KR19990082085A - Response amplification from expressed recombinant protein - Google Patents

Abstract

The present invention relates to a method for amplifying a response from a protein expressed from a recombinant papilloma vector. The present invention can be used to amplify the intended effect of a expressed protein in any method in which such expression is desired, such as, for example, in vaccination, vaccination, and gene therapy. The invention also relates to a synthetic peptide, a nucleic acid sequence, preferably a DNA sequence, which is linked to a DNA segment encoding a protein comprising a signal peptide at the N-terminus and an anchor sequence at the C-terminus. Such expression mediators are generally beneficial for expressing polypeptides in various animal cells.

Description

Response amplification from expressed recombinant protein

Prevention and treatment of various disease states, including microbial infections, have long been health problems. Many disease states and infections, particularly those caused by various viruses, cannot be easily tested with antimicrobial agents. Diseases that cannot be tested with antimicrobial agents often become resistant to treatment over time. Although progress has been made in the past few years to treat these infections, several problems still remain, such as the selection of suitable delivery system excipients and the display of proteins.

In most cases, the development of vaccines for use in various diseases involves the inclusion of adjuvant to enhance the immunogenic effect of antigenic proteins present in the vaccine.

Herpes Simplex is a viral agent that causes infections of the genital organs (commonly called the mouth, commonly called lip herpes). The incidence of herpes simplex has increased several times over the last two centuries, and effective vaccines are needed. Neutralizing antibodies against viruses have been demonstrated to recognize surface glycoproteins in tissues with two glycoproteins (glycoproteins B and D) that act as good antigens. Recombinant glycoproteins have been produced in Chinese Hamster Ovary (CHO) cells and have been known to provide protection for guinea pigs against direct challenges. The range of protection varies but depends on adjuvant coadministrated with glycoproteins. Several herpes glycoproteins can be expressed as baculoviruses (insect-based cell lines), many of which are known to be protective immune proteins when administered in Freund's adjuvant. The inflammatory properties of these adjuvant are excluded when used in humans and are greatly reduced when used in laboratory test animals. Capsid proteins from viruses can be readily expressed by recombinant scoliosis virus.

Human cytomegalovirus is a pathogen that can cause mortality as well as morbidity, especially in immunocompromised individuals. Infection of pregnant women may result in improper development of the motor nerves of the fetus. The virus is widespread with a high incidence of 1% and a reduced incidence of thousands of birth defects each year. Immunity gains in women before pregnancy should significantly reduce the incidence of adverse effects. Infected individuals have been shown to be able to obtain immune responses that include body fluids and cellular components. Tests were conducted with diluted strains of virus as immunogens. Attempts have been made to protect as a dose of protein antigen bound with adjuvant, but as a result there has been no acceptable to date.

Hepatitis weakens the constitution and is widespread as a sometimes fatal disease. Hepatitis infected with humans-There are many different types of hepatitis, such as typical A, B and C. The first recombinant vaccine product attempted to be used in humans was extracted from hepatitis B and formulated based on adjuvant. However, there is a difficulty in maximizing the immune response of these materials and ongoing attempts to improve the response to adjuvant are under study. One commercial hepatitis vaccine was made from a substance produced in yeast and mixed with alum. Since such products are expressed in mammalian host cells, there may be a lack of information given to viral glycoproteins (eg, complex glycosylation that confers specific structural properties, regulatory processing, and increased stability).

The main problem in the development of infectious disease vaccines is to accurately identify protective agents, and to effectively provide them to the host. Formulations that rely on the use of adjuvant to enhance the immune effect are at risk for the adjuvant's response, which may preclude its use. It is important to emphasize that providing an effective immune vaccine preparation only when used as one adjuvant is inherently inefficient. This critical drawback could be a major cause of the failure to commercialize many prior art vaccines.

According to a feature of the present invention, there is provided a general procedure for overcoming the drawbacks of the prior art by providing a vaccine to a host of diseases caused by microorganisms, including viruses, bacteria, protozoa and fungi. The vaccine according to the present invention provides the antigenic protein in a manner that is easy to produce an immune effect.

Several genital diseases are evident in patients who are unable to provide a quantification of one or more proteins or polypeptides. Many neurological, muscular or hematopoietic disorders and related diseases are often due to the individual's lack of ability to produce a sufficient amount of critical proteins or polypeptides. Various treatment options lie in the return of biosynthesis or the production of deficient proteins. Optionally, gene therapy techniques in which protein expression mediators are administered to patients suffering from genetic diseases represent a variety of approaches to treatment.

In the past, live papillomaviruses have been used as vaccines to effectively eradicate smallpox, and recombinant papillomaviruses expressing viral antigens are known to induce strong antibody responses while protecting immunized animals from disease (Arita, I., Nature, 1979, 279, 293-298). In addition, it has been found in animal samples that co-expression of protein immunogens involving high immunogenic papilloma virus proteins can elicit a strong immune response against specific immunogens (Moss and Flexner, Annals of the New York Academy). of Sciences, 86-103; Mackett and Smith, J. Gen. Virol., 1986, 67, 2067-2082; Houard, et al., J. Gen. Virol., 1995, 76, 421-423; Fujii, et. al., J. Gen. Virol., 1994, 75, 1339-1344; Rodrigues, et al. J. Immunol., 1994, 153, 4346-4648). Therefore, utilizing live recombinant scoliosis virus as a vaccine would be difficult. The challenges of expression and production of antigens currently encountered in the preparation of recombinant proteins in E. Coli, yeast or insect expression systems can be overcome. The panel of transfer mediators has been structured to allow the insertion of foreign genes at several sites within the 180 kb sarcoma virus genome (Earl and Moss, Current Protocols in Molecular Biology, 1993, 16.17.1-16.17.16). It has also been reported that> 25 kb of foreign DNA can be inserted into the scoliosis virus genome (Smith and Moss, Gene, 1983, 25, 21-28). Accurate treatment (Charkrabarta, et al., Nature, 1986, 320, 535-537) and appropriate post-transitional mutations (Hu, et al., Nature, 1986, 320, 537-540; Ball, et al 7., Proc. Natl Acad.Sci. USA, 1986, 83, 246-250; de la Salle, et al., Nature, 1985, 316, 268-270, transport and secretion (Ball, et al., Proc. Natl. Acad. scienc. USA, 1986, 83, 246-250 and Langford, et al., Mol. Cell. Biol., 1986, 6, 3191-3199) are indicated by the first structure of the expressed protein. In addition, recombinant scoliosis virus vaccines have the advantage that they are relatively inexpensive, easy to store, transport and produce, and are particularly important features in developing countries where malaria and other diseases are most prevalent.

The present invention relates to a method for amplifying a response of a protein expressed from an expression mediator, preferably a recombinant scleroderma mediator. The present invention can be used to amplify the intended effect of the expressed protein in any method such as, for example, vaccine preparation, vaccination and gene therapy. The present invention also relates to a synthetic peptide, a nucleic acid sequence, preferably a DNA sequence, consisting of a medium linked to a DNA segment encoding a protein comprising a signal peptide at the N-terminus and an anchor sequence at the C-terminus. Such expression mediators are generally beneficial for expressing polypeptides in various animal cells.

