JP2787926B2 - Fusion protein - Google Patents

Description

Description: TECHNICAL FIELD The present invention generally relates to fusion proteins useful as vaccines for immunological castration or reproductive function suppression of vertebrate hosts, particularly domesticated animals.

BACKGROUND ART The most popular method of preventing reproductive activity in livestock, including dogs, horses, sheep, cattle, goats and cats, is surgical ovariectomy or castration.

This method has the problem that it is irreversible and is a technically difficult operation, and therefore requires trained veterinary skills.

One alternative to surgery is to use long-term oestrus depressants in dogs (Harris & Wotchuk Am. J. Vet. Res, 24: 1003-1006, 196).
3), but unfortunately after prolonged treatment, progesters have been linked to the induction of uterine diseases, including purulent metritis, endometritis and increased incidence of benign breast tumors This is the administration of gensteroids. Their use therefore tended to be limited to short-term suppression of estrus or postponement of estrus.

In economically important breeding animals, there are no commonly available long-term contraceptives that have been found suitable for daily use in the field.

Therefore, there is a need for a non-steroidal livestock contraceptive method that is well tolerated and applicable to both male and female livestock.

One such method would be immunization against hormones that control reproductive organ development and activity.

Two gonad stimulating hormones that regulate gonad steroidogenesis and gametogenesis and are responsible for the reproductive cycle are luteinizing hormone (LH) and follicle stimulating hormone (FSH).

Luteinizing hormone-releasing hormone (LHRH, also known as GnRH) controls the synthesis and release of LH and FSH from the ventral pituitary gland. Mammalian LHRH is a decapeptide consisting of the naturally occurring amino acids set forth in the following sequence and SEQ ID NO: 1.

The N and C terminal daltamate and glycine residues are modified after translation to pyroglutamic acid and glycinamide, respectively.

A vaccine that results in the production of antibodies against LHRH by the host will suppress the production and release of endogenous LH and FSH in that host. This suppression results in reduced steroid production and impaired reproductive periodicity and fertility in the treated animals.

The resulting physiological effects are as follows.

(A) In females-(i) Pause of LH pulsation (ii) Infertility due to impaired ovulation (iii) Pause of estrous cycle due to lack of estrous hormone (iv) Regression of genital tract (v) Abortion due to regression (b) In males-Inhibition of testosterone production from Didig cells in the testicles results in reduced peripheral serum levels of circulating androgens and the following:

(I) Decrease in sexual urge (ii) Regression of auxiliary gonads (iii) Decrease in testicular volume and decrease / stop of spermatogenesis Antibodies to LHRH can be used to chemically conjugate LHRH to a suitable carrier and to provide appropriate adjuvants. It can be produced in many species by administering it in its presence (Carrelli
C. et al., 1982, Proc. Natl. Acad. Sci. USA 79 , 5392-5395).
However, chemical conjugation is difficult to control and often results in foreign and undefined products. Also, effective immunization usually requires oily adjuvants, which often lead to the formation of unacceptable side effects such as inflammation and granulomatous tissue damage.

It would be desirable to provide a means to produce good titers of antibodies to LHRH without having to use strong adjuvants.

The genetic signal of the TraT protein (TraTp) is specified by the TraT gene. TraTp is an outer membrane lipoprotein produced by certain strains of E. coli and is responsible for their resistance to serum lethality. When mice are injected intramuscularly without adjuvant,
TraTp elicits an antibody response, comparable to that obtained when injected with incomplete Freund's adjuvant. In addition, the immunogen is chemically conjugated to TraTp,
Administration of the conjugate in saline to animals results in the production of high levels of anti-immunogenic antibodies. Therefore, TraT
p can be used as a self-adjuvanting carrier for immunogens. This use of TraTp was described earlier in International Patent Application No. PCT / AU87 / 00107 (published as WO87 / 06590), where both chemical and genetic binding of TraTp to immunogenic molecules was described. Have been. The specific fusions performed and described in that specification are for large proteins. LHRH, on the other hand, is a short peptide that is inherently difficult to use without a suitable carrier. In addition, there is little change in the peptide between the mains, so it can be viewed as a self-antigen by the immune system, and consequently rebellious against stimulating the immune response.
A fusion protein consisting of an LHRH sequence and LTB (B-subunit of a heat-labile toxin produced by a strain of E. coli ) has been described (International Patent Application No. PCT /
AU86 / 00135, published as WO86 / 06635). These constructs have been prepared for the purpose of providing LHRH orally to the host immune system using the ability of LTB to bind to the mucosal epithelium. Although they are not self-adjuvant and have demonstrated reproductive arrest,
The resulting deterrence was not a strong deterrence.

PCT / EP89 / 01013 (published as WO90 / 02187) describes a fusion protein containing a peptide such as LHRH, which is not essentially antigenic by itself, as hepatitis B surface antigen, a highly antigenic and hydrophilic protein. To produce using a suitable carrier. TraTp is a membrane lipoprotein and not a highly hydrophilic protein. Further PCT / EP89 / 010
The fusion taught in 13 does not appear to be self-adjuvant.

Abbreviation LHRH: Luteinizing hormone-releasing hormone LH: Luteinizing hormone GnRh: Gonadotropin-releasing hormone (also known as LHRH) FSH: Follicle-stimulating hormone LTB: A heat-labile toxin produced by a strain of E. coli B subunit QC: Quality control QA: Quality assurance EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate SDS-PAGE: Sodium dodecyl sulfate / polyacrylamide gel electrophoresis LPS: Lipopolysaccharide EDAC: 1-ethyl-3- (3-dimethyl -Aminopropyl) carbodiimide / HCl ABTS: 2,2'-azinobis (3-ethylbenzthiazoline sulfonic acid) PEG: Polyethylene glycol BSA: Bovine serum albumin IF: Insoluble form of fusion protein SF: Soluble form of fusion protein PHA: Phytohemagglutinin MBS: n-hydroxysuccinimide ester of m-maleimidobenzoic acid A ₂₈₀ : Absorbance when set to 280 nm in a spectrophotometer ISA-20: Montanide adjuvant, SEPPIC ISA-25: Montanide adjuvant, SEPPIC sem: Standard error of average sd: Standard deviation PBS: Phosphate buffered salt solution, PH7. 2-7.4 w / w: weight vs. weight v / w: volume vs. weight DNA: deoxyribonucleic acid NSB: non-specific binding Definition TarTp is the protein product of the TraT gene. TraTp indicates a fusion protein formed as an expression product of TraT and LHRH gene fusion.

TraTp-LHRH protein fusions are designated 730p, 731p, etc., depending on the plasmid in which they were expressed.

 The plasmid is indicated as pBTA730 or the like.

The binding of E. coli / plasmid is shown as BTA1664 or the like.

A TraTp analog according to the present invention comprises an LHRH analog sequence
A molecule related to the TraTp sequence where alterations such as insertions, deletions or substitutions occur, depending on the strategy used to fuse to the sequence.

The LHRH analogs according to the present invention may be used to alter the above-identified sequence changes that occur between species, changes in post-translational modifications to particular residues that occur due to particular fusion strategies, or due to particular fusion strategies. Is a molecule related to the LHRH sequence when an amino acid difference takes into account any of the amino acid sequence changes that occur.

