WO1999067366A1

WO1999067366A1 - Surface expression system of hepatitis b virus surface antigen and core antigen of hepatitis c virus by using ice-nucleation protein

Info

Publication number: WO1999067366A1
Application number: PCT/KR1999/000339
Authority: WO
Inventors: Chul-Joong Kim; Jong-Su Lee; Kwang-Soon Shin; Jae-Gu Pan; Heung-Chae Jung
Original assignee: Korea Green Cross Corporation; Korea Research Institute Of Bioscience And Biotechnology
Priority date: 1998-06-25
Filing date: 1999-06-25
Publication date: 1999-12-29
Also published as: KR20000006463A; EP1012245A1; AU4531899A

Abstract

Disclosed is the expression of foreign proteins on the surfaces of microorganisms. Antigenic proteins originating from pathogens, including the surface HBsAg and the core antigen of HCV, can be effectively expressed on microbial cell surfaces by use of ice-nucleation proteins. The microbes which have the antigenic proteins on their surfaces can be used as attenuated, complex live vaccines.

Description

SURFACE EXPRESSION SYSTEM OF HEPATITIS B VIRUS SURFACE ANTIGEN AND CORE ANTIGEN OF HEPATITIS C VIRUS BY USING ICE-NUCLEATION PROTEIN

Technical Field

The present invention relates to the expression of foreign proteins on the surfaces of microorganisms. More particularly, the present invention relates to surface display vectors carrying ice- nucleation protein genes and method for preparing the same. Also, the present invention is concerned with bacterial strains harboring such surface display vectors and the use of the bacterial strains as complex live vaccines.

Background Art

Recently, extensive attempts have been made to stably express antigens or antigenic determinants of pathogens in the bacteria capable of mass-production by use of genetic engineering techniques. Particularly, the expression of foreign immunogens on cell surfaces has been of high interest in the vaccine developing field because the foreign proteins expressed are easy to separate and purify as well as can be allowed to retain their high immunogenecity. Generally, cell surface proteins of Gram negative bacteria have been utilized in producing foreign proteins. Foreign genes are fused to the genes encoding cell surface proteins and expressed along with the cell surface proteins after the recombinant genes are introduced in the cells. It was reported that Pho E, Lamb B and Omp A proteins, which occur on the outer membranes of bacteria, were used to express foreign proteineous antigens on cell surfaces. In addition, there were attempts to take advantage of flagella, pilli and fimbriae, with which bacteria move and perform cell junction, in expressing foreign proteins on the cell surfaces. For instance, the immunogenecity of the foot-and-mouth disease viral VPI protein was researched by use of a Pho E protein (Agterberg, et al., Vaccine, 8:85-91, 1990). According to the report, an immunogenic determinant of the viral protein is expressed together with a Pho E protein and introduced in a mouse to produce neutralizing antibodies. Another research example of utilizing such a cell surface protein is also found (Charbit, et al., AIDS, 4:545-551 , 1990), in which hybrid proteins, each consisting of a Lam B protein and one of the eight peptides originating from HIV gpl lO, are expressed on the cellular membrane and examined for the immunogenecity of the virus in rabbits. This research succeeded in identifying that one of the peptides is of antigenicity.

It was also reported that non-pathogenic salmonella bacteria were used to express the antigens of virus, bacteria or protozoa and infected to animals in order to examine their immunogenecity. The immunogens expressed on the attenuated Salmonella are of high utility because they have an advantage of being able to induce humoral immunity as well as cellular immunity and mucosal immunity. In fact, corpuscular agglutinin epitope 91 - 108 of influenza virus was fused to a flagella protein of Salmonella and this hybrid protein was expressed in Salmonella Dublin ago A mutant to obtain a live vaccine. When the live vaccine was injected into rabbits, antibodies were elicited. Also, the live vaccine showed 50% protective potency in mice (McEwen, et al, Vaccine, 10:405-41 1 , 1992). The env gene of HIV, encoding an epitope consisting of 18 amino acids, was linked to a flagella gene of Salmonella and expressed in Salmonella Dublin SL5928, a live vaccine strain. The epitope was examined for its antibody formation potency in mice (Newton et al., Res. Microbiol., 146:203-216, 1995).

Disclosure of the Invention

Therefore, it is an object of the present invention to provide a method for expressing a foreign protein on the surface of a microbe which is suitable to prepare a complex live vaccine.

More particularly, the present invention provides a method for expressing a foreign protein of interest, which comprises culturing the microbes transformed with surface display vectors carrying an ice- nucleation protein gene to induce the foreign protein. It is another object of the present invention to provide surface display vectors which take advantage of ice-nucleation proteins in displaying foreign proteins on cell surfaces.

More particularly, the present invention provides surface display vectors pKln, pKlnc-1 and pKlnc-2, in which foreign genes can be inserted, and microbial transformants harboring them. Also, the present invention provides surface display vectors pKIncH, pKlnHc and pKInH, which carry main, neutralizing antibody- formative antigenic determinants of the surface antigen of B-type hepatitis virus (HBsAg), and microbial transformants harboring them. In addition, the present invention provides a surface display vector pKInHcC which carries a main, neutralizing antibody-formative antigenic determinant of the HBsAg and a main antigenic determinant of the core antigen of C-type hepatitis virus (HCV), and a microbial transformant harboring it. The surface display vectors according to the present invention can express their foreign proteins on the surfaces of various microorganism strains. Preferable examples of the strains include E. coli, Salmonella typhi, Salmonella typhimurium, Vibrio cholera, Mycobacterium vobis, Siegella spp., Lactobacilus spp. and Listeria monocytogenes.

The novel strains, Salmonella typhi Ty21 a/pKInHc, Salmonella typhi Ty21a/pKIncH, Salmonella typhi Ty21a/pKIncWH, Salmonella typhi Ty21a/pKInH and Salmonella typhi Ty21a/pKInHcC, which were prepared by transforming the surface display vectors, pKlnHc, pKIncH, pKIncWH, pKInH and pKInHcC, into Salmonella typhi Ty21a, were deposited in Korean Collection for Type Cultures, The Korean Research Institute of Bioscience and Biotechnology on May 12, 1998 and May 27, 1999 and received Deposition Nos. KCTC 047 IBP, KCTC 0472BP, KCTC 0473BP, KCTC 0622BP and KCTC 0619BP.

