JP2002501507A - Use of secretory vectors in oral vaccination for birth control - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for birth control by oral vaccination with an attenuated Salmonella or other attenuated Gram-negative inoculum. Various expression systems capable of inducing an immune response in a specific pathway of MHC-II / CD4 or MHC-I / CD8 are utilized.

Description

DETAILED DESCRIPTION OF THE INVENTION Use of Secretion Vectors in Oral Vaccination for Birth Control The present invention relates to various expression systems capable of inducing an immune response in a specific pathway of MHC-II / CD4 or MHC-I / CD8. And methods for birth control by oral vaccination with an attenuated Salmonella or other attenuated Gram-negative inoculum. PRIOR ART It has long been known that women and men with significantly higher antibody titers to human spermatozoa often show sterility or hypofertility (Ingerslev and Ingerslev, 1989; Chen and Jones, 1981; Menge et al. , 1982; Bronson et al. , 1984). It is also known that immunization of female and male animals with whole sperm extracts can render them sterile (Kummerfeld and Foote, 1979; Munoz and Metz, 1978; Tung et al. , 1979; Menge et al. , 1979). Primakoff et al. (1988a) used a monoclonal antibody to isolate the specific antigen PH20 on the guinea pig sperm surface, and injecting this purified antigen into female or male guinea pigs induced long-lasting immunity against reproduction. (1988b). A number of other monoclonal antibodies against ejaculated human sperm or other species of isolated sperm have been shown to immunoreact with human sperm. Some responded to the cytoplasmic components of sperm and others recognized testis-derived antigens. These monoclonal antibodies have been described to immobilize or agglutinate human spermatozoa or inhibit sperm binding and invasion into zona-free hamster oocytes. Currently, several human sperm antigens, such as the 95,000 molecular weight antigen (Moore et al. , 1987); the 55 kDa antigen, which is recognized by Lee's S36-37 mAb (HSA-63) (Liu et al. , 1990)); a human homolog of the sperm receptor of ZP-C (determined by Saling and Bliel and Wassarm in mice) (Bliel, 1990; Leyton and Sling, 1989) is known. Partially identified mouse and human FA-1 antigen (Naz, 1988), and a 24 kDa antigen from rat and human testis (Shaha et al. , 1 990) is even more interesting. The antigens identified by Moore and Lee, as well as the SP-10 immunogen, have been proposed by the World Health Organization (a special field of birth control vaccines) as major vaccine candidates (Anderson et al. , 1987). Three protein components of the zona pellucida (ZP), the extracellular matrix that covers papillary processes, are also suitable as antigens. These are commonly referred to as ZPA, ZPB and ZPC (Wassarmann, 1987, Science 235, 553-560). That is, the interaction between the egg and sperm can be inhibited or affected by immunological blockade. This was also confirmed by experiments with recombinant products of the zona pellucida protein ZPC, which is required for sperm to initially bind to the zona pellucida. However, in all studies reported to date, irreversible damage to the ovaries has induced a prolonged immune state (Skinner et al. 2004). , 1984, Endocrinology 115, 2418-2432). The mechanism of this loss of ovarian function is unknown to date. Here, genes or gene fragments suitable for birth control include: (a) a complete hemolysin operon containing an hly-specific promoter and enhancer-like regulator hlyR, and (b) a major portion of the hlyA gene Inserted into a secreted vector, which has been removed, thereby (c) allowing the attenuated Salmonella or other attenuated Gram-negative inoculum to synthesize proteins or protein fragments suitable for birth control; (d) these antigens (E) oral vaccination. In a preferred embodiment of the invention, said gene or gene fragment suitable for birth control encodes a zona pellucida protein. In a preferred embodiment of the invention, said gene or gene fragment suitable for birth control encodes a zona pellucida protein. In another preferred embodiment, the secretion vector is pMOhly1. In another preferred embodiment, the Salmonella bacterium is Salmonella typhimurium. In a particularly preferred embodiment, the gene or gene fragment suitable for birth control encodes a zona pellucida protein, the secretion vector is pMOhly1, and the Salmonella typhimurium is Salmonella typhimurium. Said