A protein to be expressed by encoding a protein in an expression medium in which the N-terminus comprises a signal peptide and the C-terminus comprises an anchor sequence is significantly amplified or intended as compared to a protein that does not comprise a signal sequence or an anchor sequence. The effect is derived. The present invention can be used to amplify the effects of proteins expressed for various purposes, including vaccine preparation, vaccination and, in particular, treatment of diseases such as gene therapy.

In particular, the present invention relates to a method of amplifying a therapeutic or immunogenic effect of a protein expressed according to a gene therapy technique or a vaccine dependent on a protein or peptide as an antigenic crystal.

In embodiments related to vaccine treatment or disease prevention, the present invention relates in particular to vaccines that can be used for the prevention and treatment of various diseases by various microorganisms, including bacteria, viruses, protozoa and fungi. In particular, the methods of the invention can be used to amplify antigenic proteins expressed in vaccines against human malaria due to herpes simplex, cytomegalovirus, hepatitis and Plasmodium falciparum, among other diseases.

This invention was supported by Darpa License No. N-00014-90. The right to this invention lies with the government.

Summary of the Invention

The present invention relates to a method for enhancing the biological or pharmacological effects of a protein or peptide expressed in an animal patient. In the present invention, an expression medium comprising a gene or other nucleic acid sequence encoding a sequence of a protein or peptide exhibiting the intended pharmacological effect is inserted into a patient. The expressed protein or peptide sequence also includes an anchor peptide sequence at its C-terminus and, optionally, a signal sequence at its N-terminus. Preferably, in one particularly preferred embodiment, the expressed protein or peptide sequence is immunogenic and also comprises a signal peptide at its N-terminus and an anchor peptide sequence at its C-terminus.

After the expression mediator is inserted into an animal patient, preferably a mammal such as a human, the mediator expresses the desired protein in the patient, including signal peptides and anchor peptides, and produces the intended pharmacological effect. Unexpectedly, it was found that the inclusion of a signal peptide sequence at the N-terminus and an anchor sequence at the C-terminus of the expressed peptide sequence greatly enhanced the pharmacological effect obtained from the expressed protein. When immunogenic proteins or peptides are expressed along signal and anchor proteins, the immunogenic effect is often much greater than when the proteins or peptides are expressed without signal or anchor peptides.

A method for further enhancing the pharmacological effect of a protein expressed in a patient includes a DNA (nucleic acid) sequence encoding a peptide sequence consisting of a pharmacologically active protein or peptide that is usually expressed as not a signal peptide and / or an anchor peptide. Binding to an expression mediator in combination with an anchor peptide at the C-terminus of the expressed peptide, optionally comprising a signal peptide at the N-terminus of the expressed peptide, and administering said expression mediator to the patient . This method can be applied very widely. The present invention is applicable to enhancing the biological or pharmacological effects of virtually any protein that can bind to expression mediators and exhibits its biological or pharmacological effects outside of the cells in which the protein is expressed. This method applies to any protein or peptide in which cell surface expression is an important function case, in particular, including receptors including immunogenic proteins and peptides, insulin receptors for insulin-resistant diabetes, hormone receptors and growth factor receptors. Essentially applicable.

By following the steps of the method of the present invention, the pharmacological effect obtained from the expressed protein or peptide is much enhanced compared to that obtained from expression of the same protein without signal peptide and anchor peptide. In general, the binding of signal and anchor peptides results in at least two times more effects than the response produced by peptides that do not include signal and anchor peptides. In preferred cases, this effect is 5 times, more preferably at least 10 times, in some cases at least 100 times. For antigen, this enrichment is measured in a manner based on direct comparison of antibodies induced in recipient hosts. These antibodies are for protein / peptide nuclei and are quantified by Elisa. In a therapeutic aspect of the invention, expression of the protein is determined by the use of a specific antibody in a standard capture Elisa or, for example, by measurement of a specific biological function (eg, enzyme activity or other). Activity) can be measured.

In the expression mediator according to the invention, the nucleic acid coding for the desired protein region is expressed in the patient by the expression mediator after administration. Peptides as complete proteins or fragments thereof expressed in a patient provide the desired therapeutic effect in the patient or, in the case of vaccination, elevate the humoral and / or cell-mediated response to antigenic proteins or peptides Provides the effect of protecting the vaccinated patient from subsequent infection by the microorganism comprising the expressed protein or peptide.

Peptides expressed by the expression mediator include anchor peptides. Preferred embodiments also consist of signal peptide and anchor peptide sequences. In accordance with the present invention, it has been found that the addition of a signal and anchor peptide to the expressed peptide unexpectedly enhances the effect of treatment or immunogenicity in a patient of the expressed protein. Incorporation of signal and anchor proteins into the expressed peptides by this invention surprisingly results in a much larger therapeutic or immune response than the peptides produced alone or in the case of vaccines, alone or in combination with adjuvant.

The vaccine properties of the present invention are particularly advantageous over the prior art in that there is no need to administer with adjuvant to enhance the immune effect of antigenic proteins or peptides. In the immunogenic properties of the invention, the inclusion of a signal and an anchor peptide in the antigenic peptide sequence expressed by the expression mediator is at least 2 to 100 times greater than the immune response generated by the antigenic peptide not comprising the signal and anchor peptide sequence. Inducing an immune effect that is more than doubled is an unexpected result.

The most important problem in the development of infectious disease vaccines is to accurately identify protective antigens and provide them effectively to the host. Formulations that rely on the use of adjuvant risks side effects that may preclude their use. Formulation of vaccines that are effective only when used in conjunction with adjuvant is inherently inefficient for providing immune cells. The overall process of the present invention has been proposed to overcome these drawbacks. In vaccine production, the inclusion of genetic information for accurate pathway migration in infected host cells with means for immobilizing proteins produced in the plasma membrane of the same cell ensures utility against host immune cells even if the cells die. .

In the method of treating a disease, it is also proposed by the present invention to induce an immune response to a patient or to prevent a patient from a disease. In this method, the patient is dosed with an amount of an expression mediator, preferably a papillomavirus vaccine effective to express the present protein or peptide, so that the patient generates, or is selective for, the required protein or peptide by the therapeutic amount of the indicated disease. As a result, it begins to generate an immunogenic response to the expressed protein or peptide. In the case of a vaccine, preferably the generated immunogenic response becomes a "substantial protective agent", ie the vaccine protects the patient from more severe symptoms and physiological conditions of the disease, including death of the patient due to the disease.

The present invention also relates to dosage forms of expression media as therapeutic modalities, such as pharmaceuticals or vaccines. Dosage forms according to the present invention may, for example, provide an immunogenic response to a protein or peptide to treat a disease, such as a genetic disease in which the protein or peptide to be expressed does not normally develop, or to generate a protective effect against the disease. Can be used to induce.