DESCRIPTION OF THE INVENTION In the present invention, specific insertion sites in TraTp have been identified, thereby producing a novel fusion protein of TraTp or an analog thereof and an LHRH analog capable of eliciting a strong immune response against LHRH. Can now be obtained. The present invention has shown that all fusions of LHRH analogs with TraTp or TraTp analogs are not always suitable for producing good titers of antibodies against LHRH. In addition, among species, there was a change in the effect of various multimers of LHRH at specific positions on TraTp.

The present invention relates to a vaccine useful for fusion or control of the LHRH analog genetic coding sequence to a TraTp or TraTp analog genetic coding sequence for inhibiting or controlling the reproductive function of vertebrate hosts, particularly bred animals. Can be used to effectively provide

According to the present invention, recombinant DNA is used to create a novel fusion protein of TraTp or TraTp analog and LHRH analog.
Techniques can be used that, when administered in saline or in an adjuvant such as saponin, produce antibodies that recognize LHRH (hereinafter LHRH antibodies), which in turn suppress reproductive function in animals. It is.

The immunogen fusion studies exemplified herein show that insertion of a tandem repeat of an LHRH analog gives more immunogen fusion than insertion of one insert.

The advantages associated with making a fusion protein in E. coli compared to the chemical conjugation of TraTp and LHRH are as follows.

a) The manufacturing process is simpler than chemical bonding.

b) It is easier to chemically define the nature of the fusion protein product than conjugation, thus favoring product quality control (QC) and product quality assurance (QA).

c) Because fusions can be selected for the exact location of LHRH analog insertion into TraTp or TraTp analogs and the number of repeating epitopes to give optimal immunological response, fusion Greater specificity and flexibility than mechanical conjugation.

The present invention provides a novel fusion protein. These fusion proteins may consist of a single copy of an analog of the LHRH decapeptide inserted or fused to TraTp or an analog thereof, or a multiplex of LHRH analogs inserted at multiple positions within the TraTp or TraTp analog. It may be a copy. Particular cloning strategies include LHRH analogs or TraTp
It may also require the inclusion of nucleotides designating the genetic code in non-innate sequences, or may lead to deletion of bases from the sequence specified in the genetic code.

Preferably, the fusion is an LH as set forth in SEQ ID NO: 2.
Consists of RH analogs.

Preferably, at least one LHRH analog is inserted between amino acids 80 and 81 200 and 201 or 235 and 236 of the TraTp sequence, or a combination of these positions where amino acid 1 is Met1 of the TraTp signal sequence.

The novel fusion proteins of the present invention can be used to provide vaccines suitable for administration to livestock to suppress or modify the reproductive function of those animals.

The present invention also provides polynucleotide molecules that encode the fusion proteins of the present invention.

Preferred polynucleotide molecules are recombinant DNA molecules. More preferably, the recombinant DNA molecule consists of a plasmid vector. A preferred vector is pBTA812.
It will be appreciated that vectors other than plasmid vectors could have been used. Other vectors include expression systems, including viral, cosmid and phasmid vectors.

The present invention further provides a transformant host having the polynucleotide molecule of the present invention. Typically, the host is a bacterial host such as E. coli . Preferred hosts are
E. used with the polynucleotide molecule of the present invention .
coli strain is the N4830, where the fusion gene is under the control of the P _L promoter. Other hosts that could be used are yeast, fungi, other bacterial hosts, including insect and mammalian cell lines, and other eukaryotic hosts.

The vaccine of the invention comprises at least one fusion protein of the invention and a carrier, diluent, excipient and / or adjuvant suitable for human or veterinary use.

The amount of fusion protein that may be combined with the carrier to produce a single dosage form will depend upon the conditions induced, the host treated and the particular mode of administration.

Also, specific dosages for a particular host may vary depending on the specific fusion protein activity, age, body weight, general health, host sex and diet, administration time, route of administration, excretion rate and drug combinations. It will be understood that it depends on factors.

The vaccines of the invention may be administered orally, parenterally, rectally or vaginally in dosage units containing conventional non-toxic, pharmaceutically acceptable carriers, diluents, adjuvants and / or excipients, as desired. Can be administered.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. . Among the acceptable vehicles or solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, certain sterile oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Suitable adjuvants for vaccination of animals or humans include saponin, Mostanide ISA-20 or Montanide ISA.
Oil emulsions such as -25, Marcol 52: Montanide 888
(Marcol is a trademark of Esso; Montanide, Montanide ISA-20
And Montanide ISA-25 are trademarks of SEPPIC, Paris, Squalane or Squalene, Adjuvant 65 (Peanut oil,
Mineral gels such as mannose monooleate and aluminum monostearate), aluminum hydroxide, aluminum phosphate, calcium phosphate and alum, hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecyl ammonium bromide, N, N- Surfactants such as dioctadecyl-N ', N'-bis- (2-hydroxyethyl) propanediamine, methoxyhexadecylglycerol and pluronic polyols, pyran, dextran sulfate, DEAE-dextran, polyacrylic acid and Polyanions such as carbopol, muramyl dipeptide,
Including but not limited to peptides and amino acids such as dimethylglycine, tactosin and trehalose dimycolate. The fusion proteins of the invention may also be added to ribosomes or other microcarriers, or conjugated to polysaccharides, proteins or polymers, or to "Iscoms" (Quil-A to form an immunostimulatory complex). (Marein et al., Nat
ure, 380, 457-460 [1984]).

The route of administration, the dose to be administered, as well as the frequency of injections, are all factors that can be optimized using conventional techniques. Typically, the initial vaccination is followed by one or more “boost” vaccinations, several weeks later, the net effect of which is the production of high titers of antibodies against the immunogen. .

Suppositories for the administration of the fusion protein of the present invention rectally or vaginally include cocoa butter, which is solid at normal temperatures but liquid at rectal or vaginal temperatures.
Mix with a suitable non-irritating excipient, such as theobroma oil, glycerinated gelatin or polyethylene glycol, or with a liquid that is present in a suitable cavity and thus melts in the rectum or vagina to release the fusion protein. It can be prepared by contact.

Solid dosage forms for oral administration include capsules, tablets,
Pills, powders and granules can be included. In such solid dosage forms, the fusion protein may be sucrose,
It can be mixed with at least one inert diluent, such as lactose or starch. Such dosage forms can also comprise, as is common practice, additional substances other than inert diluents, eg, lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage form can also consist of buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, syrups, solutions, suspensions and microparticles in elixirs, including inert diluents commonly used in the art, such as water. Microcapsules can be included. Such compositions also include sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol,
Oils such as sesame oil, olive oil, soybean oil, preservatives such as alkyl parahydroxybenzoate, humectants including flavors such as strawberry flavor and seaweed, emulsifying and suspending agents, adjuvants such as sweetness, flavor and flavoring It can also consist of

The present invention further provides a method of controlling reproductive function in a vertebrate host, preferably a domestic animal, comprising administering to a host a fusion protein or vaccine of the invention and vaccinating the host. It consists of

The present invention also provides a method of inhibiting reproductive function in a vertebrate host, preferably a domestic animal, the method comprising:
Administering the fusion protein or vaccine according to the invention to a host and vaccinating the host.

The fusion proteins of the present invention are therefore generally applied to control vertebrate fertility and reproductive cycle, but are particularly useful for companion animals such as dogs and cats and cattle,
It is applied to control the reproductive activity of mammals used for commercial purposes, such as sheep, goats, pigs, and horses.

