KR20100044465A - Recombinant vector for foreign protein expression and secretion - Google Patents

Abstract

PURPOSE: A recombinant vector for a development/secretion of a foreign protein is provided to improve the secretion efficiency and to confirm the development of the foreign protein by combining and epitope. CONSTITUTION: A recombinant vector for a development/secretion of a foreign protein contains a signal peptide, a protein development verifier epitope and an insertion site for the foreign protein. The signal peptide is either an OmpW signal peptide, a StfA signal peptide or a AgfA signal peptide. The protein development verifier epitope is either a His epitope, an RGS-His epitope, a T7 epitope or a V5 epitope. The insertion site for the foreign protein comprises an EcoRI, BamHI, SalI and HindIII cutting region. The producing method of the foreign protein comprises the following steps: culturing a transformant obtained by transforming a host cell using the recombinant vector; and confirming the foreign protein at a western blot.

Description

Recombinant Vector for Foreign Protein Expression and Secretion

The present invention relates to a recombinant vector for expression and secretion of a foreign protein, and more particularly, to a recombinant vector for expression and secretion of a foreign protein containing a signal peptide, an epitope for confirming protein expression, and a foreign gene insertion site. The present invention relates to a transformed microorganism obtained by introducing into a host cell and a method for producing a secreted foreign protein using the transformed microorganism.

Traditional vaccines against existing pathogenic bacteria are mainly killed vaccines or subunit vaccines. However, the manufacturing process of these vaccines is very difficult to increase the immunity from the separation and purification of the antigen, and the vaccine should be stored stably to reduce the side effects caused by the vaccine. In addition, these vaccines have the inconvenience of immunizing each individual with a syringe. To alleviate this inconvenience, methods for inducing antibody production for mucosal and humoral immunity have been introduced by a method in which bacteria in a host cell secrete foreign proteins to bacteria.

Foreign protein secretion production methods have been tried in various ways in Salmonella . That is, a method of forming a recombinant protein in organs such as flagella and cilia that are secreted to the outside of the cell and sending the antigen to the outside of the cell, or using a system capable of secretion as a signal peptide is used. The existing signal peptide is derived from beta lactamase (β-lactamase), and this protein is secreted into the periplasmic space and has a problem of low extracellular secretion efficiency (Infection and Immunity, 70 (4), 1739-). 1749, 2002). Such a method has a problem in that current secretion efficiency is low or not stable, and thus, a large amount or stable secretion of the antigen of interest cannot be caused to induce an appropriate host immune response.

Therefore, the present inventors have made efforts to develop a secretion production method of foreign proteins with high secretion efficiency by supplementing the disadvantages of the system for secreting foreign proteins with the above-described signal peptides. It confirmed that it is high, and completed this invention.

After all, an object of the present invention is to provide a recombinant vector for expression and secretion of a foreign protein containing a signal peptide, an epitope for confirming protein expression, and a foreign gene insertion site.

Another object of the present invention is to provide a transformed microorganism obtained by introducing a recombinant vector into a host cell selected from the group consisting of bacteria, fungi and yeast.

Another object of the invention (a) culturing the transforming microorganism; And (b) to provide a method for producing a foreign protein comprising the step of obtaining a foreign protein in the culture.

The present invention provides a recombinant vector for expression and secretion of a foreign protein containing a signal peptide, an epitope for confirming protein expression, and a foreign gene insertion site.

In the present invention, the signal peptide is composed of OmpW signal peptide ( ompW SS ), StfA signal peptide ( stfA SS ), β-lactamase (β-lactamase) signal peptide ( bla SS ) and AgfA signal peptide ( agfA SS ) It may be characterized in that any one or more selected from the group.

In the present invention, the protein expression label (epitope) may be any one or more selected from the group consisting of His epitope, RGS-His epitope, T7 epitope and V5 epitope.

In the present invention, the foreign gene insertion site may be characterized by having Eco RI, Bam HI, Sal I and Hin dIII cleavage sites.