The invention can also be used in a variety of diseases including autoimmune diseases, genetic diseases that occur at reduced concentrations or none at all in microbial infections, cancer, viral infections, protozoan infections, and other microbial infections. have.

Detailed description of the present invention`

Terms used herein to describe the present invention are as follows.

The term "patient" is used in animals, including mammals, and in particular human patients administering a dosage form of the expression mediator according to the invention. In the present invention, the expression mediator is encoded for the desired protein or peptide and refers to the protein or peptide encoded in the patient. The expressed protein or peptide can provide a therapeutic or immunogenic response in the patient being treated.

The term "chimeric peptide" or "chimeric peptide sequence" is used to describe an artificial peptide sequence according to the invention consisting of the expressed protein or peptide, anchor peptide and / or signal peptide. As can be seen from the use of this term, a chimeric peptide is a synthetic peptide produced by an expression mediator comprising a desired target protein or peptide or a target protein or peptide moiety in combination with such a signal and / or anchor peptide sequence ( In fact, not commonly found in combination with signal and / or anchor sequences).

The term "expression mediator" refers to a means by which a nucleic acid comprising DNA, cDNA, RNA or a variant thereof, more preferably a DNA fragment, can be inserted into a patient or tissue of a patient to express or produce a desired protein. it means. Such mediators include mediators in which the desired protein or peptide, anchor peptide sequence, and optionally nucleic acid sequences encoding for signal peptide sequences, are inserted into the host tissue and replicated (as desired, or according to an optional agent). Preferred mediators are those capable of expressing a peptide or protein in eukaryotic cells, the limited sites of which are well known, and those having the necessary working elements for the desired protein or peptide sequence. In the present invention, the mediator is preferably a papilloma virus vaccine, adenovirus or a herpes virus that is capable of infecting a mammalian cell and expresses or synthesizes a protein using a biosynthetic mechanism of the host. In this case, it is most preferable that the viral vehicle used for production infects the cells but does not cause cytolysis due to replication (i.e., supervision selected from adenoviruses, scoliosis viruses and herpesviruses among similar species). Or partially parietal virus).

According to the mediator approach of the present invention, the mediator infects a host cell and synthesizes a protein or peptide encoded by said mediator using the biosynthetic pathway of the host cell. Any immune excipient with detailed hereditary and human history of use can be used as an expression mediator of the invention. Preferred expression mediators are virus mediators, more preferably "Current Protocols in Molecular Biology, 1993, 16.17.1-16.17" by, for example, Earl and Moss. 16) and a papilloma virus mediator as depicted in the "gene" by Smith and Moss (Gene, 1983, 25, 21-28). However, any papilloma or other viral mediator that can be used as described above is also suitable for use in the present invention. In the case of oral therapeutic products in which the expression of proteins occurs in the gastrointestinal tract, the use of bacterial expression mediators such as, for example, Salmonella expression mediators is preferred.

In order to express the desired protein or peptide, the expression mediator must comprise at least one promoter, at least one effector, at least one terminator and other sequences necessary for efficient copying and subsequent displacement of the mediator nucleic acid. . Such operating elements are already well known in the art. In a preferred embodiment of the invention, the expression mediators are present in a host tissue with at least one selectable marker and at least one promoter sequence capable of initiating a copy of the nucleic acid (preferably DNA) sequence. At least one origin of replication recognized by the invention.

The terms “expressed peptide”, “expressed polypeptide” and “expressed protein” are expressed by the expression mediator according to the invention to treat a disease or to condition or generate an immunogenic response or protective agent effect on the disease. It is used to describe the target peptide or protein. Preferred expressed proteins and peptides include any protein or peptide for which cell surface expression is an important functional outcome. All such proteins, peptides or polypeptides are not normally found in combination with (ie, in fact) signal and / or anchor peptides. The target peptides and proteins can be found in nature, but not in combination with signal and / or anchor sequences.

Preferred proteins and polypeptides according to the invention are in particular, for example, hormones, growth factors, blood clots, enzymes, neuroproteins, apolipoproteins, tumor suppressors, endogenous and exogenous tumor antigenic proteins and peptides, bacterial surface proteins And parasite, fungal surface proteins and peptides including peptides, antigens comprising viral surface proteins and peptides, protozoal cell surface proteins and peptides, and viral reverse transscriptases and related virus specific enzymes. Other preferred proteins include receptors such as insulin receptors, hormone receptors, growth factor receptors for insulin-resistant diabetes.

The term "vaccine" refers to a medicament for active immunological prophylaxis throughout the specification. In the present invention, the vaccine is an expression mediator, preferably a papillomavirus expression mediator which expresses an antigenic protein or peptide from a disease-causing microorganism after administration of the expression mediator to an animal such as a mammal. There are a variety of methods for administering the vaccines according to the invention, in particular intravenous, oral, oral, skin and nasal, but intramuscular or subcutaneous administration is the most preferred method of administration. In the course of making vaccines for humans and other animals, DNA encoding such that the desired protein or peptide is expressed is either incorporated into the expression media or as described in detail below, by any of the commonly available methods well known in the art. . The mediator is formulated in an immunogenic dose form as a vaccine and administered to the patient by one or more methods as described above to protect the patient from disease.

The terms "amino-terminus" or "N-terminus" and "amino-terminal" refer to a protein or peptide moiety expressed by the present invention (the term amino -Including the terminal amino acid), which portion is at or adjacent to the amino terminal end of the protein or peptide. It is at or near the 5 'end of the DNA sequence encoding the amizo-terminus of the expressed protein or peptide (generally just above or slightly above the 5' end of the sequence encoding the protein or peptide). , At the N-terminus of the expressed protein or peptide where sequence encoding for the signal protein or peptide is found.

Used to describe a protein or peptide moiety expressed in accordance with the present invention "carboxy-terminus" and "carboxy-terminal" (the term includes carboxy-terminal amino acids) Which is at or near the carboxy-terminus of the protein or peptide. The DNA sequence encoding the carboxy-terminus is at or near the 3 'end of the sequence encoding the expressed protein or peptide. It is close to the 3 'end of the DNA sequence encoding the carboxy-terminal region of the expressed protein or peptide (generally just below or slightly below the 3' end of the sequence encoding the protein or peptide) and the anchor protein or peptide Is at the carboxy-terminus of the expressed protein or peptide generally found.

For some vaccines, due to the degree of specificity of the immune response that can be induced for such protein monomers, the carboxy-terminus or near carboxy-terminus of the expressed protein or peptide may be responsible for the development of humoral and / or cell mediated responses. It may indicate a desired goal. Therefore, preferred immunogenic proteins or peptides for use in the vaccines of the invention may be present at the carboxy-terminus or proximal regions of the complete immunogenic protein.