In the present invention, the transformants are used to express the whole surface HBsAg or a part of the antigen and the core antigen of HCV on microbial cell surfaces.

The microbes on the cell surface of which antigen proteins originating from pathogens are expressed, can be used as complex live vaccines.

Brief Description of the Drawings

The above and other objects and aspects of the invention will become apparent from the following description of embodiments with reference to the accompanying drawings in which:

Fig. 1 is a schematic diagram illustrating the construction of the surface display vectors pKInc-1 and pKInc-2; Fig. 2 is a schematic diagram illustrating the construction of the surface display vectors pKIncH and pKLncWH;

Fig. 3 is a schematic diagram illustrating the construction of the surface display vector pKlnHc; Fig. 4 is a schematic diagram illustrating the construction of the surface display vector pKInH;

Fig. 5 is a schematic diagram illustrating the construction of the surface display vector pKInHcC;

Fig. 6 is a Western immunoblotting result illustrating the expression identification of Hbs antigen protein fused to the N- terminal and C-terminal of an ice-nucleation protein in E. coli and Salmonella typhi Ty21a with anti-S antigen antibody (A and B) and the expression identification of the core antigen protein of HCV with a specific antibody (C). In panels A and C, lane 1 is provided for pKLnc-1, lane 2 for pKInH, lane 3 for pKlnHc, lane 4 for pKIncH, lane 5 for pKInHcC, and lane 6 for pKIncWH. In panel C, lane 1 is provided for pKInc-1 and lane 2 for pKInHcC;

Fig. 7 shows FACS histograms illustrating the location determination of HBs antigens on the surfaces of E.coli and Salmonella typhi Ty21a by use of an anti-S antigen antibody;

Fig. 8 shows antibody valences against S antigen of the sera taken from mice immunized once (A) and twice (B) with Salmonella typhi Ty21a harboring surface display plasmids (□: pKInc-1 ; E_2I : pKInH; S : pKlnHc; H: pKIncH and ϋ: pKInHcC) ; and Fig. 9 shows antibody valences against core antigen of the sera taken from mice immunized once (0) and twice ( 23) with Salmonella typhi Ty21a harboring the surface display plasmid pKInHcC and from mice immunized with Salmonella typhi Ty 21a harboring the surface display plasmid pKInc-1 ([_!)

Best Modes for Carrying Out the Invention

Below, details are given of the present invention.

The present invention provides a technique of expressing an antigenic protein originating from a pathogen on a microbial surface with the aim of developing a complex live vaccine.

For this, there is needed a surface display vector which carries a foreign gene of interest in association with a gene encoding a surface protein and is able to express the hybrid protein on a cell surface. Ice-nucleation proteins, which occur on the outer membranes of Pseudomonas spp., Erwinia spp., Xanthomonas spp., etc, function to promote the formation of ice when they are in supercooled water. In a middle region of an ice-nucleation protein, there is a repeating sequence of 8, 16, or 48 amino acids, which provides a frame of allowing supercooled water molecules to be arranged into ice particles. Ice-nucleation proteins have secretory signals and target signals at their N-terminals and C-terminals, with which they can pass through the inner membranes of cells. In detail, an ice-nucleation protein consists of 1 ,200 amino acids. The repeating sequence between the C- terminal and the N-terminal is involved in the ice nucleation activity and its length can be controlled. While the N-terminal has a function of merely attaching to the outer membrane, the C-terminal plays a role in secreting and targeting the protein to the outer membrane (Green, et al., Mol. Gen. Genet., 215: 165-172, 1988). By virtue of these structural characteristics, an ice-nucleation protein can be used as a surface display recipient.

In accordance with the present invention, a surface display vector is constructed which carries an ice-nucleation protein gene. In this regard, the plasmid pGIN21 (Deposition No. KCTC 8608P) in which an ice-nucleation protein gene of Pseudomonas syringe is cloned, is used to amplify a DNA fragment coding for a partial portion of the repeating sequence, the N terminal region and the C-terminal region through a polymerase chain reaction (PCR). Then, this DNA fragment is inserted in the expression vector pKK223-3 to construct a surface display vector carrying a gene which codes for a portion of the repeating amino acid sequence, the N-terminal region which attaches to the cell surface, and the C-terminal region which serves to secrete and target the protein to the outer membrane. The expression vector pKK223-3, commercially available from Pharmacia Biotech, a potent tac promoter which is regulated by a lac repressor such that isopropyl -D-thiogalactoside [IPTG] can play a role as an inducer. Downstream the tac promoter is located the multicloning site of pUC 8, followed by a potent rrn B ribosomal terminator. In addition to the expression vector pKK223-3, various expression vectors, if they comprise a tac promoter, are available in the present invention. Also, useful are the plasmids comprising a tac promoter. For example, an ice-nucleation protein gene is inserted downstream the trc promoter and then, conjugated with a foreign gene. Upon expression, a hybrid protein comprising the ice-nucleation protein and the foreign protein can be located on the cell surface. Alternatively, vectors comprising a T5 or T7 promoter may be used in the present invention. Genes coding for the N-terminal and C-terminal regions of an ice-nucleation protein and a surface antigen gene of HBV may be inserted in a vector comprising a T5 or T7 promoter and the recombinant vector may be transformed into E. coli or Salmonella typhi Ty21a. The surface antigen can be expressed on the cell surface by culturing the transformant. The vectors comprising a tac promoter and a trc promoter are preferably exemplified by pDR540 (Pharmacia Biotech) and pTrc99A (Pharmacia Biotech), respectively. Referable Examples of the vectors comprising T7 promoter include pET (Stratagene) and pGEMEX (Promega). As a vector comprising a T5 promoter, pQE (Qiagen) is preferred.

In this expression display vector, a foreign gene may be inserted only at the end of the N-terminal, between the N-terminal and the C- terminal, only at the end of the C-terminal, or at both ends of the C- terminal and the N-terminal. From this recombinant vector, the hybrid protein can be stably expressed on the cell surface. In addition, various surface display vectors may be constructed by controlling the length of the repeating sequence of the ice-nucleation protein or by providing various restriction enzyme recognition sites to the insertion sites.