The fusion proteins of the invention are typically synthesized in E. coli after gene expression of TraTp or a chimera that assigns the genetic code to the TraTp and LHRH analogs.

There are a few possible strategies for creating such chimeric genes. These are described below, but are not limited thereto.

1. Random insertion: The LHRH analog may be located anywhere within the TraT protein or its analog using an appropriate gene construction method. The product can be tested for LHRH immunogenicity and the best composition selected on a vaccine basis. There are many possible ways.

a) D that specifies the genetic code for TraTp or its analogs
To NA, DNAse I [Lin et al., Anal. Biochem, 197, 114-119 (19
85)], and insert a DNA fragment designating the genetic code into LHRH.
Clone into E. coli . By placing the TraT gene under the control of a suitable promoter, the induction can be selected by performing an immunoassay on the colonies with the LHRH antibody for further characterization, and a set of fusion expressed percentages. This would result in a recombinant clone.

b) By inserting the coding DNA of the LHRH analog into a convenient restriction position in the TraT gene. Insertion of DNA must be tailored to be compatible with the various cohesive ends created by various restriction endonucleases, and must preserve the open reading frame for translation.

2.Designated insertion: Inserting an LHRH analog into the sequence of a TraTp or TraTp analog such that the LHRH analog is exposed to the surface of the TraTp or TraTp analog will result in minimal disruption of the TraTp or analog. And mimicking the TraTp-LHRH junction such that the LHRH is preferably located on the outer surface of the molecule will result in optimal display of the LHRH analog in the immune system. An alternative approach is TraT
Part of p will be replaced with an LHRH analog. The designated insertion is made by constructing appropriate restriction sites at specific locations in the gene sequence.

3. The LHRH analog can be inserted as a monomer at one position in the TraTp or TraTp analog, as a monomer at one or more positions, or as a multiple replication at one or more positions.

4. Substitutions, insertions or deletions at one or more positions of the LHRH analog containing amino acids can be used in any of the ways described above. The amino terminus of LHRH is py
It should be noted that roGlu cannot be formed if the amino acid sequence encoded by the LHRH gene is inside the fusion protein. Therefore,
The expression of the gene is controlled by Glu- of LHRH inside the TraTp protein.
This results in one analog. Similarly, the carboxy terminus of a native molecule is a glycinamide residue as a result of post-translational processing. When the amino acid sequence designating the genetic signal for LHRH is located in the fusion such that it is an internal residue, no post-translational processing occurs, and therefore LHRH
The Gly-10 analog results.

(A) naturally occurring variants of LHRH as described in SEQ ID NOs: 3, 4 and 5: (b) Glu-1, His-1, Pro-1 and Lys-6 analogs (SEQ ID NO: 6, 7, 8 and are described in 9) bRIEF dESCRIPTION oF tHE dRAWINGS FIG. 1: A.TraT gene map of plasmid pBTA812 to express, sequence and TraT gene BP _L promoter (SEQ IDN
o: 10) (5 'untranslated region).

Figure 2: TraT sequence designating the genetic code and derived
TraTp peptide sequence (also described in SEQ ID NO: 11), the numbers below each line refer to amino acids. The restriction positions used for insertion of LHRH analogs that specify the genetic code of the sequence are indicated.

FIG. 3: DNA and amino acid sequence of the TraTp-LHRH fusion protein (SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19 and 2)
0). For each construct, only the LHRH analog and the adjacent TraTp sequence are shown. The number is Tra
Refers to amino acids in Tp.

Figure 4: (a) DNA designating the genetic code for LHRH analogs
Fragment sequence (also described in Sequence ID Nos. 21 and 22) (b) Sequence of linker DNA (also described in Sequence ID Nos. 23 and 24) (c) LHRH analog DNA used for construction of pBTA870 (Also described in SEQ ID NOs: 25 and 26) (d) LHRH analog dimer DNA used for construction of pBTA862
FIG. 5: Sequence of the multimeric insertion of the LHRH analog into TraTp (also described in SEQ ID Nos. 29, 30 and 31) Is
As in FIG. 2, amino acids are shown in TraTp.

FIG. 6: Proliferative response of T-cells from dogs immunized with 732p in various formulations. Data were expressed as mean ± sd. The stimulation index was calculated by dividing cpm in the presence of antigen by cpm in the absence of antigen.

FIG. 7: Mean (± sem) LHRH serum antibody response from dogs immunized on days 0, 28 and 56 with 732p in various formulations. Blood samples (5-8 ml) were collected from the head or neck vein on days 0, 28, 42, 56 and 70 and the serum was subjected to the LHRH tracer binding assay (described in Example 3B).
^The ability to bind ¹²⁵ I-LHRH (at a final 1: 2000 dilution)
It was measured.

FIG. 8: TraTp-LHRH fusion protein, LHRH analogs arranged in tandem on days 0, 28 and 56 by 732p (SEQ ID NO: 30
862p with four inserts
Mean (± sem) LH from dogs immunized on day 121
RH serum antibody response. Dogs each received 500 μg of 862p in 0.05% saponin and 0.1% SDS. Blood was drawn from these dogs on days 121 (before booster # 3), 134, 141, 155, 162, 164 and 167, and the sera were analyzed for the ability to bind ¹²⁵ I-LHRH by the LHRH tracer binding assay (described in Example 3B). .

FIG. 9: TraTp-LHRH analog fusion protein 733p, 870p, 86
The average (± sem) LHRH serum antibody response was measured on days 28 and 42 from (a) dogs and (b) mice immunized on days 0 and 28 with 2p, 859p. The sera (at a final dilution of 1: 2000) were analyzed for their ability to bind ¹²⁵ I-LHRH in an LHRH tracer binding assay (described in Example 3B).

FIG. 10: Serum testosterone concentration (ng / ml; described in Example 3C) and LHRH serum antibody binding in dog # 060/10. Blood was collected on days 0, 28, 42, 56, 70 and 84 (immunized with 732p; 1000
μg) and 121 (boosting at 862p; 500 μg), 13
Collected on days 4,141,148,155,162,169 and 176. Days are for the first immunization with 732p (at day 0). About serum (at a final dilution of 1: 2000)
¹²⁵ I- in the LHRH tracer binding assay (described in Example 3B)
The ability to bind and take LHRH was analyzed.

FIG. 11: Various TraTp-L in saponin / SDS adjuvant
Proliferative response of T-cells from dogs immunized with the HRH analog fusion protein. Data are shown as means ± sd. The stimulation index was calculated by dividing cpm in the presence of antigen by cpm in the absence of antigen.

BEST MODE FOR CARRYING OUT THE INVENTION The recombinant DNA molecules and transformed hosts of the present invention were prepared using standard procedures of molecular biology such as digestion and ligation.

The fusion protein of the invention is obtained by culturing the transformed host of the invention under standard conditions as appropriate for that particular host, and separating the fusion protein from the culture by standard techniques. The fusion protein may be used in an impure form or may be purified by standard extraction techniques as appropriate for the fusion protein to be produced.