The terms "signal peptide", "signal sequence" or "signal protein" refer to 7-30 units of amino acid peptide sequence, preferably to substantially enhance the biological activity of a protein or peptide expressed in a patient by the present invention. Refers to about 15-26 units of amino acid peptide sequence generally found at or near the N-terminus of an expressed protein or peptide used in the present invention. The signal sequence generally has a hydrophobicity of 7 to 30 amino acid units, more preferably about 15 to 26 amino acid units, even more preferably 16 to 24 amino acid units, most preferably about 18 to 20 amino acid units. It contains peptide sequences and appears to be essential for targeting protein chains (generally secretory proteins) to membranes within cells. This hydrophobic sequence is of sufficient length to cross the lipid bilayer of the cell membrane. Signal sequences are used as formers for cellular traffic of macromolecules. These proteins appear to play a central role in the displacement of polypeptide chains across the membrane. In this invention, binding of the signal protein sequence to the amino terminus of the protein or peptide sequence expressed by the vaccinated patient results in substantial enhancement in the biological activity (including the therapeutic effect of immunogenicity) associated with the expressed protein or peptide. It is related. Signal sequences well known in the art can also be used in the present invention. For example, it is also possible to utilize signal sequences in yeast or sub-trophic ranks, but apparently the stray signal ranks are preferred for mammals, and the signal ranks of specific species are most preferred for the desired mammalian species to be treated. . Therefore, in order to provide a protein or peptide expressed to humans, human signal rank is most preferred.

Signals for use in the present invention generally comprise three regions, the first or C region (signal peptidase) at the carboxy-terminus of the peptide, consisting of about 5 to 7 amino acid residues which tend to be very polar but which do not open Used as a cleavage site for enzymes); The c region at the N-terminus, ie a second or h region (mainly Leu, Ala, Met, Val, Ile, Phe, and Trp amino acids, very hydrophobic and generally about 7 to 13 amino acid residues in length, Sometimes consisting of Pro, Gly, Ser or Thr amino acid residues); And length and composition are highly variable and generally span the net positive charges contributed by the N-terminus and the third or n-regions (known as negative charges contributed from acidic residues) carrying any charged residues. region c to h are small, one to three amino acid residues that tend to be unfilled (Ala, Gly, Ser, etc.). The synthetic homopolymerizable h region consisting of leucine, isoleucine, phenylalanine, valine, alanine, and tryptopine, preferably leucine, isoleucine and phenylalanine, can be used for the signal protein according to the present invention. In general, see Von Heijne's European Journal of Biochemistry, (1983), 133, pp. 17-21.

The signal sequence used in the present invention preferably comprises a eukaryotic signal sequence, preferably 7 to 30 amino acid units in length, preferably 16 to 26 units, more preferably 16 to 26 amino acids, even more preferably 18 to 20 amino acids. Include the unit. In the present invention, the c region of the signal peptide is more polar, and the boundary of the c region between the h region and residues -5 to -6, or -7 or -8 (calculated from the cleavage position of the signal sequence, ie mature agent) One amino acid or expressed protein or peptide is +1) between 1 and 3 amino acid residues that are small and tend not to be charged (Ala, Gly, Ser, etc.). Location selection of amino acid h / c is as follows:

-10 most preferably leucine or alternatively isoleucine, valine, alanine, or phenylalanine;

-9 most preferably leucine, alternatively isoleucine, alanine, valine, phenylalanine;

-8 most preferably leucine, alternatively isoleucine, alanine, valine, glycine, phenylalanine;

-7 most preferably alanine, alternatively leucine, isoleucine, alanine, valine, phenylalanine;

-6 most preferably valine, alternatively leucine, valine, isoleucine, phenylalanine, alanine;

-5 most preferably proline, alternatively glycine, alanine, leucine, valine;

-4 most preferably glycine, alternatively proline, leucine, alanine, valine;

-3 most preferably alanine, alternatively valine;

-2 most preferably leucine, alternatively phenylalanine;

-1 most preferably alanine, alternatively glycine.

In the signal sequence used in the present invention, the h region may vary in length. The n region is polar and contains the positively charged amino acids (lysine and arginine predominant) and varies over the overall length of the signal peptide as described above. c region extends from the -1 to -5 residues of the signal peptide / expressed or mature protein. The positions of the c, h and n regions are the N-terminus of the protein to be expressed or mature, the h region is from the N-terminus to the c region (the 1-3 amino acid border between the c region and the h region) and the n region is the anode From the N-terminus to the h region charged with. Overall, the n region is variable in length, generally charged with an anode (preferably +2 charge), the h region is hydrogenous and variable in length, and the c region is preferably about 5 (5-7) Phosphorus generally includes polar amino acids.

The end of the hydrogenated region (ie the border between the hydrogenated residues listed above) is preferably in the -6 / -5 position. Overall, the signal sequence consists of a 5-10 residue initial sequence (starting with methionine) followed by at least 7 residue sequences (as described above), and additional amino acids 1-10 residues in length. Representative sequences known for this region are: ILLLLAV.

The signal sequence used should be characterized by the cell type used for expression of the protein. Therefore, in veterinary applications, the signal sequence used should be mammalian in character. Most mammalian signal sequences have important efficacy in expressing proteins or peptides in other mammalian cells. Human signal sequences are preferably used for applications to humans.

The term "anchor protein" or "anchor peptide sequence" refers to a protein or peptide that is anchored to the outer surface of the plasma membrane generally by covalently binding to a glycan containing phosphatidyl inositol. This structure, which is bound to the anchor protein or peptide, is often referred to as glycosyl phosphatidylinositol. In all cells, anchor proteins covalently bound to GPIs are found on the outer surface of the cell's plasma membrane or on the lumen surface of secretory vesicles.

In the present invention, "anchor protein" or "anchor peptide" is generally expressed at the carboxy-terminus of a protein or peptide expressed by the expression mediator according to the invention (3 'end of DNA sequence expressing the desired protein or peptide). Or carboxyl terminus of the expressed protein or peptide).

In the present invention, many proteins or peptides expressed in a patient and, in particular, immunogenic proteins or peptides of the vaccine according to the invention expressed in a patient, are substantially enhanced when the anchor peptide is bound to the carboxy-terminus of the protein or peptide. Produce a biological or immunogenic response in the patient. In addition to the anchor peptide at the carboxy-terminus of the expressed protein, including a signal protein at or near the N-terminus is associated with an unexpected effect of enhancing the biological effect of the expressed protein. This is especially true if the expressed protein is in fact antigenic or immunogenic.

The carboxy-terminus of the expressed protein or peptide is modified by attachment of a glycolipid anchor, which is used to anchor the modified protein or peptide to the cell surface. The peptide residue to which the GPI anchor is added is always one of the small amino acids such as glycine, aspartic acid, asparagine, alanine, serine and cysteine. Since these occur at the carboxy-terminus of the present protein / peptide, they can be specified by binding the appropriate codons to the DNA fragments that are added to the cDNA sequence that specifies the present protein / peptide. Also, the two residues below the anchor addition site are usually small.

