. This suggests that the balance-lethal host-vector system using glmS is highly effective in the maintenance of plasmids containing genes encoding foreign proteins in animal systems.

Experimental Example 3: Salmonella glmS ⁺  Characteristics of Plasmids

3-1: Survival analysis

Salmonella spp., As well as E. coli, have been shown to aggregate in tumor transplanted into animals and have been developed to carry a wide range of anti-tumor cargo proteins (Nguyen et al ., Cancer Res ., 70: 18-23, 2010). As with the E. coli mutants, the phenotype of glmS ^- mutant Salmonella was described by culturing the mutant strain (SKS1001) in minimal medium (Fig. 9). The viability of glmS ^- mutation was reduced to 5x10 ² CFU after 12 hours at 5x10 ⁴ CFU. However, the CFU of mutant type was increased from 5x10 ⁴ to 2x10 ⁹ as wild-type when the GlcNAc is present. In addition, glmS ^- mutant Salmonella strains with ^St GlmS ⁺ plasmids proliferated to a similar extent as those of the wild-type control without GlcNAc. Interestingly, E. coli, and even though substantially the same as that of the two kinds of Salmonella GlmS protein is only enough to vary only eight of the 609 amino acids, and ^Ec GlmS ⁺ p is glmS ^- The mutation failed to compensate for S. typhimurium .

3-2: Cellular permeability assay

S. typhimurium can invade or replicate animal cells. Therefore, the characteristics of glmS ^- mutant Salmonella can be investigated with cultured Hela cells and peritoneal macrophages extracted from BALB / c mice. The peritoneal macrophages BALB having a weight 20-30 g / c and BALB / c athymic (athymic) nu ^- / nu ^- injected with 4% thioglycolate (thioglycollate) solution ㎖ 2 into the abdominal cavity of mice and 3 days And cultured in DMEM containing 10% fetal bovine serum (FBS) for 4 hours at 37 ° C. Then, unattached cells were cultured and only the attached cells were collected and used in the experiment. The Hela cells were also cultured in high glucose-DMEM medium containing 10% FBS and 1% penicillin-streptomycin. First, time course analysis was performed in a manner that counts bacteria within the cells at 3 hour intervals. A wild-type Salmonella strains and glmS cultured in the presence of GlcNAc ^- a bacterial mutant Salmonella strains were mixed with HeLa cells after cell 10 ㎍ / ㎖ gentamicin (gentamicin, Sigma Chemical) was counted in the presence (Kim et al, J.. Microbiol. Methods 64: 17-26, 2006) (Figure 10). Bacterial invasion assays were performed as described previously (Jeong et al ., J. Bacteriol ., 190: 6340-6350, 2008) Specifically, strains of S. typhimurium 14028S were grown in LB medium Lt; / RTI > The S. typhimurium was inoculated into fresh medium and cultured at 37 DEG C for 4 hours. The cells were then appropriately diluted and resuspended in cell culture medium for infection of the cell monolayers at an MOI of 1:10 for 30 minutes. 1x10 ⁵ cells were seeded in 24-well plates and cultured in DMEM medium containing 10% FBS at 5% CO _{2 and} 37 ° C in an incubator. Infected cells were washed three times with PBS (pH 7.4). DMEM containing 10 / / ml of gentamycin was added and incubated for 30 minutes. The intracellular bacteria were harvested by extraction with lysis buffer (0.05% Triton X-100 in PBS, pH 7.4) and the colonies were counted on a BHI agar plate supplemented with GlcNAc (brain-heart infusion agar plates) Times.

As a result, as shown in Figure 10, the number of bacteria entering the HeLa cells is substantially the wild type strain (white circle) glmS ^- All mutant bacteria (black circles) were approximately similar ( ^3-4 × 10 ³ CFU, T = 0 h). The number of wild-type Salmonella began to increase after 6 hours of infection and reached 10 ⁶ CFU after 24 hours of infection. Conversely, the number of glmS ^- mutant Salmonellae began to decrease after 6 hours of infection and reached less than 10 CFU after 24 hours of infection. This confirms that the essential nutrients for cell membrane synthesis and cell wall of glmS ^- mutant Salmonella are not sufficiently present in animal systems. My starting point is reduced at the beginning of the increasing number of wild-type bacteria (elapsed six hours after infection) because the cells that match glmS ^- The reduction in the number of mutant Salmonella is believed to be due to the effect of the failure of peptidoglycan synthesis.