In the present invention, the recombinant vector is asd gene, located upstream of the asd gene as asd gene in the opposite direction to the trc promoter (Ptrc), ompW signal peptide (ompW SS) located downstream of the trc promoter (Ptrc), PMMP62 comprising a His-epitope gene located downstream of the ompW SS , a foreign gene insertion site located downstream of the His-epitope gene, and a transcription termination sequence 5ST1T2 located downstream of the foreign gene insertion site. Can be.

In the present invention, the recombinant vector is asd gene, located upstream of the asd gene as asd gene in the opposite direction to the trc promoter (Ptrc), stfA signal peptide (stfA SS) located downstream of the trc promoter (Ptrc), PMMP63 comprising a His-epitope gene located downstream of the stfA SS , a foreign gene insertion site located downstream of the His-epitope gene, and a transcription termination sequence 5ST1T2 located downstream of the foreign gene insertion site. Can be.

In the present invention, the recombinant vector is asd gene, located upstream of the asd gene as asd gene in the opposite direction to the trc promoter (Ptrc), agfA signal peptide (agfA SS) located downstream of the trc promoter (Ptrc), PMMP65 including a His-epitope gene located downstream of the agfA SS , a foreign gene insertion site located downstream of the His-epitope gene, and a transcription termination sequence 5ST1T2 located downstream of the foreign gene insertion site. Can be.

In the present invention, the foreign protein may be selected from the group consisting of antigens, enzymes, polypeptides and hormones, the foreign protein may be characterized in that the PspA.

The present invention also provides a transformed microorganism obtained by introducing a recombinant vector for expression and secretion of the foreign protein into a host cell selected from the group consisting of bacteria, fungi and yeasts.

In the present invention, the host cell may be characterized in that the Salmonella.

The present invention also comprises the steps of (a) culturing the transformed microorganism; And (b) provides a method for production and secretion of foreign proteins comprising the step of identifying the foreign protein in the culture Western block.

In the present invention, the foreign protein may be selected from the group consisting of antigens, enzymes, polypeptides and hormones.

The present invention provides a recombinant vector for expression and secretion of a foreign protein into which one or more signal peptide genes selected from the group consisting of agfA , ompW, and stfA , His epitope gene, and a gene encoding a foreign protein are inserted, and host the recombinant vector. There is an effect of providing a method for producing a secreted foreign protein, characterized in that the recombinant microorganism obtained by introduction into the cell and the recombinant microorganism are cultured to express and secrete the foreign protein.

According to the present invention, it is possible to increase the secretion efficiency of foreign proteins as compared to the conventional secretion system using a signal peptide, by combining a protein expression identification label (epitope) can conveniently confirm the expression of foreign proteins, the present invention The foreign protein (antigen) obtained through can be used as a vaccine against infectious bacteria in livestock.

If foreign proteins derived from various enterobacterials are applied to the expression system of this foreign protein, it can be utilized as a multivalent vaccine system that can protect against various pathogenic enterobacteriaceae.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for producing secretion of foreign proteins for secreting excessive amounts of foreign proteins using recombinant technology. Most bacteria are secreted by type-II sec-dependent systems when they secrete proteins extracellularly and are essentially involved in signal peptides. This signal peptide is the leader peptide sequence of the secreted protein and affects the expression and secretion efficiency of the protein.

In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

The vector underlying the present invention is pYA3493 (Infection and Immunity, 70 (4), 1739-1749, 2002). The pYA3493 vector contains the aspartate semi-aldehyde dehydrogenase ( asd ) gene, which is an essential component of m-diaminopimellic acid (m-DAP), which is essential for bacterial cell wall synthesis. To synthesize the synthesized enzyme acting at the starting point.