The vaccine of the invention comprises mixing, preferably homogeneously, the fusion protein with carriers, diluents, excipients and / or adjuvants acceptable for human or veterinary use using standard methods of drug preparation. Prepared by

The amount of fusion protein required to produce a single dosage form will vary depending upon the conditions to be induced, the host to be treated and the particular mode of administration. The specific dosage for a particular fixation depends on the activity of the fusion protein used,
It will depend on a number of factors including the age, weight, general health, sex and diet of the individual, the time of administration, the route of administration, the rate of elimination and the combination of drugs.

The vaccines may be administered orally, parenterally, rectally or vaginally in unit dose formulations containing conventional non-toxic carriers, diluents, excipients and / or adjuvants, as appropriate for human or veterinary use. Can be administered.

The invention will be further described with reference to the following examples, which do not limit the scope of the invention in any way.

Example 1 Plasmids expressing various TraT-LHRH analog fusion proteins TraTp-LHRH analog fusion proteins are produced in E. coli . The gene designating the genetic code for TraTp is carried in a multiple replication plasmid vector that has been modified by inserting one or more copies of the DNA designating the genetic code into an LHRH analog. When the LHRH gene is at the 5 'end of the fusion, PyroGlu containing the fusion protein results, but when the LHRH gene is inside the TraT sequence, the TraTp-LHRH fusion containing Glu-1 as the first amino acid of the LHRH sequence. Protein is produced. Similarly, when LHRH is at the 3 'end of the fusion, glycinamide containing the fusion protein results, but when the LHRH gene is inside the TraT sequence, Gly-10 is replaced with LHRH.
A TraTp-LHRH analog fusion protein is created that contains as the last amino acid of the sequence.

The basic TraT expression vector pBTA812 is shown in FIG. 1A. This is based on plasmid pBR322 [Bolivar F. et al. (1977) Ge
ne, 2, 95-113] .
possesses an ampicillin-resistant gene that allows for the selection of E. coli (alternative selectable genes could be incorporated that would specify the genetic code for other antibiotic resistances). this is,
Further, the left promoter of lambda which promotes the transcription of TraT gene (P _L) [Ogata RT etc. (1982) J.Bacteriol., 15
1, 819-827]. Plasmid BTA812 is PCT / AU
87/00107 (published as WO87 / 06590),
It is similar to pBTA439 deposited as ATCC 67331 in the American Type Culture Collection. pBTA812 can be made as follows. p _L -lambda (Ph
armacia LKB, Uppsala, Sweden), according to the manufacturer's instructions.
Digest with restriction endonucleases SmaI and EcoRI
The linear vector was religated in the presence of polymerase I (Klenow fragment) and deoxynucleotide triphosphate. (Methodology, T. Maniatis, EFFritsch & j. Samb
rook, "Molecular Cloning: Laboratory Handbook", Cold Spr
ing Harbar Laboratory Press, 1982,). After each coupling step, an appropriate E. coli host strain (e.g., C600ramuda carrying the repressor of P _L promoter) transforming the product into. The new plasmid thus lacks one of the EcoRI, SmaI and BamHI positions. This plasmid was then cut with HpaI, treated with exonuclease such as Bal31 (Promega), and digested with the majority of 5 'untranslated N gene DNA.
After removing the DNA that specifies the genetic code from the gene, the DNA is subjected to phenol extraction and ethanol precipitation. This was then cut with XmnI and about 670 base pair DNA was separated by electrophoresis on agarose with low gelling temperature. Sma to pBTA439
Cleavage with I and SacI, then religation in the presence of the Klenow fragment and deoxynucleotide triphosphates (which removes these positions), followed by cleavage and recombination with BamHI and BglII (BamHI, SaII, PstI and BglII) To remove the position). The resulting plasmid was cut with EcoRI and XmnI and the 3054 base pair fragment was separated by electrophoresis on agarose to give the 670 base pair fragment described above in the presence of the Klenow fragment and deoxynucleotide triphosphate. Joined. Selection by growth on ampicillin ensures correct orientation of the fragments by reassembly of the β-lactamase gene. G in the first strand of the approximately 670 base pair fragment is
Next to AATTC. The recombinant plasmid is selected so that EcoRI is present, and the DNA of the positive clone is sequenced in the region at the position of EcoRI. 5 'untranslated m of the P _L promoter and pBTA812
Its sequence corresponding to the RNA is shown in FIG. 1B and in SEQ ID NO: 10.

Expression of the P _L promoter and TraTp was determined by N4830 [M. Joyce &
NDF Grindley (1983) Proc. Natl. Acad. Sci. USA, 80 , 183
0-1834], and is controlled by the temperature-sensitive repressor CI857 present in E. coli strains that have not been modified as described above.

Fig. 2 shows the entire sequence of the genetic code of TraTp (Ogata, supra)
And SEQ ID NO: 11. It is cleaved in E. coli between amino acids 20 and 21 to provide an N-terminal cysteine carrying a fatty acid modification [Perumal, NB & Minkley EG
(1984) J. Biol. Chem., 259 , 5359-5360].

Restriction positions within the TraT gene used as insertion sites for LHRH analog DNA are indicated.

An example of eight plasmid constructs expressing a TraTp-LHRH analog fusion is shown in FIG. 3 and the sequence INDo: 12, 13, 14, 15, 1
6, 17, 18, 19 and 20 are shown. Unique insertion positions were distributed throughout the TraTp molecule.

Plasmid construction: pBTA812 was prepared by extracting from an appropriate E. coli K12 host strain (eg, C600λ) and purifying on a cesium chloride density gradient.

pBTA731 is prepared according to the manufacturer's instructions, pBTA812
Is cut with the restriction endonuclease HpaI, and the linear DNA is
For example, it was constructed by purification on agarose having a low gelling temperature (Maniatis, supra).

The DNA that specifies the genetic code for the LHRH analogs shown in FIG. 4 (a) and in the sequence INDo: 21 and 22 was obtained from Beaucage SL & Caru
thers (1981) Tetrahedron Lett., 22, 1859-1862.
It was bound to TA812 and transformed into E. coli K12 of an appropriate strain. Plasmids containing cells were selected by plating on media containing ampicillin. Colonies of the plasmid with the LHRH analog insert were labeled with [ ³² P] -labeled LHRH D
Either by colony hybridization using NA as a probe (Maniatis, supra) or by picking a large number of colonies and extracting the plasmid, the SHR unique to LHRH DNA
It was identified by determining the presence of the maI restriction site. L
The correct sequence and orientation of the TraT DNA flanking the HRH analog DNA was confirmed by dideoxynucleotide sequencing.

pBTA730, 733 and 734 replace pBTA812 with limited amounts of the restriction endonucleases SspI, ScaI and RsaI, respectively, so that all enzyme positions in the plasmid are not cleaved.
By digestion. Only the plasmid that had been cut once with each enzyme was removed from the low gelling temperature agarose following electrophoresis. Fig. 4 (a) and sequence IDN
o: DNA that specifies the genetic code for the LHRH analogs shown in 21 and 22
Was ligated to this DNA, and E. coli carrying the appropriate new recombinant plasmid was identified as described above. Restriction mapping and DNA sequencing were used to show that the correct restriction positions contained the LHRH analog DNA in the correct orientation.

pBTA732, 735, 737 and 740: these required the construction of an intermediate containing a short linker fragment of DNA inserted at the chosen position. Linker (FIG. 4 (b) and sequence
ID Nos. 23 and 24) are inserted between the codons so that the DNA that specifies the genetic code for the LHRH analog is inserted into the frame for expressing the full-length fusion protein of the TraTp-LHRH analog. Provides a unique new SmaI location located.

pBTA812 was either completely cut with the restriction endonucleases EcoRV, StuI or BalI, or partially cut with linear DNA once cut with Hae III and each of these enzymes separated by agarose gel electrophoresis. Linker D
NA (FIG. 4 (b) and described in sequence ID Nos. 23 and 24)
Was ligated to each of these DNAs and inserted into E. coli . Recombinations were selected by colony hybridization (using radiolabeled linkers as probes or by restriction analysis of DNA from a large number of colonies). The linker was designed such that the position of SmaI was between the codons, depending on the orientation of the linker. The orientation of the linker may be determined by sequencing that region of DNA or by its presence when a new position is created, or by inserting the LHRH analog gene at the position of SmaI, and for the recombinant the TraTp-LHRH analog fusion. Determined by assaying the protein.