Although the three residues have less stringent steric requirements, the segmentation / anchor addition site remains in the three small amino acid residue regions. In order to ensure that functional or immunologically important amino acids are not compromised at or near the carboxy-terminus of the protein / peptide target, several additional amino acids (preferably lysine or arginine But polar amino acids such as threonine, alanine and proline) are inserted in that direction so that small, polar segments are at the carboxy-terminus. The remainder of the further signal sequence comprises 15 to 35 amino acids in the carboxy-terminal hydrophobic region. This region extends for 15-25 amino acids and will include amino acids such as valine, leucine, isoleucine, alanine, phenylalanine, but may not include proline and glycine as well as tryptophan. Examples of such representative sequences are as follows:

TACDLAPPAGTTD AAHPGRSVVPALLPLLAGTLLLLETATAP

The small sequence is shown in bold, with the left side showing the terminal and D residues of the protein, the GPI addition site, and the right side split during the GPI addition with an underlined sequence representing the hydrophobic end.

In the present invention, the anchor peptide may have a cleavable N-terminal sequence, which directs the peptide to the cell transport pathway to which endogenous reticulum and GPI anchors are added. As mentioned above, the anchor peptides also have a hydrogenous sequence that is dominant at the carboxy-terminus, generally ranging in size from about 15 to 35, preferably from about 15 to 30, more preferably from about 15 to 25 amino acid residues, and the GPI Indicates the addition of anchors, split at the same time as adding GPI. In particular any hydrogenated amino acid sequence of at least about 15 to 35, more preferably about 15 to 30 amino acid residues, may be subject to the addition of a GPI anchor. Anchor addition is generally a transamidation reaction in which the free ethanolamine amino group of the GPI precursor invades the peptide bond at the target amino acid (as a nucleophilic addition), resulting in a c-terminal amino acid.

In general, in the expressed anchor peptide sequence, immediately above the hydrophobic sequence to which the GPI anchor is added is a hydrophilic spacer comprising a hydrophilic amino acid (generally about 5-10 residues). The residue to which the GPI anchor is added ("anchor addition site") is an amino acid residue in a hydrophilic spacer selected from the group consisting of glycine, aspartic acid, arginine, asparagine, alanine, serine and cysteine. In addition, two residues below the anchor addition are generally small amino acid residues that apparently minimize steric hindrance at the anchor addition site.

Preferably, the GPI moieties are combined in advance and added as a single unit to specific amino acid residues near the carboxy-terminus of the expressed protein or peptide. The carboxy-terminal region is therefore characterized by the presence of a C-terminal signal peptide, preferably 10 to 30 amino acids in length, which provides the information necessary for entry of the GPI anchor. The actual amino acid residue to which the GPI structure is attached is referred to as the omega site, which residue is glycine, alanine, cysteine, serine, asparagine or aspartic acid. The omega +1 site (expressed and directed to the carboxy-terminus of the untreated protein) is preferably selected from glycine, alanine, cysteine, serine, asparagine, aspartic acid, glutamate, and threonine. The omega +2 site is alanine or glycine. The omega +2 site preferably leads to a hinge or spacer of an ideal 5-7 amino acid comprising filled amino acid and proline; This in turn leads to a hydrophobic sequence of amino acids, preferably terminating the carboxy signal peptide.

The overall structure of the anchor peptide is summarized as 15-35 amino acid peptide at the carboxy-terminus of the expressed protein or peptide. This anchor peptide sequence begins with a hydrophobic extension of the variable amino acid, preferably followed by a sequence of 5-7 amino acids comprising a charged residue leading from the amino acid (from the end towards the end of the amino group) (omega +2 site). Glycine or alanine); In glycine, alanine, cysteine, serine, asparagine, aspartic acid, glutamate and threonine at the +1 omega site; And glycine, alanine, cysteine, serine, aspartic acid, or asparagine at the omega site.

In the present invention, the signal VII sequence is generally found at the N-terminus (directly from the N-terminus or shifted by at least 1,000 amino acids from the N-terminus), and the anchor peptide sequence is generally the carboxy- of the expressed protein or peptide. It should be noted that the signal peptide, which is found at the terminus, can be found at or near the carboxy-terminus of the expressed target protein or peptide.

Anchor sequences well known in the art can be used in the present invention. For example, it is also possible to utilize the anchor sequences of the yeast or sub-trophic ranks, but apparently the stray anchor ranks are preferred for use in mammals, with the anchor rank of a particular species most preferred for the desired mammalian species to be treated. . Therefore, human anchor rank is most preferred for providing proteins or peptides expressed in humans.

The term "effective amount" refers to the expression or production of an amount of protein or peptide that is therapeutically effective in treating a disease or that can result in a protective immunogenic response to a disease, ie that can prevent a patient from disease. Refers to the amount or concentration of a recombinant expression mediator effective at. In general, an effective amount of expression media administered to a patient may vary depending on the patient, including whether the patient can produce a protein or peptide to be expressed and whether the patient has already been exposed to a protein or peptide for which the patient wants an immunogenic response. It depends on the factors. For vaccines, cellular and humoral immune and / or therapeutic responses can be measured prior to administration. Under normal conditions, in humans, the effective amount of recombinant expression mediators is widely variable, and in general, for viral papilloma mediators, the effective amount is from about 10 ³ to about 10 ⁸ plaque-forming units, more preferably about 10 ⁴ To 10 ⁷ plaque forming units. In the case of vaccines, the preferred effective dose range is from about 10 ⁴ to 10 ⁷ plaque-forming units, and more preferably from about 1x10 ⁶ to about 5x10 ⁶ plaque-forming units (as determined by the measurement test as described above). This range of the administered papillomavirus is chosen to enhance the possibility of producing sufficient amounts of proteins or peptides that do not show a toxic response. In the case of a vaccine, an effective concentration is one that can elicit an immunogenic response without overinfecting the patient to be vaccinated.

The chimeric peptides according to the present invention, rather than to the expression mediator, are produced in an amount that is therapeutically effective for the treatment of the disease or to produce a protective immunogenic response against the disease, ie to prevent the patient against the disease. May be administered directly.

In the use of the methods of the invention, the desired target peptide or protein is identified and sequenced to determine the specific amino acid sequence that is ultimately expressed by the expression mediator administered. Appropriate amino acid sequences can be readily obtained from isolated proteins or peptides using standard sequencing techniques commonly available in the art. Alternatively, currently available sequences can be obtained from several databases, including, for example, GCG and the Brookhaven data base. Target peptide fragments that can also be used can be readily obtained from amino acid sequences obtained from isolated proteins.

After identifying the amino acid sequence of the target protein or peptide, the DNA fragment (DNA fragment; not generic but conservative) is synthesized. This is the case in the case of peptide and yape peptide sequences to be used. Those skilled in the art can use the well-known techniques in producing a preferred nucleic acid sequence corresponding to the protein or peptide sequence to be expressed. Nucleic acid synthesizers or DNA fragment synthesis techniques are currently readily available in the art, allowing those skilled in the art to readily synthesize DNA sequences corresponding to, for example, the known amino acid sequences of the protein or peptide to be expressed.