Therefore, the host in which the vector of the present invention resides is missing the asd gene, and the asd gene present in the vector can be used as a selective marker. In addition, the pYA3493 vector has Eco RI, Bam HI, Sal I and Hin dIII cleavage sites for cloning foreign proteins, and β-lactamase in the upstream region of the foreign antigen cloning site for effective secretion of foreign proteins. ) Signal peptides ( bla SS ; pMMP62), AgfA signal peptides ( agfA SS ; pMMP63), OmpW signal peptides ( ompW SS ; pMMP64), StfA signal peptides ( stfA SS ; pMMP65) are also present. It retains six His coding residues in the stream and its expression is controlled by the Ptrc promoter. Transcription termination of the cloned foreign protein is by the ρ-independent transcription termination sequence of 5ST1T2 (5S rRNA transcription terminator T1T2).

β-lactamase is a protein secreted into the periplasmic space from Gram-negative bacteria in a SecB-independent manner by a type II Sec-dependent secretion system. AgfA and StfA are the main proteins of fimbriae that are externally secreted by the type IV Sec-dependent secretion system, and each has been reported to be different in secretion mode. Agf pimbria are secreted in a manner that has not yet been accurately identified, while Stf pimbria are secreted in a manner similar to the type II Sec dependent approach. OmpW is an outermembrane protein W that is SecB dependent by a type II Sec dependent secretion system. As described above, the signal peptide was selected due to the difference in the region or the method of secretion.

Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) are bound to regulatory sequence (s). For example, the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame.

Furthermore, pspA (Streptococcus pneumoniae surface protein A) gene or other foreign proteins were cloned into the pMMP62, 63, 64, and 65 vectors of the present invention to prepare a recombinant vector for expression and secretion of the foreign protein. The recombinant microorganism obtained by introducing into the host cell was cultured to measure the secretion efficiency of the foreign protein pspA antigen and other foreign proteins.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

In addition, in the present embodiment, as a signal peptide, OmpW signal peptide ( ompW SS ), StfA signal peptide ( stfA SS ), β-lactamase (β-lactamase) signal peptide ( bla SS ), AgfA signal peptide ( agfA SS ), protein His (Histidine) -eptitope and foreign protein PspA are exemplified as the expression confirmation epitope, but it is obvious that the present invention can be applied to other signal peptides, epitopes for protein expression and foreign proteins.

Example 1.  ompW SS Of Vectors Containing and His-epitope

ompW SS was based on Salmonella typhimurium x3339 (Gulig and Curtiss, Infect. Immun ., 55: 2891, 1987) using primers of SEQ ID NOs: 1 and 2 introduced with Bsp HI and Eco RI restriction enzyme cleavage sequences, respectively. Amplification was performed by PCR. His-epitope was prepared as the underlined portion of the primer sequence of SEQ ID NO: 2, and was automatically inserted by PCR.

The conditions of PCR were performed after initial denaturation at 94 ° C. for 3 minutes, followed by denaturation (30 seconds at 94 ° C.), binding (annealing, 30 seconds at 60 ° C.), and extension (30 at 72 ° C.). Total 30 times).

[SEQ ID NO 1] 5'-TCATGAAAAAATTTACAGTGGCGG-3 '

[SEQ ID NO 2] 5'-GAATTC ATGGTGATGGTGATGATG TCCGGCTTCGTGCGCGAACG-3 '

The amplified DNA product was cloned into a T-vector (Promega), digested with Bsp HI and Eco RI restriction enzymes, and the pYA3342 vector (Infection and Immunity, 2002. 70 (4), 1739-1749) was identified as Nco I and Cleavage with Eco RI restriction enzyme and conjugation at translation level (pMMP62; FIG. 2). As a result, the gene conjugated to the pYA3342 vector was the same as SEQ ID NO: 3. SEQ ID NO: 4 is the amino acid sequence of ompW SS and His-epitope inserted into pYA3342 vector.