With reference to Fig. 1, there are schematic diagrams illustrating the construction of basic surface display vectors, named pKIn, pKInc-1, and pKInc-2. In order that a foreign protein is synthesized in a cell, passes through the inner membrane and succeeds in attaching onto the cell surface, the corresponding foreign gene must be ligated to the ice-nucleation protein gene without disturbing its open reading frame and the resulting vector is transformed in the cell. The expression of the hybrid protein on the cell surface may be induced with the aid of an inducer, for example, IPTG.

In accordance with the present invention, a recombinant surface display vector is constructed which is available to produce a complex live vaccine. In this regard, various foreign genes, including genes encoding antigenic proteins of pathogens, are inserted in the basic surface display vectors, pKIn, pKIn-1 and pKIn-2.

In the basic surface display vector pKInc-2, the whole S antigen gene of HBV is inserted at the 3 '-end of the ice-nucleation protein gene, to obtain a surface display vector pKIncWH. First, using as a template the vector pUHBl anchoring an S antigen gene of HBV, the whole S antigen gene is amplified by PCR. This gene is inserted in the basic surface display vector pKInc-2 to construct the surface display vector pKIncWH which is, then, transformed into E. coli and Salmonella typhi Ty21a (typhoid- prophylactic vaccine strain). By culturing these strains, the surface expression of the surface HBsAg is induced.

For identifying the surface expression of the foreign protein, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and Western immunoblotting techniques are useful. Alternatively, a fluorescence-activating cell sorting (FACS) technique may be used to identify the surface expression of the foreign protein. The identifying techniques need an anti-S antigen antibody. In the present invention, the S antigen protein is expressed on cell surfaces and a fraction of the hybrid protein (about 63 kDa) reacts specifically with an anti-S antigen antibody as identified by these techniques.

In accordance with the present invention, surface display vectors, pKInH, pKlnHc and pKIncH, each of which carries a gene encoding a neutralizing antibody-formative antigenic determinant of the S antigens of HBV, are constructed from the basic surface display vectors, pKIn, pKInc-1 and pKInc-2.

First, a gene corresponding to an immunogenic determinant formative of a neutralizing antibody is amplified from a vector (pUHBl) in which the S antigen gene of HBV is cloned, by PCR. This gene is inserted at the 5'-end or the 3'-end of the ice-nucleation protein gene or between the 5 '-end and the 3'-end in pKIn, pKInc-1 and pKInc- 2.

To induce the surface expression of the S antigenic determinant ofHBV, the surface display vectors, pKInH, pKIncH and pKlnHc, are transformed into E. coli and Salmonella typhi Tylla which are then cultured. The expression of the antigenic determinant is identified by SDS-PAGE and Western immunoblotting using an antibody against the S antigen. As a consequence of these techniques, the hybrid proteins (about 29 Kda for pKInH, about 37 Kda for pKlnHc, and about 39 kDa for pKIncH) were found to react specifically with the S antibody.

In accordance with the present invention, a surface display vector, named pKInHcC, which carries a gene encoding a main antigenic determinant of the core antigen of the HCV, is constructed from the basic surface display vector pKlnHc. First, a vector (p740) in which a gene for a structural protein of C type hepatitis virus is cloned, is used as a template from which a gene corresponding to the immunogenic determinant formative of a neutralizing antibody is amplified through PCR. This amplified gene is inserted in the basic surface display vector, pKlnHc.

The resulting surface display vector pKInHcC is transformed into E. coli and Salmonella typhi Ty21a which are then cultured to induce the surface expression of an S antigenic determinant of HBV and a main antigenic determinant of C-type hepatitis viral core antigens. The expression of the antigens can be identified by SDS- PAGE and Western immunoblotting using antibodies against S antigen and HCV. As a consequence, a fraction of a hybrid protein (about 48 KDa for pKInHcC) is identified to show a specific reaction with the S antibody and HCV antibody. The vectors pKIncH, pKlnHc and pKInH are the same, except that the inserted genes for antigenic determinants have different restriction enzyme recognition sites at their ends. They are utilized in examining whether the expression of the antigenic determinants is dependent on the position of the N-terminal and C-terminal of the ice- nucleation protein. In addition, the surface expression of the antigenic determinants can be recognized with the N-terminal only. Where the N-terminal and C-terminal are utilized at the same time, it can be identified whether at least two kinds of antigens are expressed on cell surfaces. In accordance with the present invention, as live vaccines are provided microorganisms on the surface of which the foreign proteins are expressed. For this, the microorganisms are injected to animals to examine their immunogenecity. For instance, the surface display vectors are transformed into E. coli and Salmonella typhi Ty21a and the strains which are recognized to have the antigens expressed on their surfaces are injected to mice. After a predetermined period of time, sera are taken from the mice and examined for the formation of antibodies against S antigen antibodies against a core antigen with the aid of an enzyme-linked immunosorbent assay (ELISA) technique. The bacteria harvested after transformation are washed many times with buffers and injected peritoneally at a predetermined concentration in mice.

It should be noted that the strains above suggested to produce foreign proteins and live vaccines by use of ice-nucleation proteins are set forth to illustrate, but not to limit the present invention. A better understanding of the present invention may be obtained in light of the following examples which are set forth to illustrate, but are not to be construed to limit the present invention.

EXAMPLE I: Construction of Surface Display Vectors pKIn, pKInc-1 and pKInc-2

In order to construct surface display vectors which take advantage of the N-terminal or the N-terminal and C-terminal of an ice-nucleation protein, a conventional expression plasmid pGINP21 (Deposition No. KCTC 8608P) with a size of 7.1 kb was used. First, DNA fragments coding for the N-terminal region and the C-terminal region of the ice-nucleation protein were obtained. For this, primer sets, Sequence 1 (5'-CGGAAGTCATGAATCTCGACAAGGCG-3') and Sequence 2 (5'-TCCCCCGGGAATTAGATCACTGTGGTT-3') for an N-terminal region, Sequence 3 (5'-