After appropriate DNA sequences comprising restriction digest fragments are synthesized, the fragments are cloned, bound or alternatively bundled together and inserted into the clone mediators. The DNA fragments, anchor peptide sequences and signal peptide sequences corresponding to the protein or peptide to be expressed are first sequenced and then amplified and bound. Clone mediators that can be used to generate quantification of the DNA to be amplified include, for example, the following clone mediators; In particular, all commercially available pBR322, pGEM3z, pSP70, pSE420, pRSET, lambdaZAP. Such restriction digestive fragments can be obtained from clones isolated from eukaryotic or prokaryotic sources.

Appropriate DNA sequences (proteins or peptides to be expressed alone or in combination with anchor peptide sequences and optionally signal sequences) are cloned in appropriate clonal mediators and isolated, for example, using gel electrophoresis or related methods, Amplified by binding to amplification mediators. Preferred DNA sequences can also be amplified by standard polymerase chain reactions for a sufficient number of cycles to obtain the desired amount of DNA (about 5 to about 40 cycles, depending on the amount of DNA desired). The DNA encoding of the anchor peptide sequence and optionally the signal peptide sequence can be bound to the mediator comprising the desired protein or peptide to be expressed after identification of the appropriate DNA fragment in the positive clone by PCR and endonuclease digestion. Amplified appropriately using the technique.

After amplification, nucleic acid salts, preferably in the form of DNA, are bound to the transfer mediator and are produced by eukaryotic cells such as monkey kidney cells (BSC-1 cells) and wild head disease virus (WR) in order to produce recombinant papillomaviruses. ) Is infected. Recombinant scleroderma virus is purified prior to amplification. After amplification and in some cases further purification, the recombinant scoliosis virus is administered to the animal in the form of a therapeutic or immunogenic dose expressed in combination with a desired protein or peptide, preferably an anchor peptide and optionally a signal peptide. .

It should be noted that it may be desirable to combine two or more copies of the DNA sequence corresponding to the desired protein or peptide in combination with an anchor peptide sequence and optionally a signal peptide sequence with other operating elements. In this way, multiple copies of the same target protein can be expressed in combination with an anchor peptide and optionally a signal peptide. In one embodiment, multiple proteins can be expressed within a single polycistronic vector by placing expression of each desired protein or peptide and anchor peptide under the control of an internal ribosome inlet site and IRES (eg, See Molla et al., Nature, 1992, 356: 255; Jang, et al., J. of Virol., 1989, 263: 1651). Using this approach, the amount of protein expressed can be significantly increased.

Once the nucleic acid sequence coding immunogenic chimeric protein is pre-established in an appropriate expression mediator, the expression mediator can be used in a therapeutic or immunogenic dose form or alternatively the mediator can be used in an appropriate eukaryotic cell line, for example It may be used for the purpose of expressing immunogenic chimeric proteins in order to facilitate the generation of the desired peptide sequence outside of the host animal. Such eukaryotic cell systems include, for example, HeLa, L929, T2 or RMA-2, preferably T2 or RAM-2. In this method, cells comprising an expression mediator are grown to lyse to separate synthetic peptides comprising the desired protein or peptide sequence in combination with an anchor peptide sequence and, optionally, a signal sequence. The isolated peptide sequence can be used directly in the form of a therapeutic or immunogenic dose. Alternatively, expression mediators can be administered directly to a patient who will express the desired protein or peptide and anchor sequence and cause the therapeutic or immunogenic effect as intended for that patient.

The expressed proteins are subjected to standard protein purification procedures well known in the art, including gel electrophoresis, affinity and immunological affinity chromatography, discriminant precipitation, molecular filtration chromatography, isoelectric focusing and ion-exchange chromatography. After lysis, it can be obtained from cell culture. In immunological affinity chromatography, a protein or peptide can be purified by a route through a column containing a resin that is bound to an antibody specified for at least the protein or peptide moiety.

An expressed protein or peptide comprising an anchor peptide sequence and optionally a signal peptide sequence obtainable from cell culture, may be in pure form for a patient in need of such treatment by purifying the include lysate from the cell culture. It can be administered in a sufficiently pure form. Preferably, the expressed protein consists of a therapeutically or immunogenically effective amount of the expressed protein comprising an anchor peptide sequence and optionally, a signal sequence, in combination with a therapeutically acceptable additive additional carrier or excipient. Such formulations are possible in the form of unit doses by methods well known in the art. The amount of expressed protein or peptide administered will vary depending on the pharmokinetic parameters, the severity of the treated vagina or the desired immunogenic response. Of course, the dose is already exposed to the age, body weight, and, in the case of an immunogenic dose form, the free-promoting properties of the protein expressed from the therapeutic dose form of the invention as well as the microbiological response to the disease to which the patient is vaccinated. It is set by the prescribing physician taking into account relevant factors including the condition of a patient, including whether or not it has been used.

The amount of expressed protein administered by the present invention is an amount effective to produce the intended effect. For therapeutic doses, the intended effect is to sufficiently reduce or eliminate protein or peptide deficiencies and the physiological signs that these deficiencies cause. In the case of administration of immunogenic expressed proteins or peptides, the intended effect is to obtain an immunogenic response in the patient that provides a sufficient protective effect against infection.

In an embodiment according to the invention, the expressed mediator, preferably in the form of recombinant scleroderma virus, is administered directly to the patient. By directly administering the expression mediator to the patient, the mediator produces the target expressed protein or peptide directly in the patient, thus eliminating or eliminating the need to continuously administer the target protein or peptide. In this example, the amount of recombinant scoliosis virus administered to the patient is an effective amount to express a protein or peptide sufficient to provide a therapeutic or immunogenic response in the patient. The expressed protein or peptide may be selected from the anchor peptide sequence and optionally, the signal within the expression mediator to sufficiently increase the therapeutic effect and / or immunogenicity of the expressed protein or peptide as compared to the protein or peptide that does not include the signal and anchor peptide. Is bound to the peptide sequence. The therapeutic aspect of the present invention provides a therapeutic effect to sufficiently eliminate the physiological effects associated with the lack of expressed proteins or peptides, unlike the immunogenic features of the present invention which provide a protective effect against infection.

A pharmaceutical composition according to the present invention may consist of an effective amount of a protein or peptide or alternatively consists of an effective amount of a medium in a sufficiently pure form or alternatively in combination with a pharmaceutically acceptable additive, carrier or excipient. Can be. The composition may be administered directly or may be added to a pharmaceutically acceptable additive, carrier or excipient for convenience of administration. Suitable pharmaceutically acceptable carriers will be apparent to those skilled in the art and include non-polar carriers as well as low molecular weight alkanols such as ethylene glycol, polyethylene glycol, and propylene glycol, water and other polar materials including polyalkanols. Stabilizers and other additives, including antiadsorbents, may be advantageously used in the pharmaceutical compositions of the present invention.

