CN1575301A

CN1575301A - Administration of nucleic acid sequence to female animal

Info

Publication number: CN1575301A
Application number: CNA018220088A
Authority: CN
Inventors: R·J·施瓦茨; R·H·卡彭特; R·德勒吉亚－阿克里; D·R·克恩; R·G·史密斯
Original assignee: Baylor College of Medicine; Advisys Inc
Current assignee: Baylor College of Medicine; Advisys Inc
Priority date: 2000-12-12
Filing date: 2001-12-12
Publication date: 2005-02-02
Also published as: EP1364004A4; WO2002061037A3; PL366116A1; BR0116472A; WO2002061037A2; CA2430921C; AR035671A1; KR20040039187A; CA2430921A1; MXPA03005236A; EP1364004A2; AU2002248194B2; WO2002061037B1

Abstract

Growth is improved by utilizing growth enhancement potential methodology to administer a nucleic acid sequence, such as GHRH or an analog, to a female animal, preferably through a parenteral route of administration. Piglets born from sows injected with DNA encoding GHRH are larger, and effects ae demonstrated in subsequent pregnancies without additional administration(s) of the vector.

Description

To the jenny administration of nucleic acid sequence

[0001] the application requires the right of priority in the U.S. Provisional Patent Application 60/255,021 of submission on December 12nd, 2000.

Invention field

[0002] the present invention relates generally to incretology, medical science and cytobiology.More specifically, the present invention relates to the improvement of growing and showing; In the production of animal moderate stimulation tethelin, the higher level that it is more relevant than normal growth; In jenny, use the DNA of coding growth hormone releasing hormone to strengthen growth with utilization.In addition, the present invention relates to strengthen the nucleotide sequence of growing and be applied to muscle tissue,, particularly use the technology of electroporation for example by the growth hormone releasing hormone or the analogue of the adjusting of muscle specific promotor.

Background of invention

[0003] tethelin (GH) production approach comprise a series of its products be the growth necessary complementary gene.The gene of this GH approach comprises: (1) part, as GH and insulin like growth factor-1 (IGF-I); (2) precursor of transcription factor such as pit-1 (prophet) or prop-1 and pit-1; (3) agonist and antagonist are respectively as growth hormone releasing hormone (GHRH) and somatostatin; (4) acceptor is as GHRH acceptor (GHRH-R) and GH acceptor (GH-R).These genes are expressed in Different Organs and tissue, comprise hypothalamus, hypophysis, liver and bone.The expression effective and that regulated of GH approach is very crucial for optimum linear growth and carbohydrate, protein and lipometabolic stable state.GH synthetic and stimulated by GHRH and suppressed by somatostatin from the prepituitary gland secretion, both are hypothalamic hormone.GH central role in control people and other vertebrate body growths, and adjusting GH is well-known from hypophysis excretory physiology relational approach.GH is mainly in liver and also increased the production of IGF-I in other Target organs.Conversely, IGF-I and GH feed back to hypothalamus and hypophysis to suppress the release of GHRH and GH.GH has two kinds of direct and indirect effects to peripheral tissues, and it directly acts on mainly and being mediated by IGF-I.

[0004] clinical condition spectrum is widely all arranged in children and grownup, wherein linear growth (prepuberal patient) or body composition all suffer damage, and these condition responsive GH or GHRH treatment.The GHRH-GH-IGF-I axle all has function in all instances, but owing to various possible reasons are uninevitable with the suitableeest susceptibility and reactivity effect.

[0005] principal character that GH lacks among the children is of short and small stature.Can produce similar phenotype people such as (, 1995) Parks by hereditary defect at the difference of GH axle, and non-GH lack of short and small stature.Non-GH lacks and has different nosetiology, as (1) genetic diseases, and Tener (Turner) syndromes (people such as Jacobs, 1990; People such as Skuse, 1999), dyschondroplasia (people such as Tanaka, 1998; Key and Gross, 1996) and Crow grace disease (people such as Savage, 1999); (2) intrauterine growth retardance (Albanese and Stanhope, 1997; People such as Azcona, 1998); (3) chronic renal insufficiency (people such as Sohmiya, 1998; Benfield and Kohaut, 1997).What wherein the unaffected case of GH axle (patient who promptly has normal hormone, gene and acceptor) accounted for all retarded growth cases surpasses 50%.In these cases, shown that GHRH or GH treatment is effective (Gesundheit and Alexander, 1995).