DNA designating the genetic code for the LHRH analog (described in FIG. 4 and in SEQ ID NOs: 21 and 22) was inserted into the construct containing the same linker as described above for pBTA730 at the SmaI position. In determining the nucleotide sequence of the final construct, pBTA732 was found to have lost three bases at the 3 'junction of the LHRH analog / linker / TraT DNA.

pBTA609 is a TraTp-LHRH analog fusion with the LHRH analog inserted between amino acids 31 and 32. In vitro mutagenesis converted the codon to the Pvu II position (the new codons represent Gln and Leu), and the DNA that specifies the genetic code for the LHRH analog was inserted at this position. This position was chosen because the protein sequence is particularly hydrophilic in this region. The inserted peptide at this position will be exposed on the surface of the TraTp molecule and will therefore be more antigenic.

pBTA736 (host vector combination BTA1669) contains an LHRH analog inserted at two different positions in TraT,
It is a composite of 731 and pBTA732.

The plasmid was also constructed to contain multiple insertions of the LHRH analog at one position. The construct with the two LHRH analog molecules (pBTA870 and as described in sequence INDo: 29) is shown in FIG. 4 (c) and the LOH shown in SEQ ID NO: 25 and 26.
LHRH in pBTA733, which specifies the genetic code for the HRH analog
After insertion at the SmaI site of the analog and transformation, colonies with two LHRH analog inserts were fixed as described above. Although the original Sma position did not rearrange, the new LHRH analog insert carried the SmaI position at an equivalent position in the second LHRH analog gene (FIG. 5: Sequence ID
No. 29, pBTA870).

A further construct with four LHRH analog repeats (pBTA
862 and described in SEQ ID NO: 30) provide the DNA encoding the dimer of the LHRH analog (as described in FIG. 4 (d) and SEQ ID NOs: 27 and 28) with the SmaI of pBTA870.
It was made by inserting in the position of. Again, the original Sm
The aI position has been lost and a new position has been created near the end of the LHRH analog DNA. Constructs with 6 and 8 repeats of the LHRH analog were made by sequentially adding LHRH analog dimeric DNA to pBTA862. The DNA sequences of some LHRH analog genes were altered (using codon degeneracy) to avoid plasmid instability that could occur when using DNA multiple identical tandem repeats.

FIG. 5 shows plasmids pBTA870, pBTA862, pBTA859 carrying 2, 4 and 8 LHRH analog inserts between amino acids 200 and 201 of TraTp, respectively (SEQ ID NO: 29, 30 and 31). As described respectively).
Using pBTA732 and pBTA740 as starting plasmids, LHRH
PBTA870, pBTA862 and pBTA8 containing multiplexes of analog DNA
It can be seen that a plasmid similar to 59 can be constructed.

All the above plasmids were transferred to Pharmacia LKB, Uppsal
a, including the temperature-sensitive repressor CI857 [M. Joyce, NDFGrindley, supra] available from Sweden .
coli strain. Other strains carrying the CI857 repressor could have been used in place of N4830. TraT
-Expression of the LHRH analog gene supports the plasmid .
It is induced by increasing the temperature of the E. coli culture from 28 ° C to between 37 ° C and 42 ° C. Both components are of the expected size
TraTp-LHRH analog protein was produced. The output of each varied depending on the location of the LHRH analog insertion, with most yielding higher levels than TraTp alone. Following cell breakage, the fusion protein was extracted and, as in Example 2,
Purified for injection into animals.

Example 2 Purification of TraTp-LHRH analog fusion protein In the first selection experiment described in Example 4, a simple fractionation procedure was used to separate the fusion protein from the E. coli protein bal. E. coli strains containing the gene fusion plasmid for the TraTp-LHRH analog were grown in shake flasks for 30 minutes.
C. and induced at 41.degree. C. for 3 hr. Centrifugation (17,00
(0 g, 20 min), and the cells were dissolved in 0.1 M Tris-HCl pH 7.5, 10 mM EDTA using a French press. The lysed cells were then overlaid on 25% glycerol and centrifuged at 10,000 xg for 15 min to separate from the inclusion bodies. The inclusion bodies (pellets) were sonicated by 0.1 M Tris-HCl pH7 containing 5% TRITON-X-100.
5. Suspended in 50 mM EDTA. The sonicated material is 12,
Centrifugation was performed at 000 × g for 20 minutes to obtain an insoluble substance (IF). The pellet was prepared by dissolving the pellet in 0.1 M Tris-HCl pH 7.5, 10 mM
Obtained by resuspension in EDTA, 2% SDS. This material was then precipitated by adding ethanol to 50% and resuspended in saline (SF) prior to injection. Insolubles (IF) were also suspended in saline before injection.

In a later test, which required a higher amount of a more pure immunogen, E. coli strains containing the gene fusion plasmid for the TraTp-LHRH analog were grown at 30 ° C. in shake flasks, 41
Induced for 3 hours at ° C. Centrifuge bacteria (17,000g, 20min)
Bacteria were dissolved in 0.1 M Tris-HCl pH 7.5, 50 mM EDTA using an APG Gaulin 15 MR homogenizer (7 passes at 9,000 psi). Centrifugation (20mi
n, 10,000 xg), the insoluble pellet fraction containing the fusion protein was washed once with lysis buffer, and the protein was then washed with 10% SDS, 0.1 M Tris-MCl pH 7.5, 25 mM EDTA.
Was solubilized. Centrifuge this substance (20min x 15,000g)
The supernatant was washed with 2% SDS, 50 mM Tris-HCl pH7.5, 2
It was applied to a Sephacryl S-200 HR column equilibrated with 5 mM EDTA. The column is eluted with this buffer and the fraction containing the fusion protein (analyzed by SDS-PAGE) is precipitated with 50% ethanol. The pellet was extracted with 1% Zwittergent 3-12, 0.1 M Tris-HCl pH 7.5, 25 mM EDTA, followed by 2% SD
Re-solubilized in S. This material was applied to a hydroxyapatite column equilibrated with 50 mM sodium phosphate buffer pH 6.5, 0.5% SDS and eluted with a 0.05-0.5 M sodium phosphate gradient in 0.5% SDS pH 6.5. Fractions containing the fusion protein were pooled and analyzed for purity on SDS-PAGE. Protein concentration was measured by _A280 and amino acid analysis and showed that the lipopolysaccharide (LPS) content was less than 1% (w / w). The final product was precipitated with 50% ethanol and resolubilized in 0.1% SDS before compounding.