In producing the pharmaceutical compositions according to the invention, the active ingredient is generally in a uniform or constant relationship with the pharmaceutical additives, excipients or carriers, and generally in unit doses such as liquids, suspensions or solids. For example, solutions or suspensions administered parenterally, such as intramuscular, intravenous, subcutaneous or intraperitoneal, are generally isotonic in the patient's blood or receptor. Such formulations may be prepared by dissolving the active ingredient in water, including physiologically compatible substances such as sodium chloride, other salts, amino acids, etc., and buffering the pH of the solution compatible with the physiological water. Such doses are administered in sterile form. Sterile solutions can be prepared by filtering the solution through a filter designed to remove any microorganisms that may contaminate the preparation without substantially impacting the components or effects of the pharmaceutical composition.

The present invention may be administered in a manner that controls the promotion of activity, particularly in relation to the administration of directly expressed proteins (ie not in the form of expression mediators that produce proteins or peptides at the receptor). Controlled liberalization preparations are possible by using pharmaceutically compatible polymers or mixtures of polymers to complex or absorb proteins or peptides or extracts thereof, or in particular by binding to microcapsules, liposomes, albumin microspheres and microemulsions. Do.

Oral preparations are formulated by combining the active agent with representative oral carriers such as sucrose, lactose, magnesium stearate, starch, gum arabic, algin and carrageenan, and in particular cellulose ethers such as methyl cellulose and hydroproxy cellulose.

Expression media administration can be administered intramuscularly, subcutaneously, intraperitoneally and even orally, but preferred is via the vein. The dose of expression mediator, preferably the recombinant papillomavirus according to the invention, which is administered in conjunction with the carrier, is often approximately equal to the amount administered alone (not administered together). Expression media can be administered in substantially pure form or in combination with a material having an affinity for nucleic acid salts and an internalization factor that binds to a material having affinity for nucleic acid salts (eg, Wu, et al. , J. Biol. Chem., 262, 4429 (1987) and Wagner, et al., Proc. Natl., Acad. Sci. USA, 87: 3655 (1990)). Internalization factors, such as ligands having specificity for specific receptors on a cell, can be used to guide expression mediators to desired target cells when administered. Internalization factors include, for example, specific antibodies to cell surface proteins for which transferrin and expression mediators are preferably targeted.

Doses of the administered expression mediators, including whether they have already been exposed to the disease-causing microorganism for the patient to be vaccinated, and whether they have already been exposed to the glass-promoting properties of the protein expressed from the therapeutic dosage form of the present invention, It is set by the prescribing physician taking into account relevant factors including the age, weight and conditions of the patient.

In the immunization or vaccine features of the present invention, the dose of the expression mediator, which is preferably the sarcoma virus, depends on the dosage form. For example, the vaccine is decided, and more preferably about 10 ⁴ to 10 ⁷ plaque-forming units in accordance with a preferred level of the expressed immunogenic protein is common to include a virus concentration of about 1x10 ⁶ to about 5x10 ⁶ plaque-forming units . This range of the administered papillomavirus is chosen to enhance the possibility of producing sufficient amounts of proteins or peptides that do not show a toxic response. In the case of a vaccine, an effective concentration is one that can elicit an immunogenic response without overinfecting the patient to be vaccinated. Therefore, the concentration or amount of the sarcoidosis virus included in the vaccine of the present invention generally falls within this range; However, the amount of recombinant scleroderma virus used in any vaccine form depends on the intensity of the immunogenic response elicited.

In the therapeutic features of the present invention, the dose of the expression mediator, which is preferably a scleroderma virus, is in a range that depends on the desired concentration of the expression peptide and the number or concentration of copies of the protein or peptide produced by any mediator. In general, for such a therapeutic feature of the present invention, in the case of a papilloma virus expression medium, the concentration is generally about 10 ⁴ to 10 ¹⁰ plaque forming units, more preferably about 10 ⁶ to about 10 ⁷ plaque forming units. . In this therapeutic nature of the invention, the concentration of the expression mediator, of course, depends on the desired concentration range of the expressed protein or peptide and can be higher in vaccine properties.

In determining the amount of expression mediator, in particular, the papilloma virus of a given vaccine can use the following method. Vaccine dosage forms may include standard pharmaceutical carriers as described above. The proportion of virus included in the vaccine depends on the expected dose, as well as the chemical nature, solubility, and stability of the virus. For parenteral administration or injection via parenteral routes such as the peritoneum, intramuscular, intramammary, subcutaneous or other routes, sterile solutions of the scleroderma virus are prepared. Preferably the vaccine according to the invention is administered via subcutaneous roots.

The dose and treatment regimen of the vaccines used follow the conventions commonly adopted for other vaccination or treatment regimens in which this general method has been adopted. Initially, two or more therapeutic doses or vaccines are not expected to be required, but for vaccines, the potential for providing an efficacy promoter dose is not expected. Preferably, the dose plan for immunization for most microorganisms is to subcutaneously inject at least about 1 × 10 ⁶ plaque forming units of the myelopathy virus.

In the treatment method according to the present invention, expression of a target protein or peptide occurs in combination with an anchor peptide sequence and optionally with a signal peptide sequence by administering to the human patient an effective amount of an expression mediator, preferably a recombinant papillomavirus. do. In some cases, additional potency promoters of recombinant scleroderma virus may be given to promote a therapeutic or immunogenic response. If desired, additional doses of recombinant scoliosis virus can be provided to promote the efficacy of the initial inoculation.

The present invention devised to bind both the signal peptide sequence and anchor peptide sequence to the expressed protein or peptide, as in the preferred embodiment. Such combinations have been found to be particularly beneficial for generating therapeutic and immunogenic responses to target proteins or peptides. The signal peptide sequence is preferably bound to or near the amino group end of the target protein or peptide, and the anchor peptide is generally bound to or near the carboxy terminus of the protein or peptide. After administration of the recombinant expression mediator, preferably the recombinant scoliosis virus, the protein or peptide is properly expressed by the mediator and comprises a signal peptide sequence and an anchor peptide sequence. Therefore, in the present invention, the signal and anchor peptide are preferably expressed at the amino group and the carboxy terminus of the expressed protein or peptide, respectively. Thus, in a preferred embodiment, the signal peptide sequence is located above from the expressed protein or peptide and the anchor peptide is located below from the protein or peptide.

The following examples are provided for illustrative purposes and do not limit the scope of the present invention.

Example

Example 1-Herpes Simplex

Neutralizing antibodies against viruses have been designed to recognize surface glycoproteins in tissues as two glycoproteins (glycoproteins B and D) that function as dominant antigens. Recombinant glycoproteins have been produced in Chinese Hamster Ovary (CHO) cells and have been known to provide protection for guinea pigs against direct challenges. The range of protection varies, but generally depends on Freund's adjuvant, which is excluded for human use.