Then, the present inventors transformed glmS switch to ^St GlmS ⁺ p ^- were analyzed in the invasion and cell proliferation of the mutant S. typhimurium (Fig. 11). Post-infection 0 hours and 24 hours after the result of counting the bacteria cells, glmS the glmS ⁺ gene is compensated as shown in Figure 11A ^- mutant Salmonella strain is that it has grown in the cell, such as breaking into HeLa cells and the wild type strain Show. This was also the same in experiments with peritoneal macrophages (Fig. 11B).

3-3: Check whether plasmid is maintained

g balanced using lmS-accuracy (fidelity) of the lethal system is glmS held by the wild type strain and ^St GlmS ⁺ p in vitro ^conditions was investigated by using the mutant Salmonella strain (Fig. 12). As a result, as shown in Figure 12, glmS ^- mutant strain for (SKS1002, Fig. A 12), when cultured in a GlcNAc supplemented medium (white circles) at least 99% of the day 6 ^St GlmS ⁺ p disappeared . However, when cultured in medium lacking GlcNAc (black circle), it was strictly maintained. For the wild-type strain (SHJ2037, B of FIG. 12), ^St GlmS ⁺ p is, but is held there when the antibiotic is present (black circles), in the absence of antibiotic (white circle) was rapidly lost.

Finally, Salmonella glmS balanced-lethal system for glmS have to ^St GlmS ⁺ p ^- Mutant Salmonella strains and wild type Salmonella strains were tested in animal models. However, since S. typhimurium is toxic in rodents, an attenuated S. typhimurium mutant lacking ΔppGpp synthesis has been used (Jeong et al., J. Bacteriol., 190: 6340-6350, 2008 ). The synthesis of ppGpp by relA and spoT is required for the toxicity of S. typhimurium (Song et al ., J. Biol. Chem. , 279: 34183-34190, 2004). Kanamycin (kanamycin) and chloramphenicol (chloramphenicol) both ppGpp-null mutant having resistance to both (relA :: kan, spot :: cat ) strain and glmS ^{- St} for ppGpp-null mutant strains have mutations with ampicillin resistance 0.0 & ^gt ; GlmS ⁺ p & lt ^{; /} RTI > Was injected intravenously to each mouse via the Salmonella strain to 3x10 ⁷ CFU tail vein ^-, CT26 cells to establish an implanted tumor mouse model after 14 days, or ΔppGpp ΔppGpp / glmS holds the GlmS ⁺ pLux as described above. Tumor tissues were harvested at the indicated dates and homogenized and plated on LB plate media supplemented with GlcNAc, which essentially contained kanamycin and chloramphenicol and optionally did not contain ampicillin. The total number of bacteria (Kan ^R Cat ^R ) and the number of bacteria carrying the ampicillin resistant plasmid (Amp ^R ) were then measured (Figure 13). As a result, as shown in FIG. 13, ^St GlmS ⁺ p to have glmS ^- mutant strain for (SKS1002, Figure A of 13) the total number of cells was the same (black bars) and ampicillin-resistant bacteria (white bars), infection days 10 to ⁷⁵ days after on the other hand, after lapse of showing a tendency to gradually decrease after, since this increased to 5x10 ^9, the case of ^St GlmS ⁺ p wild-type Salmonella strains (SHJ2037, B in Fig. 13) held by the total number of cells is the glmS ^- same pattern with mutant strain But the number of ampicillin - resistant cells was 50 times less than the total number of cells after 1 day of infection, 5000 times less after 5 days, and less than 10000 times after 12 days. The results show that the Salmonella glmS ^- balance-lethal host-vector system ensures the maintenance of the plasmid even in the absence of a selective determinant in the animal.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. I will understand. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> NDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Novel bacterial vector system based on glmS <130> PD12-0449 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 1 <400> 1 ttactcaacc gtaaccgatt ttgccaggtt acgcggctgg tcaacgtcgg tgccttgatt 60 gtgtaggctg gagctgcttc gaa 83 <210> 2 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 2 <400> 2 ctgaatcctt atatgaatat cctccttcgt tcc 83 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 3 <400> 3 ggaagcttat gtgtggaatt gttggcg 27 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 4 <400> 4 gggtcgactt actcaaccgt aacagatttt g 31 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 5 <400> 5 gggctagaat gtgtggaatt gttggc 26 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 6 <400> 6 gggaagcttt tactctacgg taaccgattt c 31 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR primer7 <400> 7 ggcccggggt gagttagctc actcattag 29 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PCR primer8 <400> 8 aaacccgggg aattctagag tcgccgc 27 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> PCR primer 9 (lux1 primer) <400> 9 gggaattcta taccgaaact acatac 26 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> PCR primer 10 (lux2 primer) <400> 10 gggtctagag cacttaatgc cgctact 27 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 11 <400> 11 gggaattcca tggtcatagc tgtttc 26 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 12 <400> 12 gtgagctcgg gatcctctag agtcga 26 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 13 <400> 13 aagtcgacat gtgtggaatt gttggcg 27 <210> 14 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer14 <400> 14 gggtcgactt actcaaccgt aacagatttt g 31 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 15 <400> 15 aaagtcgaca tgtgtggaat tgttggc 27 <210> 16 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 16 <400> 16 ggggtcgact tactctacgg taaccgattt c 31