Example 2.  stfA SS Of Vectors Containing and His-epitope

stfA SS was amplified by PCR using the primers of SEQ ID NOs: 5 and 6 using Salmonella typhimurium x3339 as a template and introduced Bsp HI and Eco RI restriction enzyme cleavage sequences, respectively. His-epitope was prepared with an underlined portion in the primer of SEQ ID NO: 6 and was automatically inserted by PCR.

The conditions of PCR were performed after initial denaturation at 94 ° C. for 3 minutes, followed by denaturation (30 seconds at 94 ° C.), binding (annealing, 30 seconds at 60 ° C.), and extension (30 at 72 ° C.). Total 30 times).

[SEQ ID NO 5] 5'-TCATGAATACAGCAGTAAAAGC TG-3 '

[SEQ ID NO: 6] 5'-GAATTC ATGGTGATGGTGATGATG ACCGGTAAAAGTCACCGTAC-3 '

The amplified DNA product was cloned into T-vector (Promega), cleaved with Bsp HI and Eco RI restriction enzymes, and pYA3342 vector was cleaved with Nco I and Eco RI restriction enzymes and conjugated at translation level (pMMP63; FIG. 3). As a result, the gene conjugated to the pYA3342 vector was the same as SEQ ID NO: 7. SEQ ID NO: 8 is the amino acid sequence of the stfA SS and His-epitope inserted into the pYA3342 vector.

Example 3. bla SS Of Vectors Containing and His-epitope

To add His-epitope to bla SS (Kang, et al ., Infection and Immunity, 70 (4): 1739, 2002), plasmid pYA3493 was used as a template and Bsp HI and Eco RI restriction enzyme cleavage sequences were introduced, respectively. PCR was performed using the primers of SEQ ID NOs: 9 and 10 to amplify.

His-epitope was prepared as the underlined portion of the primer sequence of SEQ ID NO: 10 and inserted automatically by PCR. The conditions of PCR were performed after initial denaturation at 94 ° C. for 3 minutes, followed by denaturation (30 seconds at 94 ° C.), binding (annealing, 30 seconds at 60 ° C.), and extension (30 at 72 ° C.). Total 30 times).

[SEQ ID NO 9] 5'-TCATGAGTATTCAACATTTCCGTG-3 '

[SEQ ID NO 10] 5'-GAATTC ATGGTGATGGTGATGATG TTCAGCATCTTTTACTTTCA-3 '

The amplified DNA product was cloned into T-vector (Promega), digested with Bsp HI and Eco RI restriction enzymes, and pYA3342 vector was digested with Nco I and Eco RI restriction enzymes and conjugated at translation level (pMMP64; FIG. 4). As a result, the gene conjugated to the pYA3342 vector was the same as SEQ ID NO: 11. SEQ ID NO: 12 is the amino acid sequence of bla SS and His-epitope inserted into a pYA3342 vector.

Example 4. agfA SS Of Vectors Containing and His-epitope

The agfA SS was amplified by PCR using the primers of SEQ ID NOs: 13 and 14 using Salmonella typhimurium x3339 as a template and introduced Bsp HI and Eco RI restriction enzyme cleavage sequences, respectively. His-epitope was prepared as underlined in SEQ ID NO: 14, and automatically inserted by PCR.

The conditions of PCR were performed after initial denaturation at 94 ° C. for 3 minutes, followed by denaturation (30 seconds at 94 ° C.), binding (annealing, 30 seconds at 60 ° C.), and extension (30 at 72 ° C.). Total 30 times).

[SEQ ID NO 13] 5'-TCATGAAACTTTTAAAAGTGGCAG-3 '

[SEQ ID NO 14] 5'-GAATTC ATGGTGATGGTGATGATG GCCGCCGTTATGATTACCGC-3 '

The amplified DNA product was cloned into T-vector (Promega), cleaved with Bsp HI and Eco RI restriction enzymes, and pYA3342 vector was cleaved with Nco I and Eco RI restriction enzymes and conjugated at translation level (pMMP65; 5). As a result, the gene conjugated to the pYA3342 vector was the same as SEQ ID NO: 15. SEQ ID NO: 16 is the amino acid sequence of the agfA SS and His-epitope inserted into the pYA3342 vector.

