Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Hormone or other secreted growth regulatory factor,...
Reexamination Certificate
1994-06-22
2001-11-13
Achutamurthy, Ponnathapura (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Hormone or other secreted growth regulatory factor,...
C424S185100, C435S069400, C530S311000
Reexamination Certificate
active
06316004
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of gene and protein engineering, in particular to the preparation of chimeric proteins, the desired component of which for one or another reason cannot be obtained in a free form by microbial synthesis, and also because of its small size only has the properties of a hapten, i.e., is capable of inducing the formation of antibodies only after being joined to high-molecular carriers. In particular, tetradecapeptide somatostatin-14 having an amino acid sequence corresponding to amino acid positions 233 to 246 in Seq. ID Nos. 1 and 2, and amino acid positions 242 to 255 in Seq. ID No. 3 relates to such a genus of oligopeptides. The use of a chimeric somatostatin-comprising protein in an immunogenic composition to increase the productivity of farm animals is also proposed.
BACKGROUND OF THE INVENTION
Acceleration of the growth of farm animals at a lower cost per 1 kg of weight gain is one of the main problems of stock raising. It is known that the productivity of farm animals can be increased by giving them somatostatin, some anabolic hormones or antibiotics. However, the high cost of somatostatin does not always make this method profitable, and furthermore, the use of hormonal preparations especially anabolic ones in the production of foodstuffs is not received with enthusiasm by the public. For these reasons somatotropic preparations have not yet found wide use in stock raising and anabolic hormones are prohibited in animal husbandry. However, it is possible to increase the concentration of endogenic anabolic factors by acting on their inhibitor—somatostatin, which has good prospects for use in agriculture and in medicine (Muromtsev G. S. et al., 1990, “Basics of agricultural biotechnology”, Agropromizdat, Moscow; Reichlin S., ed., 1987, Somatostatin, Basic and Clinical Status, Plenum Press, New York, Weil C., ed., 1992, Basic and Clinical aspects of neuroscience, v. 4 Somatostatin, Springer-Verlag; Spencer G. S., 1985, Hormonal systems regulating Growth, Review, Livestock Production Science, 12, 31-46).
Somatostatin, a biologically active tetradecapeptide having the following amino acid sequence—AGCKNFFWKTFTSC, is produced in the hypothalamus and the gastrointestinal tract. The sequence of somatostatin-14 is highly conservative among vertebrata, while in mammals in general it does not have a specific species. Somatostatin has a strong inhibiting effect on a large number of hormones and related thereto functions of the organism: somatostatin, the thyrotrophic hormone, insulin, glucogen, secretin, gastrin, pepsin, maletin and a number of regulatory peptides. The wide range of action of somatostatin on the factors necessary for growth and utilization of food provides a good outlook for its use as a means for controlling the growth of animals, for reduction of expenditures on foodstuff, etc. Therefore, the autoimmune reaction to somatostatin, resulting in a reduction of the concentration of this peptide in the blood, and a result induction of anabolic factors and acceleration of the growth of the animals, is one of great interest. Active or passive immunization of animals, as a result of which antisomatostatin antibodies (Reichlin, 1987; Spencer, 1985; Baile C. A. et al.) The neurophysiological control of growth, In: Control and Manipulation of Animal Growth, Buttery P. J. et al., ed., 1986, Butterworths, London, pp. 105-118) appear in the blood, is used to reduce the concentration of endogenic somatostatin.
Somatostatin is a low-molecular protein-hapten, its half-life in the blood stream is several minutes. In view of this somatostatin conjugates with various proteins are used for immunization with somatostatin. It should be underlined that this approach makes it possible to obtain ecologically pure food products, since it does not include the use of any preparations of direct hormonal effect or antibiotics, but is based on small changes in the concentration of endogenous protein anabolic factors, characteristic for elite, highly-productive animals (Reichlin, 1987; Buttery et al., 1986).
A large number of studies have shown that animals immunized with somatostatin have an average daily weight gain of 10-20%, an appetite reduced by 9% and an 11% increase in the efficiency of food utilization. Wherein improved absorption of food components and a slower passage of food through the gastrointestinal tract with sluggish peristalsis is observed. Animals immunized with somatostatin, and also their offspring, have correct proportions, and the distribution of the weight of the animals between the muscles, bones and fat is the same as in the control (Reichlin, 1987). Immunization of gestated goats results in an increase in the weight of newly-born by 10% and an increase in milk yield.
However, the wide use of somatostatin-14, in particular, to stimulate the growth of animals by means of immunocorrection using antisomatostatin antibodies (Muromtsev G. S. et al. and Reichlin, 1987) is not possible because of its high price, since the main way to obtain somatostatin is by chemical synthesis (38 U.S.A. dollars for 1 mg of a somatostatin preparation, Sigma catalog, U.S. A., 1992), and this does not make it possible to realize this approach in practice from an economical point of view. The development of gene engineering methods has made it possible to prepare a number of protein and peptide hormones by synthesis in the cells of microorganisms. However, it is not possible to effect the direct microbial syntheses of somatostatin using recombinant DNA technology because of its small size (14 amino acid residues) (Itakura R. et al., 1977), “Expression in
E. coli
of a chemically synthesized gene of the hormone somatostatin”, Science, 1986, 1056-1063). Several methods for obtaining somatostatin in the form of chimeric proteins with subsequent specific cleavage of the product of interest have been described (Itakura R. et al., 1977; Russian patent application No. 4921158/13 of Mar. 26, 1991).
The first research in respect of obtaining somatostatin-14 by use of gene engineering technology was conducted in 1977 by Itakura. The authors constructed a hybride gene on the basis of &bgr;-galactosidase
E. coli,
in the C′-end region of which a chemically synthesized sequence of somatostatin is engineered. Later, effective producers of chimeric proteins were created on the basis of that process with a sequence of somatostatin introduced into chimeric trpE, trpD and recA
E. coli
genes. The level of expression of chimeric proteins reached 15-30% of the total amount of proteins, while the output of the somatostatin approached the requirements of industrial production (Itakura R. et al.).
A recombinant plasmid DNA encoding somatostatin and a strain
E. coli—
a producer of somatostatin, are taught in Russian patent application No. 4921158/13 with priority from Mar. 26, 1991. The claimed plasmid determines the constitutive synthesis of a hybride protein of chloramphenicol acetyl transferase-somatostatin-14 under the control of its own promoter in cells of
E. coli
MKD3207 with a reduction of the level of degradation of anomalous proteins. However, the gene engineering constructions described above ensure the preparation of chimeric proteins, the immunogenic activity of which with respect to somatostatin is extremely low, and therefore these proteins are not yet being used in agriculture or medicine.
SUMMARY OF THE INVENTION
The present invention relates to chimeric immunogenic proteins including somatostatin-14, coupled with a protein-carrier through a spacer (Sp)
n
ensuring the positioning of the somatostatin on the surface of the carrier. Wherein, the number of monomeric proteins of the spacer (n) is preferably from 1 to 8, while the spacer (Sp) includes a sequence of amino acids consisting of an alkaline amino acid and amino acid ensuring a rigid &bgr;-structure, in particular, Lys-Pro or Arg-Pro. The invention relates to recombinant DNA molecules encoding the aforementioned chimeric proteins, to a process for preparing immunogenic chimer
Bader Leila Bakievna
Karpov Vladimir Abramovich
Khodun Marat-Vladimir Leonidovich
Lunin Vladimir Glebovich
Sergienko Olga Vasileivna
Achutamurthy Ponnathapura
Bui Phuong T.
Tikhonenko T.
Townsend & Townsend & Crew LLP
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