gene or gene fragment suitable for birth control is defined as any gene or gene fragment encoding a protein or protein fragment suitable for birth control. Most proteins that are transported through the inner membrane of Gram-negative bacteria into the periplasm have a signal peptide at the amino terminus that is cleaved during a sec-dependent transport process (Pugsley, 1993) . In contrast, E. coli hemolysin (HlyA) is secreted by the secretion system through the inner and outer membranes into the extracellular medium. In contrast to the standard N-terminal transport signal in the sec-dependent transport process, HlyA has a transport signal consisting of 50-60 amino acids (HlyA-S) at the C-terminus (Hess et al. , 1 990; Jarchau et al. , 1994). This HlyA signal is not cleaved during secretion and itself has little antigenicity. The hemolysin secretion system of Escherichia coli is composed of three membrane proteins. Two of these proteins, HlyB (Gentschev & Goebel, 1990) and HlyD (Schulein et al. , 1992) are present in the inner membrane and are encoded by each gene of the hemolysin gene group. This group of genes consists of an operon containing the four genes hlyC, hlyA, hlyB and hlyD (Wagner et al. Hess et al., 1983; , 1986). The third protein in this transport system, TolC, is located on the outer membrane (Wandersman & Delepelaire, 1990). Transport of HlyA across both membranes of Gram-negative bacteria requires energy in the form of ATP and the membrane potential of the inner membrane (Koronakis et al. , 1995). Various studies have already shown that fusion of HlyA-S with other proteins or protein fragments and secretion of these fusion proteins by the hemolysin secretion system (Blight & Holland 1994; Gentschev et al. al. , 1994). In such cases, the efficiency of secretion depends on the folding and conformation of the reporter protein. The hemolysin secretion system of E. coli also functions in attenuated Salmonella, which is used as an inoculum, and is used to secrete the fusion protein (Gentschev et al. 2002). Su et al., 1992; , 1992). The genetic system used in this case was previously based on a plasmid carrying two elements: the genes required for transport (hlyB and hlyD) and a plasmid allowing the expression of the fusion protein (Gentsch ev et al. al. Hess et al., 1992; , 1990). Secretion vector pMOhly1 (Gentschev et al. , 1995) describe an hly-specific promoter and enhancer-like regulator hlyR (Vogel et al., 1995). , 1988), containing the complete hemolysin operon (Goebel & Hedgpeth, 1982). Since the major part of hlyA has been removed, only the amino terminal 34 amino acids and the carboxy terminal 61 amino acids (HlyA-S) are encoded as HlyA. At the single-NsiI site between the amino terminal residue and the carboxy residue of HlyA, a heterologous gene or gene fragment can be inserted in-frame with HlyA-S. Into this secretion vector pMOhly1, an antigen gene consisting of 10-1000 amino acids can be inserted and used to attenuate Salmonella and other attenuated Gram-negative inoculants (for example, E. coli). Escherichia coli, Vibrio cholerae, and Yersinia enterocolitica (Yersinia enterocolitica) can secrete these antigens. Thus, in contrast to other secretion systems, transport of heterologous fusion proteins by a single plasmid is possible. Compared to transport systems previously used for antigen presentation and most often only to transport relatively short peptides outside bacterial cells (Cardenas & Clements, 1992), the hemolysin secretion system Another important advantage is that the size of the protein that can be transported is quite large. By manipulating the HlyA secretion system, the same antigen can be produced in a suitable attenuated Gram-negative inoculum in cytoplasmic, surface-bound or secreted form. Furthermore, by inserting into the Listeriolysin (listeriolysin) secreted by Salmonella (listeriolysin) (from Listeria monocytogenes) and infecting the inoculated Salmonella producing the antigen, the same antigen, in the phagosome of antigen-presenting macrophage cells, Or it can be processed in the cytoplasm (Gentschev et al. , 1995). In this case, it is possible to induce an enhanced CD4 + or CD8 + T cell response (Hess et al. , 1996). Using this vector system, it is also possible to secrete cytokines and soluble cytokine receptors separately or together with antigens produced by attenuated Salmonella (Schulein, 1993; Gentschev, unpublished). The immune response