Example 5. Confirmation of Expression Using PspA Protein

Restriction enzymes to the vectors pMMP62, 63, 64, and 65 prepared with the pspA ( Streptococcus pneumoniae surface protein A) gene for the expression of external proteins through the signal peptide and His-epitope of the vectors constructed in Examples 1 to 4 above Ligation was performed with Eco RI and Hin dIII.

The ligated product was transformed into an asd mutant E. coli x6212 (Infection and Immunity, 70 (4), 1739-1749, 2002) and digested with Eco RI and Hin dIII to confirm the insert. These recombinants cloned were named pMMP66, 67, 68, 70, respectively.

Western blot was performed to measure the expression level of PspA in the transformed E. coli x6212. Primary antibodies are antibodies expressed against PspA in mice (Ho Young Kang, et al. , Infection and Immunity , 70 (4): 1739, 2002) and His-epitope antibodies (Ig) expressed in commercially available mice. Therapy Co.) was used as the primary antibody. As a secondary antibody, an antibody conjugated to horseradish peroxidase (HRP) was used for anti mouse IgG expressed in goat (Stressgen Co.). As a result, as shown in Figure 6, the PspA antibody and His-epitope antibody was detected at a similar level, it can be seen that the expression of the external protein (PspA) occurs stably through this expression vector. As described above, the external antigens cloned in the present vector can be easily identified using His-epitope antibodies without being identified by individual antibodies. Also, compared with pYA3494 (Infection and Immunity, 70 (4), 1739-1749, 2002), which cloned the pspA gene into pYA3493, pMMP68 (derived from pMMP64) with His-epitope added to pYA3493 was similar to that of pYA3494. It was found to express. Therefore, hereinafter, pYA3494 does not have His-epitope, and thus, expression levels were compared using pMMP68 as an internal control without using pYA3494.

Example 6. Confirmation of secretion ability according to the signal peptide

In order to determine the degree of improvement of the external protein secretion ability according to the type of the signal peptide of Examples 1 to 5, it was confirmed using PspA protein. PMMP66, 67, 68, 70 were transformed into S. typhimurium x8554 ( asd A16) (Infection and Immunity, 70 (4), 1739-1749, 2002) to determine the possibility of applying the external antigen transport system to live Salmonella vaccine. It was. The strain may survive when the asd gene is mutated to provide a DAP externally or when a vector capable of expressing the asd gene is transformed.

In order to determine the expression level of total PspA, transformed strains were transformed into S. typhimurium x8554 by incubating the transformed strains with 0.8 L at 37 ° C. and absorbance at 600 nm at 37 ° C., and then aliquoting the culture solution at 10 μl each for 100 ° C. After boiling for 5 minutes, electrophoresis was performed to perform Weston block. In order to confirm the secretion capacity, the supernatant was centrifuged for 5 minutes at 12,000 rpm and cultured in the same culture method, and the supernatant was completely sterilized through a 0.2 μm filter. Trichloroacetic acid (TCA) was added to the sterilized solution to a final concentration of 10%, and then left at 4 ° C for one hour. The precipitated solution was centrifuged at 12,000 rpm for 15 minutes and resuspended in SDS loading buffer (precipitate concentrated 37.5 times compared to the stock solution), and 6 µl (225 µl in terms of stock solution) was loaded onto SDS polyacrylamide gel. After electrophoresis, Weston block was performed.

As a result, as shown in FIG. 7, the expression level of the total PspA was 3.47 times higher than that of the OmpW signal peptide when the Bla signal peptide was 1.00. The signal peptide showed a high expression rate of 1.40 fold.

As a result of measurement of secretion capacity, the amount of PspA secreted was 2.86 times higher than that of β-lactamase signal peptide when the bla signal peptide (β-lactamase signal peptide) was 1.00, and the AgfA signal peptide was β-lactamase. The secretion rate of 1.08 fold and StfA signal peptide were 0.13 fold similar to that of the signal peptide.