AAACTGCAGGCTGGTAAGAACCGTGTC-3') and Sequence 4 (5'- CCCAAGCTTCTTTACCTCTATCCAGTG-3') for a C-terminal region, and Sequence 5 (5'- TCCCCCGGGGGCGGGCATGACTGCACC-3') and Sequence 6 (5'- AACTGCAGCTTTACCTCTATCCAGTG-3') for a C-terminal region, were synthesized and used to amplify the DNA fragments corresponding to the regions from the expression plasmid through PCR. In order to insert the amplified DNA fragments in cloning plasmids, restriction enzyme recognition sites present in the plasmids were provided to them. Upon the synthesis of the primers, Sequence 1 was designed to be provided with an EcoRI recognition site, Sequence 2 with a Smal recognition site, Sequence 3 with a Pstl, Sequence 4 with a Hindlll recognition site, Sequence 5 with a Smal recognition site, and Sequence 6 with a Pstl recognition site. These restriction enzyme recognition sites all were also present in the expression vector pKK223-3. Using these primer sets, there was obtained a DNA fragment about 620 bp long with a code for an N-terminal region of an ice-nucleation protein while two DNA fragments about 240 bp and 300 bp long, respectively, were amplified with a code for N-terminal regions of the ice-nucleation protein.

After being digested with appropriate restriction enzymes (EcoRI and Sma I, Pstl and Hindlll, and Smal and Pstl), the DNA fragments were inserted in pKK223-3 which was also treated with the same restriction enzymes. As a result, there were obtained a vector about 4.7 kb in size, which carries a DNA fragment corresponding to an ice-nucleation peptide stretch from the N-terminal region to a partial potion of the repeating sequence, and two vectors about 5.0 kb in size, which each carries a DNA fragment corresponding to a C- terminal region of the ice-nucleation. The basic surface display vectors were named pKIn, pKInc-1 and pKInc-2, respectively. In the case of pKInc-2, Bglll, Ncol, BamHI and EcoRI recognition sites, together with a linker (GAT CAA CAA GGA GGA), were provided to the back of the Inpc gene in order to allow the easy insertion of foreign genes. The construction procedures for pKIn, pKInc-1 and pKInc-2 are illustrated in Fig. 1.

EXAMPLE II: Construction of Surface Display Vector pKIncWH

There was constructed a surface display vector, named pKIncWH, which is able to display the whole S HBsAg on bacterial surfaces by taking advantage of the N-terminal and the C-terminal of an ice-nucleation protein. A gene for the whole S HBsAg was obtained by amplifying a 1.4 kb gene cloned in a general vector pUC8 (pUBl) through PCR with a primer set of Sequence 7 (5'- CATGCCATGGATGGAGAACATCACATA-3') and Sequence 8 (5'- CCGGAATTCTTAAATGTATACACCCAGA-3'). The amplified gene was about 680 bp in size which corresponds to the whole S antigen gene.

Sequences 7 and 8 were designed to have Ncol and EcoRI recognition sites, respectively, which are also in the multicloning site of the expression vector pKK223-3. Thus, this PCR product containing the whole S antigen gene was digested with Ncol and EcoRI and ligated to the ice-nucleation gene of pKK223-3 at C- terminal without disturbing the open reading frame. The construction procedure of pKIncWH is illustrated in Fig. 2.

EXAMPLE III: Surface Expression of Whole S HBsAg on E. coli.

An examination was made on the expression of the viral whole S antigen on the cell surface of bacteria. First, the surface display vector pKIncWH was transformed into E. coli which was, then, mass-cultured in a 500 ml flask containing 50 ml of an LB medium (Yeast Extract 5g/L, Trypton lOg/L, NaCl 5g/L, pH 7.0) added with 100 mg/1 of ampicillin.

The hybrid protein in which the S antigen was fused between the N-terminal and the C-terminal of the ice nucleation protein, was found to be expressed on the cell surface of E. coli as identified by SDS- PAGE and Western immunoblotting techniques using an anti-S antigen antibody. In this regard, proteins were obtained from the same concentrations of cultured bacteria, then denatured and analyzed by SDS-PAGE, after which the separated proteins were transferred to a PVDF membrane. Then, the blotted PVDF membrane was treated for 1 hour in a blocking buffer (50 mM Tris-Cl, 5% skim milk, ph 8.0) with shaking and reacted with a primary anti-S antigen goat polyclonal antibody 1,000-folds diluted in the blocking buffer for 16 hours. After completion of the reaction, the membrane was washed with a PBS buffer, treated with avidin-biotin reagent for 1 hour and washed again. A coloring reaction was conducted in which H₂0₂ and a DAB solution were used as a substrate and coloring reagent, showing the specific binding of the anti-S antigen goat antibody to the hybrid protein as illustrated in Fig. 6. the hybrid protein expressed from the pKIncWH plasmid was measured to be about 63 KDa in size as analyzed by the Western immunoblotting.

In addition, the surface expression of the S antigen with the aid of the N-terminal and C-terminal of the ice-nucleation protein was identified by FACS flow cytometry. For immunofluorescence staining, the bacteria cultured were harvested at the same concentrations, washed many times with PBS buffer (pH 7.4), suspended in 1 ml of a solution of 1% bovine serum albumin in PBS buffer and treated with a 1, 000-fold diluted solution of a biotin- conjugated, secondary antibody at 4 C for 3 hours. Thereafter, the cells were washed many times with PBS buffer, resuspended in 1 ml of a solution of 1% bovine serum albumin in PBS buffer and treated with a 1, 000-fold diluted solution of biotin-specific streptavidin-R- phycoerythrin staining reagent. After being washed many times with PBS buffer, the E. coli was subjected to FACS flow cytometry. In consequence, as shown in Fig. 7, the transformed E. coli with the surface display vector was identified to have the S antigen protein on its cell surface as compared with the control E. coli , non-transformed.

EXAMPLE IV: Surface Expression of Whole S HBsAg on Salmonella An examination was made on the expression of the viral whole S antigen on the cell surface of Salmonella. To this end, first, the surface display vector pKIncWH was transformed into Salmonella typhi Ty21a which was, then, mass-cultured in a 500 ml flask containing 50 ml of a tryptic soy medium (exclusive of dextrose) added with 100 mg/1 of ampicillin.