can be altered (for TH1 or TH2). Due to the efficient secretion of proteins or protein fragments / HlyA-S fusions suitable for birth control, and the high stability of this vector, a completely new, highly attenuated Salmonella or other attenuated Gram-negative inoculum is used. Specific contraceptive vaccination is possible. When the recombinant Salmonella is orally administered, the antigen (protein or protein fragment suitable for birth control) comes into contact with the host immune system via secretion of a protein or protein fragment / HlyA-S fusion suitable for birth control. And activate B cells and / or T cells, depending on the epitope of the protein and the secretory pathway. Since HlyA-S has poor antigenicity for B cells and T cells, the antibodies and T cells induced by the HlyA-S fusion are mainly directed to the reporter antigen portion. Attenuated Salmonella and Yersinia bacteria that secrete antigens are particularly suitable for inducing a humoral, mucosal immune response and are therefore preferred. When mucosal immunity is induced, antigen-specific antibodies are produced even on distant mucosal surfaces. Thus, B cell responses at the site of infection stimulate mucosal immunity in the genital tract. The invention disclosed herein makes it possible for the first time to produce a recombinant protein or a part thereof directly in situ in order to generate a considerable immune response which leads to contraception, without using expensive production steps. Was. If a considerable vector is used, the protein or a part thereof in the phagosome can be expressed intracellularly or extracellularly. Thus, a humoral or cellular immune response can be intentionally induced. DESCRIPTION OF THE FIGURES FIG. 1 shows a map of the plasmid pMOhly1, a secretion vector. FIG. 2 shows Western blots examining the expression and secretion of various pMOhly1 / (hu) ZPA constructs. After overnight culture of E. coli DH5α transformed with various pMOhly1 / (hu) ZPA constructs, 2 ml of the supernatant was precipitated on ice for 4 hours with 10% (v / v) TCA (trichloroacetic acid) and centrifuged. The resulting pellet was resuspended in SDS-sample buffer. The protein in this supernatant was separated by SDS-PAGE using a 15% polyacrylamide gel, transferred to nitrocellulose, and then examined for expression and secretion by a polyclonal antibody against the hemolysin portion of the fusion protein. A1 indicates huZPA-1 construct (see FIG. 7); A4 indicates huZPA-4 construct (see FIG. 7); pMO indicates vector control; B4 indicates huZPB-4 construct (see FIG. 8). FIG. 3 shows Western blots examining the expression and secretion of various pMOhly1 / (hu) ZPB constructs. After overnight culture of E. coli DH5α transformed with various pMOhly1 / (hu) ZPB constructs, 2 ml of the supernatant was precipitated on ice for 4 hours with 10% (v / v) TCA (trichloroacetic acid) and centrifuged. The resulting pellet was resuspended in SDS-sample buffer. The protein in the supernatant was separated by SDS-PAGE using a 15% polyacrylamide gel, transferred to nitrocellulose, and then examined for expression and secretion by a polyclonal antibody against the hemolysin portion of the fusion protein. B1 is a huZP B-1 construct (see FIG. 8); B2 is a huZPB-2 construct (see FIG. 8); B3 is a huZPB-3 construct (see FIG. 8); B4 is a huZPB-4 construct B5 indicates huZPB-5 construct (see FIG. 8); pMO indicates vector control. FIG. 4 shows Western blots examining the expression and secretion of various pMOhly1 / (hu) ZPC constructs. After overnight culture of E. coli DH5α transformed with the various pMOhly1 / (hu) ZPC constructs, 2 ml of the supernatant was precipitated on ice for 4 hours with 10% (v / v) TCA (trichloroacetic acid) and centrifuged. The resulting pellet was resuspended in SDS-sample buffer. The protein in the supernatant was separated by SDS-PAGE using a 15% polyacrylamide gel, transferred to nitrocellulose, and then examined for expression and secretion by a polyclonal antibody against the hemolysin portion of the fusion protein. C1 is a huZPC-1 construct (see FIG. 9); C2 is a huZPC-2 construct (see FIG. 9); C3 is a huZPC-3 construct (see FIG. 9); FIG. 5 shows Western blots examining the expression and secretion of various pMOhly1 / (m) ZPB constructs. After overnight culture of E. coli DH5α transformed with various pMOhly1 / (m) ZPB constructs, 2 ml of the supernatant was precipitated on ice for 4 hours with 10% (v / v) TCA (trichloroacetic acid) and centrifuged. The resulting pellet was resuspended in SDS-sample buffer. The protein in the supernatant was separated by SDS-PAGE using a 15% polyacrylamide gel, transferred to nitrocellulose, and then examined for expression and secretion by a polyclonal antibody against the hemolysin portion of the fusion protein. B1 is an mZPB-1 construct (see FIG. 10); B2 is an mZPB-2 construct (see FIG. 10); B3 is an mZPB-3 construct (see FIG. 10); B4 is an mZPB-4 construct (See FIG. 10); pMO indicates vector control. FIG. 6 shows Western blots examining the expression and secretion of various pMOhly1 / (hu) ZPB constructs. S. transformed with various pMOhly1 / (hu) ZPB constructs. After overnight culture of typhimurium SL7207, 2 ml of the supernatant was precipitated on ice for 4 hours with 10% (v / v) TCA (trichloroacetic acid), and the pellet obtained by centrifugation was resuspended in SDS-sample buffer. did. The protein in the supernatant was separated by SDS-PAGE using a 15% polyacrylamide gel, transferred to nitrocellulose, and then examined for expression and secretion by a polyclonal antibody against the hemolysin portion of the fusion protein. B1 is a huZPB-1 construct (see FIG. 8); B2 is a huZPB-2 construct (see FIG. 8); B3 is a huZPB-3 construct (see FIG. 8); B4 is a huZPB-4 construct B5 indicates huZPB-5 construct (see FIG. 8); pMO indicates vector control. FIG. 7 shows Western blots examining the expression and secretion of various pMOhly1 / (hu) ZPC constructs. After overnight culture of Yersinia en terocolitica Wap Yv-515 transformed with various pMOhly1 / (hu) ZPC constructs, 2 ml of the supernatant was treated with 10% (v / v) TCA (trichloroacetic acid) for 4 hours on ice. The pellet obtained by sedimentation and centrifugation was resuspended in SDS-sample buffer. The protein in the supernatant was separated by SDS-PAGE using a 15% polyacrylamide gel, transferred to nitrocellulose, and then examined for expression and secretion by a polyclonal antibody against the hemolysin portion of the fusion protein. C1 is a huZPC-1 construct (see FIG. 12); C2d is a huZPC-2 construct with a double insert (see FIG. 12); C3d is a huZPC-3 construct with a double insert (See FIG. 12); C4 indicates huZPC-4 construct (see FIG. 12); pMO indicates vector control. FIG. 8 shows a gene map of cDNA of huZPA. The arrow indicates the position of the primer for PCR amplification (Table 1), and the number there indicates the start position of the 5 'end of the primer with reference to the start codon of the cDNA sequence of the present gene. The solid square indicates the amino-terminal transport signal, a putative fusion cleft, and a strongly hydrophobic region on the C-terminus that can function as a membrane anchor. In the lower part, a fragment obtained by cutting the PCR amplification product with Nsi-I restriction endonuclease is shown together with the start position and the end position. Each fragment is referred to as hu ZPA-1, huZPA-2, and the like. Further, the length of each fragment is indicated by both the number of base pairs (bp) and the number of amino acids (aa). The calculated molecular weight of this fragment / HlyA fusion protein in kDa is shown for each fragment of the zona pellucida protein and in parentheses. Finally, the results of detection of the secretion of the fusion protein by an antibody against the HlyA portion are shown. FIG. 9 shows a gene map of huZPB cDNA. The arrow indicates the position of the primer for PCR amplification (Table 1), and the number there indicates the start position of the 5 'end of the primer with reference to the start codon of the cDNA sequence of the present gene. The solid square indicates the amino-terminal transport signal, a putative fusion cleft, and a strongly hydrophobic region on the C-terminus that can function as a membrane anchor. In the lower part, a fragment obtained by cutting the PCR amplification product with Nsi-I restriction endonuclease is shown together with the start position and the end position. Each fragment is referred to as huZPB-1, huZPB-2, and the like. Further, the length of each fragment is indicated by both the number of base pairs (bp) and the number of amino acids (aa). For each fragment of this zona pellucida protein, and in parentheses, the calculated molecular weight of this fragment / HlyA fusion protein is shown in kDa. Finally, the results of detection of the secretion of the fusion protein by an