[0006] reducing the GH secretion from prepituitary gland causes from the loss of 25 years old skeletal muscle mass to the aging course.The GHRH-GH-IGF-I axle in aging course and in the older, experience significant the variation (D ' people such as Costa, 1993) along with the GH productivity that reduces and GH transformation period, IGF-I stimulate the reactivity that reduces to cause the reduction (Bartke, 1998) of loss (sacropenia), osteoporosis and the fat increase and the lean mass of skeletal muscle mass to GH and GHRH.The research of front has been presented at the normal year elder philtrum of squillion, and the level the when level of GH and IGFs is with respect to they teenager in the serum has reduced 70-80% (people such as Corpas, 1993 significantly; People such as Iranmanesh, 1991).The development that sacropenia has been described can be treated by GH and be remedied.Yet because its cost and frequent side effect, this remains controversial treatment in the older.

[0007] production of recombinant protein provides the useful tool for the treatment of these situations.Although GH replaces treatment and is widely used in the patient with growth shortage and satisfied growth is provided, and may have positive psychology effect (Rosenbaum and Saigal, 1996 to the children of treatment; , Erling, 1999), but this treatment has several shortcomings, comprises unpractical GH (people such as Monti, 1997 of frequently using of needs; People such as Heptulla, 1997) and undesirable second-order effect (people such as Blethen, 1996; Watkins, 1996; People such as Shalet, 1997; People such as Allen, 1997).

[0008] determines well, as the exocrine GHRH of head of the molecule of sophisticated peptide or brachymemma (as with islet cell tumor and be positioned to be seen in the carcinoid of different positions) often have biologic activity and even can produce acromegaly (people such as Esch, 1982; People such as Thorner, 1984).The GHRH of reorganization is applied to level that children that GH-lacks or grownup increased IGF-I, has increased the secretion of GH in proportion with respect to the dosage of GHRH, but still caused reaction (Bercu and Walker, 1997) the GHRH heavy dose.Thereby GHRH uses and has represented the another kind of physiology that increases subnormal GH and IGF-I level to select people such as (, 1993) Corpas.

[0009] although the GHRH protein therapeutic causes and stimulated normal circulation GH secretion and in fact have no side effect, the transformation period that GHRH is short in the body needs using of (needing high 300-times dosage) in frequent (every day 1-3 time) intravenously, the subcutaneous or nose.Thereby as chronic treatment, GHRH uses unrealistic.Yet, the exocrine GHRH of head, as the kinds of protein (Tyr1-40 or Tyr1-Leu44) of processing or even as shorter brachymemma molecule, have biologic activity people such as (, 1984) Thorner.Importantly, low-level GHRH (100pg/ml) can stimulate GH secretion people such as (, 1993) Corpas and make GHRH become the fabulous material standed for of gene therapy expression in blood supply.Plasmid DNA transgenosis at present directly is the basis of many emerging strategies in gene therapy, and does not need virogene or lipid granule (people such as Muramatsu, 1998 like this; Aihara and Miyazaki, 1998).Skeletal muscle is the selected objective target tissue, because myofiber has long life-span and can be by cyclic DNA plasmid transduction (people such as Davis, 1993 of expression for years in the immunocompetence host; People such as Tripathy, 1996).The person of good sense GHRH cDNA that reports of front can send into muscle by injection-type myogenic expression vector in mouse, and it stimulates GH secretion in the period in 2 week momently to suitable degree people such as (, 1997) Draghia-Akli there.