From the above results, it was found that in the presence of a signal peptide, the expression and secretion ability of the external protein increased as a leader sequence and due to the role of the signal peptide. Ability and secretion ability was found to be inconsistent.

Therefore, since the difference in the expression ability according to the sequence of the amino acid in each external antigen may be attributed to each signal peptide, it was found that the vector could be appropriately selected according to the expression amount and the external secretion purpose.

The specific parts of the present invention have been described in detail above, and it should be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a schematic diagram showing a method of constructing a vector containing each signal peptide and His-epitope gene.

Figure 2 shows a genetic map of pMMP62 containing ompW SS and His-epitope.

3 contains stfA SS and His-epitope The genetic map of pMMP63 is shown.

4 contains bla SS and His-epitope Gene map of pMMP64 is shown.

5 contains agfA SS and His-epitope Genetic map of pMMP65 is shown.

6 is a clone of the pspA gene in each vector and its expression is examined. The left figure is Western blotting using PspA antibody, and the right is western blotting using His-epitope antibody.

7 is a comparison of the expression and secretion ability of the foreign protein according to the presence of each signal peptide, the left side confirmed all expressed PspA through western blotting, the right side confirmed the secreted PspA through western blotting. Both were confirmed using His-epitope antibody.

<110> Industrial Cooperation Foundation Chonbuk National University <120> Recombinant Vector for Foreign Protein Expression and Secretion <130> P08-E277 <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tcatgaaaaa atttacagtg gcgg 24 <210> 2 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gaattcatgg tgatggtgat gatgtccggc ttcgtgcgcg aacg 44 <210> 3 <211> 101 <212> DNA <213> Artificial Sequence <220> <223> ompW SS and His-epitope <400> 3 ccatgaaaaa atttacagtg gcggcactgg cgttaacaac tcttctctca ggcagcgcgt 60 tcgcgcacga agccggacat catcaccatc accatgaatt c 101 <210> 4 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> ompW SS and His-epitope <400> 4 Met Lys Lys Phe Thr Val Ala Ala Leu Ala Leu Thr Thr Leu Leu Ser   1 5 10 15 Gly Ser Ala Phe Ala His Glu Ala Gly His His His His His His              20 25 30 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tcatgaatac agcagtaaaa gctg 24 <210> 6 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gaattcatgg tgatggtgat gatgaccggt aaaagtcacc gtac 44 <210> 7 <211> 132 <212> DNA <213> Artificial Sequence <220> <223> stfA SS and His-epitope <400> 7 ccatgaatac agcagtaaaa gctgcggttg ctgccgcact ggttatgggt gtttccagct 60 ttgccaatgc tgcgggcagt aatactggta cggtgacttt taccggtcat catcaccatc 120 accatgaatt cg 132 <210> 8 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> stfA SS and His-epitope <400> 8 Met Asn Thr Ala Val Lys Ala Ala Val Ala Ala Ala Leu Val Met Gly   1 5 10 15 Val Ser Ser Phe Ala Asn Ala Ala Gly Ser Asn Thr Gly Thr Val Thr              20 25 30 Phe Thr Gly His His His His His His          35 40 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tcatgagtat tcaacatttc cgtg 24 <210> 10 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gaattcatgg tgatggtgat gatgttcagc atcttttact ttca 44 <210> 11 <211> 131 <212> DNA <213> Artificial Sequence <220> <223> bla SS and His-epitope <400> 11 ccatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 60 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaacat catcaccatc 120 accatgaatt c 131 <210> 12 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> bla SS and His-epitope <400> 12 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala   1 5 10 15 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys              20 25 30 Asp Ala Glu His His His His His His          35 40 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tcatgaaact tttaaaagtg gcag 24 <210> 14 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gaattcatgg tgatggtgat gatggccgcc gttatgatta ccgc 44 <210> 15 <211> 131 <212> DNA <213> Artificial Sequence <220> <223> agfA SS and His-epitope <400> 15 ccatgaaact tttaaaagtg gcagcattcg cagcaatcgt agtttctggc agtgctctgg 60 ctggcgtcgt tccacaatgg ggcggcggcg gtaatcataa cggcggccat catcaccatc 120 accatgaatt c 131 <210> 16 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> agfA SS and His-epitope <400> 16 Met Lys Leu Leu Lys Val Ala Ala Phe Ala Ala Ile Val Val Ser Gly   1 5 10 15 Ser Ala Leu Ala Gly Val Val Pro Gln Trp Gly Gly Gly Gly Asn His              20 25 30 Asn Gly Gly His His His His His His          35 40  