The surface expression of the viral whole S antigen fused to the ice-nucleation protein on Salmonella typhi Ty21a was identified by conducting the SDS-PAGE and Western blotting and the FACS flow cytometry in a similar manner to that of Example III. In the FACS flow cytometry, the Salmonella typhi Ty21a cultured was treated with an anti-S antigen polyclonal antibody, a biotin-conjugated, secondary antibody, and biotin-specific streptavidin-R-phycoerythrin, in sequence. As apparent from Fig. 7, the transformed Salmonella typhi Ty21a with the surface display vector was identified to have the S antigen protein on its cell surface as compared with the control Salmonella, non-transformed.

EXAMPLE V: Construction of Surface Display Vector pKIncH

There was constructed a surface display vector, named pKIncH, which is able to display a neutralizing antibody-formative antigenic determinant of the S HBsAg on bacterial surfaces by taking advantage of the N-terminal and the C-terminal of an ice-nucleation protein. A gene for the neutralizing antibody-formative antigenic determinant of the S HBsAg was obtained by amplifying a gene ofHBV cloned in a general vector pUC8 (pUBl) through PCR with a primer set of Sequence 9 (5'-GGAAGATCTCAAGGTATGTTGCCCGTT-3') and Sequence 10 (5^*-GGAAGATCTTTACCAGGACGATGGGAT-3'). The amplified gene was about 168 bp in size which corresponds to the neutralizing antibody-formative gene ofHBV.

Sequences 9 and 10, both, were designed to have a Bglll recognition site which is also in the multicloning site of the expression vector pKK223-3. Thus, this PCR product containing the neutralizing antibody-formative gene was digested with Bglll and ligated to the ice- nucleation gene of pKInc-2 at C-terminal without disturbing the open reading frame. The construction procedure of the resulting vector pKIncH is illustrated in Fig. 2. EXAMPLE VI: Surface Expression of Neutralizing Antibody- Formative Antigenic Determinant of HBV on Bacterial Cell

An examination was made on the expression of the neutralizing antibody-formative antigenic determinant on bacterial cell surfaces. To this end, first, the surface display vector pKIncH was transformed into E. coli and Salmonella typhi Ty21a which were, then, mass- cultured following the same procedures as illustrated in Examples III and IV, respectively. The surface expression of the hybrid protein on E. coli and

Salmonella typhi Ty21a was identified by conducting SDS-PAGE and Western immunoblotting in a similar manner to that of Example III. For the Western immunoblotting, an anti-S antigen antibody was used. The result is shown in Fig. 6. A band for the hybrid protein expressed from the pKIncH was observed at a molecular weight of about 40 KDa as shown in Fig. 6. The same FACS flow cytometry as in Examples III and IV was carried out to confirm the surface expression of the hybrid protein on E. coli and Salmonella typhi Ty21a. As a result, the neutralizing antibody-formative antigenic determinant of the S antigen was expressed in a hybrid form fused to the N-terminal and C-terminal of the ice-nucleation protein, as shown in Fig. 7.

EXAMPLE VII: Construction of Surface Display Vector pKlnHc

There was constructed a surface display vector, named pKlnHc, which is able to display a main neutralizing antibody-formative antigenic determinant of the S HBsAg on bacterial surfaces by taking advantage of the N-terminal and the C-terminal of an ice-nucleation protein. A gene for the neutralizing antibody-formative antigenic determinant of the S HBsAg was obtained by amplifying a gene ofHBV cloned in a general vector pUC8 (pUBl) through PCR with a primer set of Sequence 11 (5'-

TCCCCCGGGCAAGGTATGTTGCCCGTT-3') and Sequence 12 (5'- GGTTCTGCAGCCAGGACGATGGGATGGG-3'). The PCR product thus obtained was about 168 bp in size which corresponds to the neutralizing antibody-formative gene ofHBV.

Sequences 9 and 10, both, were was designed to have Smal and Pstl recognition sites, respectively, which are also in the multicloning site of the expression vector pKK223-3. Thus, this PCR product containing the neutralizing antibody-formative gene was digested with Smal and Pstl and ligated to the ice-nucleation gene of pKInc-1 between the N-terminal and C-terminal without disturbing the open reading frame. The construction procedure of the resulting vector pKlnHc is illustrated in Fig. 3.

EXAMPLE VIII: Surface Expression of Neutralizing Antibody- Formative Antigenic Determinant ofHBV on Bacterial Cell

Salmonella typhi Ty21a was identified by conducting SDS-PAGE and Western immunoblotting in a similar manner to that of Example III. For the Western immunoblotting, an anti-S antigen antibody was used. The result is shown in Fig. 6. A band for the hybrid protein expressed from the pKlnHc was observed at a molecular weight of about 37.7 KDa as shown in Fig. 6. The same FACS flow cytometry as in Example III and IV was carried out to confirm the surface expression of the hybrid protein on E. coli and Salmonella typhi Ty21a. As a result, the neutralizing antibody-formative antigenic determinant of the S antigen was expressed in a hybrid form, as apparent in Fig. 7.

EXAMPLE IX: Construction of Surface Display Vector pKInH

There was constructed a surface display vector, named pKInH, which is able to display a main, neutralizing antibody-formative antigenic determinant of the S HBsAg on bacterial surfaces by taking advantage of the N-terminal of an ice-nucleation protein. A gene for the neutralizing antibody-formative antigenic determinant of the S HBsAg was obtained by amplifying a gene ofHBV cloned in a general vector pUC8 (pUBl) through PCR with a primer set of Sequence 1 1 (5*-TCCCCCGGGCAAGGTATGTTGCCCGTT-3') and Sequence 13 (5'-CCCAAGCTTTTACCAGGACGATGGGAT-3'). The PCR product thus obtained was about 168 bp in size which corresponds to the neutralizing antibody-formative gene ofHBV. Sequences 1 1 and 13, both, were designed to have Smal and Hindlll recognition sites, respectively, which are also in the multicloning site of the expression vector pKK223-3. Thus, this PCR product containing the neutralizing antibody-formative gene was digested with Smal and Pstl and ligated to the ice-nucleation gene of pKIn at the N-terminal without disturbing the open reading frame. The construction procedure of the resulting vector pKInH is illustrated in Fig. 4.

EXAMPLE X: Surface Expression of Neutralizing Antibody- Formative Antigenic Determinant ofHBV on Bacterial Cell

An examination was made on the expression of the neutralizing antibody-formative antigenic determinant on bacterial cell surfaces. To this end, first, the surface display vector pKInH was transformed into E. coli and Salmonella typhi Ty21a which were, then, mass- cultured following the same procedures as illustrated in Examples III and IV, respectively.