Claims (20)

Use of antibiotics containing as an active ingredient a transgenic, attenuated Gram-negative bacillus transfected with a plasmid containing a glmS gene and a foreign gene operably linked to the promoter as a selection marker for the attenuated Gram-negative bacteria in which the glmS gene has been deleted A composition for preventing the loss of the plasmid in a transformed attenuated Gram-negative organism, The method according to claim 1,
The attenuation may be selected from the group consisting of pab, nadA, pncB, pmi, rpsL, ompR, htrA, hemA, rfc, poxA, galU, aro, galE, cya, cyp, crp, cdt, pur, phoP, phoQ, ssa, guaA, guaB, clpP, clpX fliD, flgK, flgL, relA or spoT gene. The method according to claim 1,
The Gram-negative bacteria may be selected from the group consisting of Salmonella spp., Helicobacter spp., Shigella spp., Enterobacter spp., Serratia spp. seusok (Stenotrophomonas spp.), genus Vibrio as beudel (Bdellovibrio spp.), Legionella genus (Legionella spp.), Pseudomonas species (Pseudomonas spp.), morak Cellar in (Moraxella spp.), Proteus in (Proteus spp.), Wherein the composition belongs to Escherichia spp. Or Yersinia spp. The method according to claim 1,
Wherein the foreign gene is a gene encoding a therapeutic gene, an imaging gene, or an antigen protein. delete 5. The method of claim 4,
Wherein the therapeutic gene is a gene encoding an anti-cancer protein. delete delete The method according to claim 1,
Wherein the promoter is a homeostatic promoter or an inducible promoter. 10. The method of claim 9,
_Wherein the inducible promoter is P _BAD . The method according to claim 6,
Wherein the anticancer protein is a protein toxin, an antibody specific for a cancer antigen or a fragment thereof, a tumor suppressor gene or an antiangiogenic factor. delete delete delete Transgenic attenuated Gram-negative bacteria transfected with a plasmid containing a foreign gene operably linked to the glmS gene and the promoter as selection markers for attenuated Gram-negative bacteria in which the glmS gene has been deleted can be administered to animals other than humans without administration of antibiotics Wherein said method comprises administering to said animal an effective amount of said plasmid. 16. The method of claim 15,
Wherein the foreign gene is a gene encoding a therapeutic gene, an imaging gene, or an antigen protein. delete delete 17. The method of claim 16,
Wherein said antigenic protein is an immunogenic protein derived from a bacterium, fungus, parasite or virus. delete

Images