Clones of herpes glycoproteins (gB1 / gB2) effective as known antigens are available. A cloned cDNA encoding information about the glycoprotein B of the herpes virus adds a mammalian signal sequence to the 5 'end of the DNA (for effective insertion at the N-terminus of the expressed glycoprotein) and at the 3' end of the DNA. It is demonstrated by adding mammalian anchor sequences. Alternatively, cDNA encoding for herpes protein gK may also be used. This is made possible by the beginning of the 5 'sequence through the use of specific oligonucleotides with homology as the cleavage site defined for the 5' sequence present and restriction endonucleases. Similar methods are used to attach a sequence for encoding an amino acid (anchor sequence) recognized by an enzymatic system that attaches a glycylphosphatidyl inositol chain to the carboxyl terminus of a protein at the 3 'end of the cDNA. In the latter case, the restriction site is located at the 3 'end of the sequence. The treated cDNA structure is then ligated to the appropriate transfer mediator or host tissue using techniques known in the art. Suitable transfer mediators are, for example, those that can be used for the production of a papilloma virus such as pSC65. Suitable host tissues can be used as receptors for the preparation of excipients suitable for direct oral administration.

Recombinant scleroderma virus is a mammalian host cell (BSC) with a sequence that encodes a selectable marker and a sequence homologous to the non-essential region of the scleroderma genome in addition to the wild type scleroderma virus and the present cDNA structure. Such as -1), preferably by co-infecting human host cells. Recombinant virus can be isolated using standard methods. This infects cells in inoculation of human hosts and causes the production of herpes glycoproteins to maximize their interaction with human immune system cells. Myelopathy acts as a "local supplement" signal to host immune cells. The addition of these signals and anchors to the protein allows for the proper transition of the cell secretion pathway and the placement of the final molecular product (eg glycoprotein B) on the surface of the infected cells in the proper direction for interaction with immune cells. To ensure that.

Example 2 Cytomegalovirus

Obtaining immunity prior to conception should significantly reduce the incidence of side effects. It was established that infected individuals can initiate an immune response, including both humoral and cellular components; Tests were performed with attenuated strains of virus as immunogens. Attempts have been made to provide protection by administration of protein antigens in combination with adjuvant.

In this example, the recombinant mediator is constructed to include a combination of signal and anchor sequences shown for herpes, in addition to genetic information about the viral protein, which ensures accurate trafficking and access to the host immune system. .

The protein of choice for binding to the expression mediator is gB. This has the advantage of providing a well-known immune response and being well preserved in different strains of virus. CDNA is available for these proteins and has already been successfully expressed in insect cells.

The general method described above for herpes simplex also applies to gB cDNA. The region at the 5 'end for the viral signal sequence is removed and replaced with that for encoding the mammalian signal sequence. This is a further modification at the 3 'end of the sequence that provides the information necessary for nuclease digestion, ligation with synthetic oligonucleotides including the present sequence and the necessary duplication, selection of recombinant product and attachment of GPI anchors to the finished protein. Is made by. This is done in much the same way as cleavage of DNA at the usual 3 'end, addition of oligonucleotides encoding the desired sequence, and binding of the signal sequence by a range of unmodified normal 3' sequences.

The final structure (frontal or oral virus, such as a bacterial virus) comprises the coding sequences for the 5 'and 3' additional gB proteins, respectively, of the signal and anchor sequences. The media chosen for administration may be viruses or bacteria. The former is suitable for subcutaneous administration and the latter is suitable for oral administration.

Example 3 Hepatitis

Recombinant clones of hepatitis B expressing antigen (rHBs- in particular HBsAg) cDNA are available and form a starting material for the preparation of reprocessed vaccines. Alternatively, another use of the grafting and binding techniques described above results in a structure in which the production tissue contains, as its genome portion, DNA encoding 5 'and 3' additional rHBs of signal and anchor sequences, respectively. . Vaccination of a host animal results in a product directed in the rHBs in the extracellular domain on the surface of infected cells as a recombinant medium (in veterinary or human). This provides improved protection by minimizing interaction with the host immune system.

This general approach can be used to provide recombinant expression mediators for immunogenic surface antigens (proteins or peptides) from hepatitis A and C as well as other hepatitis strains.

The general approach as described above can, in fact, be used for any protein or peptide sequence to be expressed, even for proteins or peptides that have not yet been isolated.

While the invention has been described in its preferred embodiments, the terms set forth in the description are not to be construed as limiting terms, and furthermore, without departing from the spirit and scope of the invention in a broader sense. Modifications can be made within the scope.

Claims (18)

a) a target protein or peptide; And b) anchor sequence peptide; And optionally c) signal sequence peptide Chimeric peptide sequence consisting of. The method of claim 1, wherein the target protein or peptide sequence is a receptor, hormone, growth factor, blood factor, enzyme, neuroprotein, apolipoprotein, tumor suppressor, antigen, tumor antigenic protein and peptide, bacterial surface protein and peptide. , Peptide surface selected from the group consisting of viral surface proteins and peptides, protozoal cell surface proteins and peptides, fungal surface proteins and peptides and viral enzymes. The peptide sequence of claim 2, wherein the receptor is selected from the group consisting of insulin receptors, hormone receptors, and growth factor receptors. The peptide sequence of claim 2, wherein the enzyme is a viral enzyme. 5. The peptide sequence of claim 4, wherein said viral enzyme is reverse transscriptase. The peptide sequence of claim 1, wherein the target protein or peptide is a viral surface protein. 7. The peptide sequence of claim 6, wherein the viral surface protein is gB or an immunogenic sequence of cytomegalovirus. 7. The peptide sequence of claim 6, wherein the viral surface protein is rHB of hepatitis B or an immunogenic sequence thereof. 7. The peptide sequence of claim 6, wherein the viral surface protein is a glycoprotein B of herpes simplex virus or an immunogenic sequence thereof. A pharmaceutical composition consisting of the peptide sequence of claim 1 in pure or substantially pure form. The composition of claim 9, wherein the peptide sequence is formulated in combination with a pharmaceutically acceptable additive, excipient or carrier. A nucleotide sequence encoding a peptide to be expressed, which is a sequence for encoding a mammalian signal peptide or a mammalian anchor peptide at the C-terminus of an expressed protein sequence and for administering the expression medium to a patient, binds to the expression medium to be administered to the patient A method for greatly enhancing the pharmacological effect produced by a peptide to be expressed in a patient consisting of. The method of claim 12, wherein the nucleotide sequence comprises both a signal peptide and an anchor peptide. 13. The method of claim 12, wherein said expression mediator is recombinant scoliosis virus. The method of claim 12, wherein the peptide is an antigenic peptide obtained from bacteria, viruses, protozoa or fungi. The method of claim 15, wherein said peptide is a surface antigen. The method of claim 12, wherein the peptide is a surface antigen protein of a virus. 18. The method of claim 17, wherein the virus is herpes simplex, cytomegalovirus or hepatitis virus.