The surface expression of the hybrid protein on E. coli and Salmonella typhi Ty21a was identified by conducting SDS-PAGE and Western immunoblotting in a similar manner to that of Example III. For the Western immunoblotting, an anti-S antigen antibody was used. The result is shown in Fig. 6. A band for the hybrid protein expressed from the pKlnHc was observed at a molecular weight of about 29 KDa as shown in Fig. 6. The same FACS flow cytometry as in Example III and IV was carried out to confirm the surface expression of the hybrid protein on E. coli and Salmonella typhi Ty21a. As a result, the neutralizing antibody-formative antigenic determinant of the S antigen was expressed, as apparent in Fig. 7.

EXAMPLE XI: Construction of Surface Display Vector pKInHcC

There was constructed a surface display vector, named pKInHcC, which is able to display a main, neutralizing antibody- formative antigenic determinant of the S antigens of HBV and a main, antigenic determinant of the core antigen of HCV on bacterial surfaces by taking advantage of the N-terminal and the C-terminal of an ice- nucleation protein. A gene for the antigenic determinant of the core antigen of HCV was obtained by amplifying a plasmid p740 carrying the gene of interest through PCR with a primer set of Sequence 14 (5'- CCCAAGCTTGATCCAGGAAGCACAAATCCTAAA-3') and

Sequence 15 (5'-CCCAAGCTTACCCAAATTACGCGACCT-3'). The PCR product thus obtained was about 342 bp in length which corresponds to the main antigenic determinant gene of the core antigen of HCV.

Sequences 14 and 15, both, were designed to have a Hindlll recognition site, which is also in the multicloning site of the expression vector pKK223-3. Thus, this PCR product containing the neutralizing antibody-formative gene was digested with Hindlll and ligated to the ice-nucleation gene of pKlnHc at C-terminal without disturbing the open reading frame. The construction procedure of the resulting vector pKInHcC is illustrated in Fig. 5.

EXAMPLE XII: Surface Expression of Neufralizing Antibody- Formative Antigenic Determinant of HBV and Antigenic Determinant of Core Antigen of HCV on Bacterial Cell

An examination was made on the expression of the antigenic determinants anchored in pKInHcC on bacterial cell surfaces. To this end, first, the surface display vector pKInHcC was transformed into E. coli and Salmonella typhi Ty21a which were, then, mass-cultured following the same procedures as illustrated in Examples III and IV, respectively.

The surface expression of the hybrid proteins on E. coli and Salmonella typhi Ty21a was identified by conducting SDS-PAGE and Western immunoblotting in a similar manner to that of Example III. For the Western immunoblotting, an anti-S antigen antibody and an anti-core antigen antibody were used. The result is shown in Fig. 6. A band for the hybrid protein expressed from the pKInHcC was observed at a molecular weight of about 48 KDa as shown in Fig. 6. The same FACS flow cytometry as in Example III and IV was carried out to confirm the surface expression of the hybrid protein on E. coli and Salmonella typhi Ty21a. The result is shown in Fig. 7.

EXAMPLE XII: Bioassay for Complex Live Vaccine

Into Salmonella typhi Ty21a were transformed the surface display vectors constructed in Examples II, V, VII, IX and XI, followed by culturing the bacteria to induce the expression of the proteins of interest in the same manner as that of Example IV. Thereafter, the whole S HBsAg, the neutralizing antibody-formative antigenic determinant of the S antigen, and the antigenic determinant of the core antigen of C-type, all being fused to an ice-nucleation protein, were assayed for antigenicity in vivo.

In this regard, the surface display vectors, pKIncWH, pKIncH, pKInH and pKInHcC, were transformed into Salmonella typhi Ty21a, after which the antigen expression was identified. The Salmonella typhi Ty21a which had the antigens on its cell surface was injected at a predetermined dose to BALB/c mice. At an interval of a predetermined period of time, blood was taken from the BALB/c mice. Before injection, the Salmonella harboring the plasmids was washed many times with PBS buffer (pH 7.4). The bacteria were peritoneally injected at a dose of 2xl0⁹ cells to each of the BALB/c mice. They were divided into two groups after the injection. From one group, blood was taken at 2, 3 and 5 weeks after the injection. On the other hand, the other group was re-administered with the bacteria at one week after the primary injection and blood was taken at 2, 3 and 5 weeks later. The blood samples were subjected to ELISA. Positive results were obtained when the antibody valences for the S antigen and the Core antigen were compared with those for a control, as shown in Figs. 8 and 9.

Industrial Applicability

As described hereinbefore, the present invention is very advantageous in mass-producing, separating and purifying vaccines because antigenic proteins originating from pathogens can be effectively expressed on microbial cell surfaces by use of ice- nucleation proteins. In addition, the microbes which have the antigenic proteins on their surfaces can be used as attenuated, complex live vaccines. Further, the surface display vectors and expression techniques according to the present invention can be applied for various fields, including the screening of various antigens and antibodies and the immobilization of enzymes.

The present invention has been described in an illustrative manner, and it is to be understood the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described. 3Ut>A.<>EST -3J ATY OV THE IXTE^ VATIONA RECOGMT!0N OF "T E DEPOSIT OF JCXOORGAMSMS FOR THE PURPOSE OP "ATEXT PROCEDURE

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7 1 O- r rri. C ui- oorg

Han oolADt. 103-3C 1. Shir-surig-ccrig , Y'-isoπg-.-cu. Tae_jon 305-345 PSΌU DJJC of Korea

I. IDENTIFICATION OF THE MICROORGANISM

[denπncaπon reference given by the Accession numoer given oy the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY

Salmonella typhi Ty21a/pK7nHc

KCTC 047 IBP π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompanied by:

[ x ] a scientific description

[ ] a proposed taxonomic designation

(Mark with a cross where appkcaoie)

El. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I aoove. whicn was received oy it on May 12 1998. rv. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this Intemaπonal Depositary Authority on and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on

V INTERNATIONAL DEPOSITARY AUTHORITY

Name: K orea R esearch Institute o f Sιgnature(s) of person(s) navmg the power to B tαscien ce and B io tech no lo gy reσresent the International Depositarv K orean Colle ction fα rTy pe C ulture s Authority or of authorized officιal(s):

Address: KCTC. KRTBB

452, Oun-dong. Yusong-ku.