Claims (13)

Recombinant vector for expression and secretion of a foreign protein containing a signal peptide, an epitope for confirming protein expression, and a foreign gene insertion site. The method of claim 1, And said signal peptide is any one or more selected from the group consisting of an OmpW signal peptide ( ompW SS ), a StfA signal peptide ( stfA SS ), and an AgfA signal peptide ( agfA SS ). The method of claim 1, The protein expression label (epitope) is any one or more selected from the group consisting of His epitope, RGS-His epitope, T7 epitope and V5 epitope recombinant vector expression and secretion of the foreign protein. The method of claim 1, A recombinant vector expression and secretion recombinant vector, wherein said foreign gene insertion site has Eco RI, Bam HI, Sal I and Hin dIII cleavage sites. The method according to any one of claims 1 to 4, The recombinant vector is asd gene, the asd ompW signal peptide located downstream of the trc promoter (Ptrc) in the asd gene in the opposite direction to the upstream of the genes, the trc promoter (Ptrc) (ompW SS), of the ompW SS-down Expression of a foreign protein comprising a His-epitope gene located in a stream, a foreign gene insertion site located downstream of the His-epitope gene, and a transcription termination sequence 5ST1T2 located downstream of the foreign gene insertion site; Secretion recombinant vector. The method according to any one of claims 1 to 4, The recombinant vector is asd gene, the asd stfA signal peptide located downstream of the trc promoter (Ptrc) in the asd gene in the opposite direction to the upstream of the genes, the trc promoter (Ptrc) (stfA SS), of the stfA SS-down Expression of a foreign protein comprising a His-epitope gene located in a stream, a foreign gene insertion site located downstream of the His-epitope gene, and a transcription termination sequence 5ST1T2 located downstream of the foreign gene insertion site; Cost Reassembly Vector. The method according to any one of claims 1 to 4, The recombinant vector is asd gene, the asd agfA signal peptide located downstream of the trc promoter (Ptrc) in the asd gene in the opposite direction to the upstream of the genes, the trc promoter (Ptrc) (agfA SS), of the agfA SS-down Expression of a foreign protein comprising a His-epitope gene located in a stream, a foreign gene insertion site located downstream of the His-epitope gene, and a transcription termination sequence 5ST1T2 located downstream of the foreign gene insertion site; Secretion recombinant vector. The method of claim 1, And said foreign protein is selected from the group consisting of antigens, enzymes, polypeptides and hormones. The method of claim 8, The recombinant protein expression and secretion vector of the foreign protein, characterized in that the foreign protein is PspA. A transformed microorganism obtained by introducing a recombinant vector for expression and secretion of the foreign protein of any one of claims 1 to 4 into a host cell selected from the group consisting of bacteria, fungi and yeasts. The method of claim 10, The host cell is a transforming microorganism, characterized in that Salmonella. Method of producing foreign protein comprising the following steps: (a) culturing the transgenic microorganism of claim 10; And (b) identifying the foreign protein in the culture by Weston Block. The method of claim 12, The foreign protein production method of the foreign protein, characterized in that selected from the group consisting of antigens, enzymes, polypeptides and hormones.

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