antibody against the HlyA portion are shown. FIG. 10 shows a gene map of huZPC cDNA. The arrow indicates the position of the primer for PCR amplification (Table 1), and the number there indicates the start position of the 5 'end of the primer with reference to the start codon of the cDNA sequence of the present gene. The solid square indicates the amino-terminal transport signal, a putative fusion cleft, and a strongly hydrophobic region on the C-terminus that can function as a membrane anchor. In the lower part, a fragment obtained by cutting the PCR amplification product with Nsi-I restriction endonuclease is shown together with the start position and the end position. Each fragment is described as hu ZPC-1, huZPC-2, and the like. Further, the length of each fragment is indicated by both the number of base pairs (bp) and the number of amino acids (aa). For each fragment of this zona pellucida protein, and in parentheses, the calculated molecular weight of this fragment / HlyA fusion protein is shown in kDa. Finally, the results of detection of the secretion of the fusion protein by an antibody against the HlyA portion are shown. FIG. 11 shows a gene map of cDNA of mZPB. The arrow indicates the position of the primer for PCR amplification (Table 1), and the number there indicates the start position of the 5 'end of the primer with reference to the start codon of the cDNA sequence of the present gene. The solid square indicates the amino-terminal transport signal, a putative fusion cleft, and a strongly hydrophobic region on the C-terminus that can function as a membrane anchor. In the lower part, a fragment obtained by cutting the PCR amplification product with Nsi-I restriction endonuclease is shown together with the start position and the end position. Each fragment is referred to as mZPB-1, mZPB-2, and the like. Further, the length of each fragment is indicated by both the number of base pairs (bp) and the number of amino acids (aa). For each fragment of this zona pellucida protein, and in parentheses, the calculated molecular weight of this fragment / HlyA fusion protein is shown in kDa. Finally, the results of detection of the secretion of this fusion protein by an antibody against the HlyA portion are shown. FIG. 12 shows a gene map of cDNA of huZPC. The arrow indicates the position of the primer for PCR amplification (Table 1), and the number there indicates the start position of the 5 'end of the primer based on the start codon of the cDNA sequence of the present gene. The solid square indicates the amino-terminal transport signal, a putative fusion cleft, and a strongly hydrophobic region on the C-terminus that may function as a membrane anchor. In the lower part, a fragment obtained by cutting the PCR amplification product with Nsi-I restriction endonuclease is shown together with the start position and the end position. Each fragment is described as hu ZPC-1, huZPC-2, and the like. Further, the length of each fragment is indicated by both the number of base pairs (bp) and the number of amino acids (aa). For each fragment of this zona pellucida protein, and in parentheses, the calculated molecular weight of this fragment / HlyA fusion protein is shown in kDa. Finally, the results of detection of the secretion of the fusion protein by an antibody against the HlyA portion are shown. One skilled in the art can practice the present invention within the scope of the present invention based on the above description. Many existing techniques and methods for manipulating nucleic acids, such as mutagenesis, sequencing, nucleic acid transfer into cells, or protein analysis, are described in Short Protocols in Molecular Biology, 2nd Ed. , Ausrubel et al. Eds. , John Wiley & Sons, 1992 and Molecular Cloning: A Laboratory Manual, 2nd Ed, Sambrock et al. 1989, Cold Spring Harbor Laboratory Press. The contents of the above references are incorporated herein by reference. The present invention includes pharmaceutical formulations used for all the uses and methods described above. Thus, the invention includes, for example, pharmaceutical preparations comprising attenuated bacteria having the secretion vector of the invention. This oral administration is preferably in the form of a capsule such as a biodegradable polymer, for example PLPG. The following examples are set forth to further illustrate and demonstrate that the present invention is feasible. This example is not intended to limit the present invention. Example 1 E. coli and S. Cloning and Expression of huZPA, huZPB, huZPC, and mZPB Fragments in typhimurium Schering AG), as well as mouse ovarian mRNA, the source of cDNA for the mouse ZPB gene, are the starting materials for cloning. From mRNA isolated from the ovary of a superovulating mouse (Uwe Eberspacher, Schring AG), the corresponding cDNA was synthesized as described below. Incubate about 5 μg of RNA in 32 μl of DEPC-treated water with 3 μl of oligo dT primer at 65 ° C. for 5 minutes. The solution is cooled at room temperature for 10 minutes and the following reagents are added: 5 μl of synthesis buffer, 5 μl of 0. 