Taejόn 305-333. Kyung Sook Bae. Curator Republic of Korea Date: May 16 1998

Form BP 4 fKCTCForm 17) BUDAPEST ON "KE RECOOTTiCV OP THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PAΪΪ.VT PROCEDURE

ΓNTERNATION.AL FORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued Dursuant to Rule ^" 1

TO:_, rLrn, C ui-Jcong

HanwoolA.pt. 103-301. Shinsurt -dcng, Yuscng-^'r , Tae_jon 305-345, Reuuoic of Korea

[. IDENTIFICATION OF THE MICROORGANISM

Identiπcation reference given oy me accession numoer given ov the

DEPOSITOR. INTERNATIONAL DEPOSIT.ARY

AUTHORΓΓΎ

Salmonella typhi Ty21a/pKIιιcH

KCTC 0472BP π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED T.AXONΌMIC DESIGNATION

The microorganism identified under I aoove was accomcanied by:

[ x ] a scientific description

[ j a proposed taxonomic designation

(Man: wth a cross where applicable) m. RECEIPT .AND ACCEPTANCE

This International Depositary Authoπty accents the microorganism lαenαπed under I aoove. wnicn was received by it on May 12 1998. rv. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I aoove was received oy mis International Depositary Authoπty on and a request to convert the oπginal deoosit to a deposit under the Budapest Treatv was received bv it on

V. INTERNATIONAL DEPOSIT.ARY AUTHORITY

Name: Korea Research Institute of 3ιgnature(s)of person(s) having the power to

Bϊoscieπce and Biotechnology represent tne international Depositary

Korean Collection forType Cultures Authoπty or of authorized offϊcιal(s):

Address: KCTC. KKTBB 52. Oun-dong, Yusong- rα.

Taejon 305-333, Kyung Sook 3ae. Curator

Republic of Korea , Date: May 16 1998

rora BPW (KC C Form IT) wuc 0»s« 3UDΛJΕST TREATY ON THE -STERNATIONAI. R≤CCCMTIO.V Or THE OEPOSrT OF flCROORCAjVLSMS FOR THE PURfCSE OF 'ATENT 'ROCΞDURE

ΓNTERNATIONAL FORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 O- Kim. C ul-Joong

HanwcolApt. 103-301, S ms'-ng-cong, Y song- . Taejoπ 305-345, RεrjuDiic of Korea

ΓDENTTFTCATION OFTHΞΛHCRCORG.AMSM

Identification reference given by the Accession number given oy the

DEPOSITOR: INTERNATIONAL DEPOSITARY

AUTHORITY Salmonella typhi Ty21a/pK7ncWΗ

KCTC 0473BP

EL SCIENTIFIC DESCRIPTION .AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompanied by:

[ x ] a scientific description

[ ] a proposed taxonomic designation

(Mark: -with a cross wnere applicable) m. RECEIPT AND ACCEPTANCE

This intemauonal Depositary Authoπty accepts the microorganism identified under I above, wnicn was received by it on May 12 1998.

HZ. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authoπty on and a request to convert the oπginal deposit to a deposit under the Budat>est Treaty was received by it on

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: orea Research Institute of Signarurε(s)of person(s) having the power to

8ioscιeπce and Biotechnology reoresent the International Depositary orean Collection far Type Cultures Authoπty or of authorized officials):

Address: KCTC. KRIBB 52, Oun-dong. Yusόng- u,

Taejόn 305-333. Kyung Sook 3ae. Curator

Republic of Korea Date: May 16 199β

Form BP (XCTCForm IT) soic ptge BUDAPEST TΠEΛTΎ ON THE INTERNATIONAL RECOGNITION OF TΠF ΠFPOSIT CF

PROCEDURE

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 71

KIM Cιιi-jccr_g

1-ιan'λτsH Act 103-301, Ξhins'j g-c ng,

jcr.2C5-245, ' cpualic of Korea

DENTIFICΛTION OF THE MICROORGANISM

Accession number given by the

Idenci ic;:."- re'V eπce i en by the INTERNATIONAL DEPOSITARY DP.PCSiT-R AUTHORITY^"

Salmonella typhi

KCTC 0619RP Ty21a/p InIIcC

π SCIENTIFIC DESCRIPTION ANTVOR PROPOSED T.AXCN^'0.\ιI3 DE lGX.V'TO.N

T micrccrganisrn identified under I above was ac om anie by

[ x ] a scientific description

[ J ϋ μr-iTKJHed laxυπυmic designation

(Mark with a cross where applicable) RCF:?T AND ACCEPTANCE

L his Inter-, .noπal Depositary Authoπty accepts the microorganism idenrified under ! above which ^>.v-= received by it on May 27 1999. rv τt?.c.?: ~ OF REQUEST FOR CONVERSION

The. micr -organism identified under I above was received by "his Intern a onal Depositary Aurhurr.- z" and a request to convert ^;1v ^r.έv'J¹ C-' ΠM. > _c c epυsit under tr.e Budapest Treaty was received by it on

L\TE?..\^'ATIONAL DEPOSITARY AUTHORITY

N^',im«- Korean Collection for Type Cultures

of person(s) having the power to represent the International Depositary Author.v o: authorized oπiciaJ ¹

Vddress Korea Research Institute of 3;oscιence and Biotechnology KRIBB) *5-i, Oun-dcπg, lusong-ku,

Tacjcn 305-333, BAE y -,τ ?' k, Director Re ubiic o:^' Korea Date Mav 3) 1 f 3

RP J K.~ 3UDATΕST TTtEATY ON THE LNTOINATIONAI. RECOGNITION OF THE DEPOSIT OF MICaOORGANISMK FOR THE ?UWOSE OF PATENT PKOCBUURS

INTERNATIONAL FORM

RECEIPT N THE CASE OF AN ORIGINAJL DEPOSIT issued pursuant to Rule 7.1 : KTΛl, C'πul-Joong

H nvv^'∞l Apt. 1G3--801, Shinsung-doπg, Yuscng-ku, Taejon 3C6-345, "Republic of Korea

I . IDENTIFICATION OF THE MICROORGANISM

Identi fication reference given by the Accession number given by the INTERN ATIONΛL DEPOSITARY DEPOSITOR AUTHORITY.