Example 3 Method for Measuring Antigen and Testosterone Production in Serum A. ELISA “Immulon 2” Micro Elisa Plate (Dynatech)
2μ of ovalbumin-LHRH (prepared by EDAC conjugation) in 0.1M carbonate / bicarbonate pH 9.6 (100μ per well)
Incubate overnight at 4 ° C. with g / ml solution or 0.25 μg / ml TraTp. After each step, the plate was washed 5 times with a phosphate buffered saline solution containing 0.05% Tween. Plates were prepared with a 1% gelatin solution in 0.1 M carbonate / bicarbonate (Davis Gelatine Com.
pany Aust. Pty. Ltd.)
Blocked for 1 hr at ° C. Plates were washed as described above. Dilute serum 1: 200 in PBS / T and add 100 μl
Diluted 2-fold in PBS / T. Serum was incubated for 1 hr at 37 ° C.

After washing in PBS / T, the conjugate conjugated to peroxidase was added to the plate at a ratio of 1/2000 in PBS / T (100μ per well) and incubated at 37 ° C for 40-45 min. The joint is G
oat anti-rat IgG, (KPL) Rabbit anti-mouse IgG,
(KPL), Rabbit anti-dog IgG, (Nordic), Goat anti
-Bovine IgG, (KPL) and Rabbit anti-sheep IgG,
(Dako).

After washing, 100 μl of peroxidase substrate was added to each well. The substrate is 0.1M citrate phosphate buffer pH
It has become a 0.5mg / ml2,2'- azinobis in 4.0 (3-ethyl-benzine thiazoline sulfonic acid (ABTS), which was added 0.1% H ₂ O ₂ immediately prior to addition to the plate. If stated otherwise All chemicals and reagents used are Sigma Ch
Analytical Great from emical Company.

B. LHRH Tracer Binding Assay Serum was assayed at 0.01 M with 0.5% w / v bovine serum albumin.
Phosphate buffered saline (PBS / BSA: hereinafter referred to as buffer)
Medium, diluted 1: 500. 100 μl of this solution was added to 200
3 ml polypropylene radioimmunoassay tube containing μ (John
s) in addition to this, the final dilution of the antiserum was 1: 2000
As a result, 10,000 cpm (approximately) of I ¹²⁵ LHRH (Amersha
m / Dupont) was added.

The tubes were incubated overnight at room temperature (14-20 ° C). Second antibody (Sheepauti-mouse IgG or Sheep auti-dog IgG; Si
lenus) was diluted 1:20 with buffer, 100 μl was added per tube, and then incubated for 1 hr at room temperature.

In each tube, polyethylene glycol (1 ml) molecular weight 6000
77500 (PEG, BDH) was added (excluding all counts), and the tube was vortexed, followed by centrifugation at 2,500 rpm for 30 minutes. The supernatant was decanted and the tube drained.

Multi-channel gamma counter (Clinigamma counter,
The pellet was counted for 1 min in LKB). The results were expressed as a percentage (%) of the total radioactivity count "minus non-specific binding (NSB)" added, giving the% of LHRH antibody in serum.

Unless otherwise stated, all chemicals and reagents used were Si
Analytical grade from gma Chemical Company.

C. Measurement of testosterone in dog serum “Direct Testosterone commerical kit” (SPECTRIA,
Testosterone was measured using Farmos Diagnostica (Finland), and the supplied tubes were pre-coated with a second antibody. After adding dog serum to the two pre-coated tubes, ¹²⁵ I-testosterone (200μ),
Testosterone antiserum (200 ml, raised in rabbits) was added and incubated at 37 ° C. for exactly 2 hours. Castrated dog serum was added to the standard and QC curves to compensate for the effect of the serum in the radioimmunoassay. Decant the supernatant without centrifugation, dab with absorbent paper, wash with 1 ml of washing solution (phosphate buffer, supplied) and drain water, then multi-channel gamma counter (Clinigamma counter, LKB)
Counted in for 1 min. Data was expressed as ng / ml testosterone.

Example 4 Immunization of Mice with TraTp-LHRH Analog Fusion Protein To determine which TraTp-LHRH analog fusion protein is most active in eliciting LHRH antibodies, several groups of female Swiss mice were (N = 5: 18 ~
22g) was immunized with 9 different TraTp-LHRH analog fusion protein constructs without adjuvant (730p-737p and 740p) and compared insoluble (IF) and soluble (SF) of the protein . Control group (N = 5)
Was immunized with TraTp prepared in the same way.

For mice, 150 μg protein in 100 μ saline
Was injected into each thigh muscle on days 0 and 28.

Blood samples were collected from the retro-orbital network on days 0, 28 and 42. Aliquots of serum from individual mice were pooled and analyzed for LHRH antibody titers by ELISA and LHRH tracer binding assay. The data of the blood collection on day 42 are shown in Table 2.

The data show that only some of the fusion protein constructs, especially proteins 732p, 733 and 740p, were advantageous for growing LHRH antibodies when administered in saline. The location of the LHRH analog insert producing a valid antigen could not be predicted by examination of the constructed construct. When assayed by measuring% binding to LHRH (and ELISA) and concomitant effects on fertility, effective IFN was obtained with both IF and SF substances from various components (second table). The fusion proteins used in these experiments yield a single, well-defined chemical entity; therefore, they have a greater stability, productivity, quality control and quality assurance compared to LHRH chemical conjugation. The advantage is added.

Example 5 T-cell Proliferation and Antibody Responses of Dogs Immunized with TraTp-LHRH Analog Fusion Protein (732p) in Various Formulations This experiment demonstrates the target species, such as dogs, where the fusion protein 732p is an antibody against LHRH A plan was designed to determine not only the response but also whether T-cells could be elicited. A T-cell response may represent a desirable effector mechanism in the immune castration process itself and / or may help the T-cell in producing LHRH antibodies.

Immunization of dogs with fusion protein and T-cell proliferation and antibody response Fifteen dogs of mixed age, gender and breeding were randomly divided into three groups of five. In one group,
Give 1 mg of pBTA732 fusion protein in alhydrogel,
Another group received 1 mg of the fusion protein in Mantanide ISA-20 and a third group was injected with 1 mg of the fusion protein construct in saponin. All three formulations contained 0.1% SDS. Animals have 0,
On days 28 and 56, the muscle was injected.

AT-Cell Proliferation T-cell responses following immunization with a fusion protein derived from pBTA732 were measured as follows. Briefly, blood samples (5-10 ml) were collected from the head or neck vessels (before day 42) and the T-cell enriched cell fraction was prepared as follows. About 10 heparinized blood
ml on a Ficoll-Pague (Pharmacia) 6 ml, 400 ml
T-cells were separated by g, 30-40 min gradient centrifugation. The yield of T-cells recovered from 10 ml of heparinized blood was 15-20 × 10 ⁶ . T-cells (RPMI1640 medium containing 10% fetal calf serum (Flow Laboratories Inc., M
clean, Va, USA) 10 ⁵ in 0.2 ml) is a flat-bottom culture plates, various amounts of TraTp, by LHRH or PHA, and cultured for 3-5 days at 37 ° C.. 16-18 hr prior to storage, cells were labeled with 0.5 μCi of tritiated thymidine, stored, and counted in a liquid scintillation counter. The results were expressed as stimulation indices, which correspond to c. In the presence of antigen.
It is calculated by dividing pm by cpm when no antigen is present.