1 M DTT, 1 μl RNase inhibitor, 3 μl 25 mM dNTPs, and 1 μl MMLV reverse transcriptase (20 U / μl). This is incubated at 37 ° C. for 1 hour. Gene fragments were amplified by PCR from the corresponding cDNAs using specific oligonucleotides (Table 1). This PCR fragment has an NsiI restriction site at the 5 'and 3' (FIGS. 8-12 show these ZP gene maps, primer positions, and product gene fragments). PCR amplification (Saiki et al.) Using Thermocycler 60/2 (Bio-Med, Theres, Germany). , 1988). For this, the corresponding 5 ′ and 3 ′ primers (1 μmol each final concentration), dNTPs (200 μmol each), and 2. CDNA was amplified in a 100 μl reaction using 5 U of Taq DNA-polymerase (Promega) and a dedicated buffer. Prior to the first cycle, denaturation was performed at 91 ° C. for 3 minutes. Subsequently, 30 cycles of 91 ° C. for 1 minute (denaturation), 55 ° C. for 1 minute (annealing) and 72 ° C. for 1 minute (extension) were performed. Reaction products were separated by electrophoresis in a 2% agarose gel and observed by ethidium bromide staining. These PCR products were first inserted into the SmaI site of the vector pUC18 with blunt ends. Next, a ZP fragment obtained by cutting this with NsiI was inserted into the expression vector pMOhly1 (FIG. 1). Escherichia coli DH5α was transformed with various pMOhly1 / ZP constructs, and the transformants were cultured overnight. 2 ml of this supernatant was precipitated on ice for 4 hours with 10% (v / v) TCA, and the pellet obtained by centrifugation was resuspended in SDS-sample buffer. The proteins in the supernatant were separated by SDS-PAGE using a 15% polyacrylamide gel and transferred to nitrocellulose, and then the secretion of zona pellucida / hemolysin fusion protein was examined. Expression and secretion of two huZPA fragments (A1 and A4, FIG. 2) and four huZPB fragments (B1-B4, FIG. 3) were detected by a polyclonal antibody against the hemolysin portion of the fusion protein. In the case of huZPC, a domain that could be transported was identified (C1, FIG. 4). Like huZPB, the secretion of three protein fragments derived from mZPB was also demonstrated by this transport system (B1-B3, FIG. 5). Next, transformation with the pMOhly1 derivative, S. This was performed using typhimurium LB5000 strain. Since this strain has no restriction system, E. coli DNA can be effectively introduced. This S. S. typhimurium LB5000 isolated recombinant expression vector, S. typhimurium SL7207 strain was successfully transformed. As already mentioned, this strain is suitable as an inoculum. Secretion of the ZP / Hly fusion protein was also performed using this strain. FIG. 6 shows the expression and secretion of huZPB fragments in Salmonella, as in the Examples. Orally administered to mice, Salmonella expressing ZP / Hly will induce specific mucosal immunity against the ZP portion of the fusion protein. Example 2 Y. Cloning and Expression of huZPA, huZPB, huZPC and mZPB Fragments in enterocolitica Plasmids pGEX-KG-huZPA, pGEX-KG-huZPB and pGEX-KG-huZPC (Peter Bringmann Schering AG), as well as mouse ovarian mRNA, the source of cDNA for the mouse ZPB gene, are the starting materials for cloning. From mRNA isolated from the ovary of a superovulating mouse (Uwe Eberspacher, Schring AG), the corresponding cDNA was synthesized as described below. Incubate about 5 μg RNA in 32 μl DEPC-treated water with 3 μl oligo dT primer at 65 ° C. for 5 minutes. The solution is cooled at room temperature for 10 minutes and the following reagents are added: 5 μl of synthesis buffer, 5 μl of 0. 