Salmonella typhi

KCTC 0622BP Ty21a/pkInH

0 SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was -xxomparued by:

I x ] a scientific: description

[ ] a proposed taxonomic designation

(Mark with a cross where applicable)

m RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I above, which was received by on May 27 1999.

IV^'. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this Intemauonal Depositary Authority on and a request to convert the original deposit ro a deposit under the Budapest Treaty was received by it on

V . INTERNATIONAL DEPOSITARY AUTHORITY

Name. Korean Collection for Type Cultures Signacure(s) of person ⁽s) h;ιvintf the power to represent the International Depositary Authority of authorized official (s):

Address^' Korea Research Institute of Biuicience and Biotechnology (KRIBR)

=52, Oun-dong, Yusong-ku,

Taejon 305-333, BAΞ Kyung Sook, Director Republic of Korea Date May 31 1999

BP/-1 (KCTC Form 1?⁾ ola a^e

Claims

1. A surface display vector, derived from a base vector comprising a tac promoter, a trc promoter, a T7 promoter, or a T5 promoter, wherein the base vector carries a gene for an ice-nucleation protein, said gene coding for a portion of the middle repeating sequence of the ice nucleation protein, a region of functioning to attach the protein onto a cell surface, and/or a region of functioning to secrete and target the protein to an outer membrane.

2. A surface display vector as set forth in claim 1 , wherein the base vector comprising the tac promoter is pKK223-3 or pDR540.

3. A surface display vector as set forth in claim 1 or 2, wherein the base vector comprising the tac promoter is pKK223-3.

4. A surface display vector as set forth in claim 1 , wherein the base vector comprising the trc promoter is pTrc99A.

5. A surface display vector as set forth in claim 1 , wherein the base vector comprising the T7 promoter is pET or pGEMEX.

6. A surface display vector as set forth in claim 1 , wherein the base vector comprising the T5 promoter is pQE.

7. A surface display vector as set forth in claim 1 , wherein the ice-nucleation protein comes from Pseudomonas spp. Erwinia spp. and Xanthomonas spp.

8. A surface display vector as set forth in claim 3, wherein said surface display vector is the vector pKLnc-1 shown in a gene map of Fig. 1 and is prepared by treating an N-terminal gene of the ice- nucleation protein with EcoRI and Smal, a C-terminal gene of the ice- nucleation protein with Pstl and Hindlll and the base vector pKK223-3 with EcoRI, Smal, Pstl and Hindlll and inserting the truncated N- terminal gene and C-terminal gene in the truncated pKK223-3.

9. A surface display vector as set forth in claim 3, wherein said surface display vector is the vector pKLnc-2 shown in a gene map of Fig. 1 and is prepared by treating an N-terminal gene of the ice- nucleation protein with EcoRI and Smal, a C-terminal gene of the ice- nucleation protein with Smal and Pstl and the base vector pKK223-3 with EcoRI, Smal and Pstl and inserting the truncated N-terminal gene and C-terminal gene in the truncated pKK223-3.

10. A surface display vector as set forth in claim 9, wherein said surface display vector is the vector pKLnWH in which a whole S antigen gene ofHBV is inserted at the NcoI-EcoRI site of the C- terminal gene of the pKlnc-2 vector.

1 1. A surface display vector as set forth in claim 9, wherein said surface display vector is the vector pKLncH in which a gene encoding a main, neutralizing antibody-formative antigenic determinant of the S HBsAg is inserted at a Bglll site of the C-terminal gene of the pKlnc-2 vector.

12. A surface display vector as set forth in claim 8, wherein said surface display vector is the vector pKLnHc in which a gene encoding a main, neutralizing antibody-formative antigenic determinant of the S HBsAg is inserted at a Smal-Pstl site between the C-terminal gene and the N-terminal gene in the pKlnc-1 vector.

13. A surface display vector as set forth in claim 3, wherein said surface display vector is the vector pKLnH in which a gene encoding a main, neutralizing antibody-formative antigenic determinant of the S HBsAg is inserted at a Smal-Hindlll site of the N-terminal gene of the pKln-1 vector.

14. A surface display vector as set forth in claim 12, wherein said surface display vector is the vector pKLnHcC in which a gene encoding a main antigenic determinant of the core antigen ofHCV is inserted at a Hindlll site of the C-terminal gene of the pKlnHc vector.

15. A trans╧èbrmant, which is prepared by transforming a surface display vector of one of claims 10 to 14 into a microorganism selected from the group consisting of E. coli, Salmonella typhi, Salmonella typhimurium, Vibrio cholera, Mycobacterium vobis, Shigella spp. Lactobacilus spp. and Listeria monocytogenes.

16. A transformant as set forth in claim 15, wherein the transformant is the Salmonella typhi Ty21a/pKlnWH (KCTC 0473 BP) which is transformed with the surface display vector pKInWH of claim 15.

17. A transformant as set forth in claim 15, wherein the transformant is the Salmonella typhi Ty21a/pKlncH (KCTC 0472 BP) which is fransformed with the surface display vector pKIncH of claim 1 1.

18. A transformant as set forth in claim 15, wherein the transformant is the Salmonella typhi Ty21a/pKlnHc (KCTC 0471 BP) which is transformed with the surface display vector pKlnHc of claim 12.

19. A transformant as set forth in claim 15, wherein the transformant is the Salmonella typhi Ty21a/pKlnH (KCTC 0622 BP) which is fransformed with the surface display vector pKInH of claim 15.

20. A transformant as set forth in claim 15, wherein the transformant is the Salmonella typhi Ty21a/pKlnHcC (KCTC 0619 BP) which is fransformed with the surface display vector pKInHcC of claim 14.

21. A method for displaying a foreign protein on a microbial cell surface, in which the transformant Salmonella typhi Ty21 a of one of claims 16 to 20 is cultured to induce the expression of the foreign protein, said protein comprising an antigen protein originating from a pathogen.

22. A vaccine, utilizing a microbe, in which the microbe has a foreign protein on its surface, said foreign protein comprising an antigen protein originating from a pathogen.