According to the data in FIG. 6, all three groups elicit strong T-cell responses to both TraTp and LHRH. Protein 732p blended in saponin, especially against LHRH, Montanide ISA-2
It appeared to be more effective at stimulating T-cells than in zero or alhydrogel. The resulting strong T-cell responses correlated reasonably well with the antibody response to LHRH (determined by binding assay).

B. Antibody response 1. LH generated after immunization with fusion protein 732p
To determine the level of RH, blood samples (5-8 ml) were taken from head and neck vessels on days 0, 28, 42, 56 and 70 and subjected to the LHRH tracer binding assay (described in Example 3B). Was)
Was analyzed for the ability to bind ¹²⁵ I-LHRH in serum (dilution 1: 200 final).

According to the data in FIG. 7, the antibody against LHRH is SDS /
Anti-LHRH responses were elicited much lower in animals given the fusion protein in aldehyde gel or Montanide ISA-20, while elicited in dogs immunized with the 732p fusion protein in saponin formulation. Therefore, to elicit antibodies against LHRH, use Alhydrogel or Montanide ISA-2.
Saponin appears to be a more effective adjuvant for this fusion protein than 0.

2. To assay the efficacy of the multiplex LHRH analog construct (862p) to stimulate LHRH antibodies, 5 dogs, 4
One is the “saponin group” and one is Montanide ISA-20
The group was further tested as follows. On day 121 (for the first immunization), all five dogs had TraTp- containing four inserts of the LHRH analog [as described in SEQ ID NO: 30] arranged in tandem. A further booster injection of the LHRH analog fusion protein (862p) (Booster # 3 in FIG. 8) was given. That is, each dog received 500 μg of this fusion protein construct in 0.05% saponin and 0.1% SDS. From these dogs, 121 (before booster # 3), 134, 141, 148, 155, 162, 164 and 167
Blood was drawn on the day. According to the data in FIG. 8, high levels of LHRH antibodies were elicited in these five dogs in response to a TraTp fusion protein construct containing four inserts of the LHRH analog. The LHRH antibody response to 732p (booster 1 and booster 2 in FIG. 8) is also shown for comparison.

These results indicate that immunization of dogs with fusion proteins containing multiple mimics of LHRH analogs in saponins and SDS can elicit strong LHRH antibody responses.
As a result of this response, total testosterone was completely reduced, with concomitant castration effects exhibited by testicular and prostate weight loss. That is, at comparable body weights in the range of 12.0-17.0 kg, the testes weighed 3.2 g versus 9.6 in the control dog and the prostate grew 1.5 g against 9.2 in the control dog (Figure 10). These data would demonstrate that a mixture of fusion proteins, eg, 862p and 732p or a derivative of 732p, were more potent than each administered alone.

The immunogenicity of the TraTp-LHRH analog fusion protein formulated in saponin was determined by alhydrosol or Montanide IS.
All subsequent studies involving the fusion protein construct as superior to that in A-20 were performed using the saponin / SDS formulation.

Example 6 T-Cell Proliferative Response of Dogs Immunized with TraTp-LHRH Analog Fusion Protein Containing LHRH Analog Multiple Inserts In an attempt to increase the immunogenicity of the LHRH analog fusion protein, we published A construct was prepared that was conditioned on a TraTp-LHRH analog fusion protein containing 1-8 LHRH analog epitopes located in. After purification, the fusion protein immunogen was tested in outbred mice and dogs.

According to the results of FIG. 9 (a), in dogs, a fusion protein containing multiple inserts of the LHRH analog generated a higher anti-LHRH response than the construct containing one insert (
Measured by binding of ¹²⁵ I-LHRH, serum dilution 1: 2000 final). In fact, there was an increase in LHRH binding corresponding to an increase in the number of LHRH analog inserts per TraTp molecule. For T-cell proliferation, there was an increase in response to LHRH (in terms of stimulation index) in vitro, corresponding to the number of LHRH analog units in the carrier molecule (FIG. 11). Thus, the T-cell data confirms the trend seen in the antibody response to LHRH (FIG. 11). TraTp-LHRH analog fusion 733p, 8
70p, 862p and 859p refer to fusion proteins containing one, two, four and eight LHRH analog inserts, respectively, at the same position in TraTp.

Two positions of outbred dogs (n = 5 per group) were immunized intramuscularly (0.5 ml per position) on days 0 and 28, while outbred mice (ARC Switzerland,
Intramuscular immunization was also performed at two locations at n = 10 per group, but the dose was 1/10 of the dose administered to dogs (per location).
0.05 ml). The fusion proteins used are as follows.

Group: 733 (750 μ in 0.075% saponin and 0.1% SDS
g), Group 2: 870p (0.079% saponin and 7% in 0.1% SDS)
90μg), group 3: 862p (0.086% saponin and 0.1% SD
860 μg in S), group 4: 859p (0.1% saponin and 0.1%
1 mg in SDS). Mice are 1/10 this dose. In dogs, blood samples with and without heparin (5-10
ml) were collected from cervical vessels on days 28 and 42, T-cell proliferation on day 42 (measured as described in the previous section) and LHRH antibody response on days 28 and 42 (performed (Same as described in Example 4). In mice, only LHRH antibody response was measured in serum (0.4 m thick blood via retro-orbital reticular route).
l) Measured in

According to the results shown in FIG. 9 (a), LHRH was obtained on day 42 of the dog.
Fusion proteins containing multiple inserts of the analog were significantly more immunogenic (evoke a higher anti-LHRH response) than constructs containing one insert. In fact, there was a gradual increase in LHRH binding, corresponding to the number of LHRH analog inserts per molecule of TraTp. In contrast, with a mouse, the 870p
Peak binding was seen in the sera of animals receiving the TraTp-LHRH analog fusion protein, while the 862p and 859p proteins elicited somewhat lower levels of the LHRH binding response (FIG. 9 (b)). In mice, at day 28 (prior to the first booster), the level of LHRH binding was low in both species, although there was a suggestion that the binding response decreased with increasing numbers of LHRH analog inserts in the TraTp molecule. On the other hand, in dogs, a slight increase in LHRH antibody levels corresponded to an increase in LHRH analog units in the fusion protein construct. These observations indicate that the introduction of multiple repeats of the peptide into the TraTp molecule significantly enhances the immunogenicity (ability to elicit a higher anti-peptide response) of the inserted peptide. Furthermore, the most effective immunogen for a particular species could not be established on a logical basis. Nevertheless, the principles and procedures now established by the present invention provide a means to apply this technology to other species and fusion proteins of commercial interest.

INDUSTRIAL APPLICABILITY The fusion protein of the present invention can be administered in a carrier such as saline or saponin to immunize a vertebrate host against endogenous LHRH so as to inhibit the reproductive function of the host. It is used to provide a self-adjuvant immunogen that can be administered to a host.