1 M DTT, 1 μl RNase inhibitor, 3 μl 25 mM dNTPs, and 1 μl MMLV reverse transcriptase (20 U / μl). This is incubated at 37 ° C. for 1 hour. Gene fragments were amplified by PCR from the corresponding cDNAs using specific oligonucleotides (Table 1). This PCR fragment has an NsiI restriction site at the 5 ′ and 3 ′ (FIGS. 8 to 12 show these ZP gene maps, primer positions, and product gene fragments). PCR amplification using Thermocycler 60/2 (Bio-Med, Theres, Germany) (Saiki et al. , 1988). For this, the corresponding 5 ′ and 3 ′ primers (1 μmol each final concentration), dNTPs (200 μmol each), and 2. CDNA was amplified in a 100 μl reaction using 5 U of Taq DNA-polymerase (Promega) and a dedicated buffer. Prior to the first cycle, denaturation was performed at 91 ° C. for 3 minutes. Subsequently, 30 cycles of 91 ° C. for 1 minute (denaturation), 55 ° C. for 1 minute (annealing) and 72 ° C. for 1 minute (extension) were performed. Reaction products were separated by electrophoresis in a 2% agarose gel and observed by ethidium bromide staining. These PCR products were first inserted into the SmaI site of the vector pUC18 with blunt ends. Next, the ZP fragment obtained by digesting this with NsiI was inserted into the expression vector pMOhlyl (FIG. 1). Escherichia coli DH5α was transformed with various pMOhly1 / ZP constructs, and the transformants were cultured overnight. 2 ml of this supernatant was precipitated on ice for 4 hours with 10% (v / v) TCA, and the pellet obtained by centrifugation was resuspended in SDS-sample buffer. The proteins in the supernatant were separated by SDS-PAGE using a 15% polyacrylamide gel and transferred to nitrocellulose, and then the secretion of zona pellucida / hemolysin fusion protein was examined. The expression and secretion of huZPA fragment, huZPB fragment and huZPC were detected by the polyclonal antibody against the hemolysin part of the fusion protein (see above). Next, transformation with the pMOhly1 derivative, Y. Enterocolitica LB5000 strain was used. Since this strain has no restriction system, E. coli DNA can be effectively introduced. This Y. Enterocolitica LB5000 isolated recombinant expression vector, Y. Enterocolitica Wap Yv-515 strain was successfully transformed. As already mentioned, this strain is suitable as an inoculum. Secretion of the ZP / Hly fusion protein was also performed using this strain. As in the example, FIG. Figure 2 shows expression and secretion of huZPC fragments in enterocolitica. Orally administered to mice, Yersinia cerevisiae expressing ZP / Hly will induce specific mucosal immunity against the ZP portion of the fusion protein. Table 1: Primers for PCR amplification of ZP gene fragment and expected PCR product size Underlined bases indicate changes in each ZP sequence made to introduce an NsiI site required for cloning.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), AL, AM, A U, AZ, BA, BB, BG, BR, BY, CA, CN , CU, CZ, EE, GE, GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, L R, LS, LT, LV, MD, MG, MK, MN, MW , MX, NO, NZ, PL, RO, RU, SD, SG, SI, SK, SL, TJ, TM, TR, TT, UA, U G, US, UZ, VN, YU, ZW (72) Inventor Genchev, Iphiro             Germany, Day 97270 Kist,             Bowbake 5 (72) Inventor Hess, Jürgen             Germany, Day 10318 Berli             , Riknitstrasse 31 (72) Inventor Kaufman, Stephan             Germany, Day 13465 Berli             , Am Rosenanger 57 ar

Claims (1)

[Claims]   1. (A) a gene or gene fragment suitable for birth control is inserted, (B) contains hly-specific promoter and enhancer-like regulator hlyR Has the complete hemolysin operon, and (C) the main part of the hlyA gene has been removed, Secretion vector, (D) Birth control by attenuated Salmonella or other attenuated Gram-negative inoculum A suitable protein or protein fragment is synthesized, and (E) by secreting these antigens, (F) oral vaccination; To be used for   2. The gene or gene fragment suitable for birth control encodes a zona pellucida protein. The use according to claim 1, wherein the use is performed.   3. The use according to claim 1, wherein the secretion vector is pMOhly1.   4. The Salmonella bacterium is Salmonella typhimurium urium).   5. The strain is Yersinia enterocolitica The use according to claim 1, wherein   6. (A) a gene or gene fragment suitable for birth control is inserted, (B) contains hly-specific promoter and enhancer-like regulator hlyR Has the complete hemolysin operon, and (C) the main part of the hlyA gene has been removed, Secretion vector, (D) Birth control by attenuated Salmonella or other attenuated Gram-negative inoculum A suitable protein or protein fragment is synthesized, and (E) by secreting these antigens, (F) oral vaccination; How to control birth by using it.