Despite the unique uses exemplified herein, LH
The approach used here for the RH analog fusion is TraT
Suggests a means of providing a fusion protein consisting of p and another immunogenic epitope, ie, an epitope that encompasses a fragment of the protein with a peptide from natural or synthetic sources. The protein is a hormone or growth factor such as LHRH, LH, FSH, chorionic gonadotropin (CG), corticotropin (ACTH), somatotropin, somatostatin, insulin-like growth factor, inhibin, activin, follistatin and its variants. They may be factors, and they may be proteins of biological interest, such as semen antigens such as zona pellucida antigens or oocyte antigens. Alternatively, antigens may be derived from proteins of protozoa, nematodes, tapeworms, insects and parasites, including mites, and may be bacteria or viruses, especially cholera, AIDS, rabies, tetanus. Antigens from those that can protect against mammalian diseases, including smallpox, palsy, diphtheria, and others of commercial importance. According to the present invention, it has been found that fusion of TraTp and LHRH analog sequences can be used to provide a vaccine for immunizing against LHRH. Believe that, based on the teachings contained herein, it may be possible to extrapolate collectively to the further immunogenic epitopes described above.

Deposits of strains The strains of E. coli were established on August 21, 1990 under the Budapest Agreement under the following accession numbers, New
Commonweal at South Wales 2073, Pymble, Suakin Street1
th Department of Administrative Services, New South
Australian Governm at Wales Regional Laboratory
ent Analytical Laboratories.

BTA 1349 for possession of a bacterial species number enroll number BTA 1665 N90 / 031366 BTA 1666 N90 / 031367 BTA 1907 N90 / 031368 pBTA439 , in accordance with the provisions of the Budapest Agreement, on March 2, 1987, the subscriber number of ATCC 67331 US MD 20852, Rockville, Parklawn Drive 1230
1 American Type Culture Collection.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI // C12P 21/00 A61K 37/43 AEX (C12N 1/21 C12R 1:19) (C12P 21/00 C12R 1:19) ( 72) Inventor Stewart, Andrew George Australia, New South Wales, 2073, Pimble, 26 Bobbin Head Road (72) Inventor Tonis, Con George Australia, New South Wales, 2114, Denniston, 8 Viewmont Avenue ( 56) References Tokuyo Sho 64-500117 (JP, A) Tokuyo Heihei 1-500900 (JP, A)

Claims (24)

(57) [Claims] 1. The method according to claim 1, comprising TraT and at least one luteinizing hormone-releasing hormone analogue, wherein at least one luteinizing hormone-releasing hormone analogue comprises 3
Inserted into at least one of the
between amino acid residues 80 and 81, and residues 200 and 201 in the sequence of aT
Between 235 and 236 and elicits the production of antibodies to luteinizing hormone-releasing hormone when administered to a vertebrate host. 2. Amino acid sequence: Glu His Trp Ser Tyr
The fusion protein of claim 1, comprising a luteinizing hormone releasing hormone analog having Gly Leu Arg Pro Gly. 3. The amino acid sequence Glu His Trp Ser Tyr
Fusion protein 73 containing Gly Leu Arg Pro Gly and containing a luteinizing hormone-releasing hormone analog inserted between amino acid residues 80 and 81 in the amino acid sequence of TraT
2p. 4. Amino acid sequence Glu His Trp Ser Tyr
A fusion protein comprising Gly Leu Arg Pro Gly and containing a luteinizing hormone releasing hormone analog inserted between amino acid residues 200 and 201 in the amino acid sequence of TraT
733p. 5. The amino acid sequence Glu His Trp Ser Tyr
A fusion protein having Gly Leu Arg Pro Gly and containing a luteinizing hormone releasing hormone analog inserted between amino acid residues 235 and 236 in the amino acid sequence of TraT
740p. 6. An amino acid sequence consisting of eight luteinizing hormone-releasing hormone analogs, wherein amino acid residues 200 and 2 in the TraT amino acid sequence.
Amino acid sequence inserted between 01 Glu His Trp
Ser Tyr Gly Leu Arg Pro Gly Glu His Trp
Ser Tyr Gly Leu Arg Pro Gly Thr His Trp
Ser Tyr Gly Leu Arg Pro Gly Glu His Trp
Ser Tyr Gly Leu Arg Pro Gly Glu His Trp
Ser Tyr Gly Leu Arg Pro Gly Glu His Trp
Ser Tyr Gly Leu Arg Pro Gly Glu His Trp
Ser Tyr Gly Leu Arg Pro Gly Glu His Trp
Fusion protein 859p containing one insert with Ser Tyr Gly Leu Arg Pro Gly. 7. An amino acid sequence consisting of four luteinizing hormone-releasing hormone analogs, wherein the amino acid residue 200
Amino acid sequence Glu His T
rp Ser Tyr Gly Leu Arg Pro Gly Glu His T
rp Ser Tyr Gly Leu Arg Pro Gly Glu His T
rp Ser Tyr Gly Leu Arg Pro Gly Glu His T
Fusion protein 862p containing one insert with rp Ser Tyr Gly Leu Arg Pro Gly. 8. A luteinizing hormone-releasing hormone analog comprising two amino acids at 200 amino acid residues in the amino acid sequence of TraT.
Amino acid sequence Glu His T
rp Ser Tyr Gly Leu Arg Pro Gly Glu His T
Fusion protein 870p containing one insert with rp Ser Tyr Gly Leu Arg Pro Gly. 9. A polynucleotide comprising a sequence of nucleotides encoding the fusion protein of any one of claims 1 to 8. 10. A recombinant DNA molecule comprising at least one polynucleotide of claim 9 and vector DNA. 11. The recombinant DNA molecule of claim 10, wherein the vector DNA comprises phage, virus or plasmid DNA. 12. A plasmid pBTA732 which encodes the fusion protein of claim 3 and is deposited in an E. coli strain having a deposit No. N90 / 031366, which has been deposited with the Analytical Laboratories of the Australian Government. 13. A plasmid pBTA733 which encodes the fusion protein according to claim 4 and is deposited in an E. coli strain having a deposit No. N90 / 031367, which has been deposited with the Analytical Laboratories of the Australian Government. 14. A plasmid pBTA740 which encodes the fusion protein of claim 5 and is deposited in an E. coli strain deposited with the Australian Government Analytical Laboratories and has a deposit No. N90 / 031368. 15. Containing at least one effective dose of a fusion protein according to any one of claims 1 to 8 together with at least one carrier, diluent, excipient or adjuvant for medical or veterinary use. A vaccine for inhibiting or controlling reproductive function in a non-human vertebrate host. 16. The adjuvant is saponin.
Vaccine. 17. A bacterial host to be cultured transformed with at least one recombinant DNA molecule according to any one of claims 10 to 14. 18. The method according to claim 1, wherein the host is an E. coli strain.
7 bacterial hosts. (19) The plasmid containing the plasmid according to (12), which has been deposited at the Australian Government's Analytical Laboratories.
Host cell BTA1665 having No. N90 / 031366. 20. A deposit comprising the plasmid of claim 13 and deposited at the Australian Government's Analytical Laboratories.
Host cell BTA1666 having No. N90 / 031367. 21. A deposit comprising the plasmid according to claim 14 and deposited at the Australian Government's Analytical Laboratories.
Host cell BTA1907 having No. N90 / 031368. 22. A reproductive function in a non-human vertebrate host comprising immunizing the host with at least one fusion protein of any one of claims 1 to 8 or the vaccine of claim 15 or 16. How to manage. 23. A reproductive function in a non-human vertebrate host comprising immunizing the host with at least one fusion protein of any one of claims 1 to 8 or the vaccine of claim 15 or 16. How to prevent. 24. The animal according to claim 22, wherein the host is a domestic animal.
the method of.