[0010] wild-type GHRH all has the relative short transformation period in the recycle system of people people such as (, 1984) Frohman and farm-animals.Incubation is after 60 minutes in blood plasma, 95% GHRH (1-44) NH ₂Be degraded, and under simulated condition incubation during the hormone of short (1-40) OH form, show the degraded of having only 77% peptide people such as (, 1989) Frohman behind 60 minutes the incubation.CDNA in conjunction with coding special proteolytic enzyme-resistance GHRH analogue in gene therapy vector causes having longer serum half-life, increases the molecule of usefulness, and provides bigger GH to discharge (people such as Draghia-Akli, 1999 in the animal of plasmid injection; Quote as a reference) herein.Prolonged the serum half-life of hGHRH molecule via sudden change to the amino acid whose amino acid replacement of proteolytic enzyme susceptibility.In addition, the enhancing of GHRH biologic activity reaches by the analogue that uses superactivity, and this analogue can increase its bonding force to special receptor people such as (, 1999) Draghia-Akli.

[0001] patent of having issued has been stated to increase tethelin and has been released to purpose and uses novel GHRH analogue protein (U.S. Patent No. 5,847,066; 5,846,936; 5,792,747; 5,776,901; 5,696,089; 5,486,505; 5,137,872; 5,084,442; 5,036,045; 5,023,322; 4,839,344; 4,410,512; RE33,699) or synthetic or naturally occurring GHRH peptide fragment (U.S. Patent No. 4,833,166; 4,228,158; 4,228,156; 4,226,857; 4,224,316; 4,223,021; 4,223,020; 4,223,019).Reported the GHRH analogue (U.S. Patent No. 5,846,936) that contains following sudden change: the Tyr of position 1 becomes His; The Ala of position 2 becomes Val, Leu or other; The Asn of position 8 becomes Gln, Ser or Thr; The Gly of position 15 becomes Ala or Leu; The Met of position 27 becomes Nle or Leu; Become Asn with the Ser of position 28.As U.S. Patent application series No.60/145,624 theme and the GHRH analogue of quoting herein as a reference do not contain in U.S. Patent No. 5,846, active necessary all amino acid replacements of report in 936.U.S. Patent application sequence No.60/145,624 invention and U.S. Patent No. 5,756,264 difference aspect 2.At first, U.S. Patent application sequence No.60/145,624 invention relates to a kind of analogue of growth hormone releasing hormone, this analogue is different with wild-type with significant modification, these modifications improved its as the GH secretogogue function: the stability that the susceptibility of proteolytic enzyme is reduced and increases, this will prolong its ability that plays therapeutic action, and increase biologic activity, and the latter will strengthen the ability of therapeutic action.U.S. Patent application series No.60/145,624 analogue lacks and is present in U.S. Patent No. 5,756, position 8 substituting to Gln, Ser or Thr in 264 the GHRH analogue.In addition, at U.S. Patent application sequence No.60/145, an aspect of 624 invention, this invention utilizes the DNA of coding in conjunction with the GHRH analogue on unique synthetic promoter, this promotor is called SPc5-12 (people such as Li, 1999), it contains from the nearside serum response element (SRE) of bone α-Ji Dongdanbai, multiple MEF-2 site, MEF-1 site and TEF-1 binding site, and surpasses the transcriptional capability of natural myogenic promotor widely.The uniqueness of such synthetic promoter is to the remarkable improvement as the myogenic expression system (for example U.S. Patent No. 5,298,422) that relates to myogenic promotor and uses thereof (for example U.S. Patent No. 5,374,544) or nucleotide sequence of the patent issued.

[0012] U.S. Patent No. 5,061, and 690 provide the hGRF of 10-20 days significant quantities or one kind analogue and directly at increasing birth weight and milk production by the female mammal to pregnancy.To the application of analogue through lactation period.Yet, introduction be multiple using, and not about use the disclosure of growth hormone releasing hormone (or factor) with the dna molecular form, as relevant with gene therapy technology.

[0013] U.S. Patent No. 5,134, and 120 and 5,292,721 similarly do not provide about use the instruction of growth hormone releasing hormone with dna form.In addition, these patents relate to multiple administered recombinant protein G H in last 2 week of gestation and postnatal 3 week specially.Equally, do not provide discussion, as providing in the present invention about any non-wild-type.

[0014] uses tethelin (GH) to farm-animals and strengthened lean tissue accumulation and/or milk production (people such as Etherton, 1986 simultaneously having increased feeding efficiency; People such as Klindt, 1998).Many researchs have shown that GH has reduced the carcass fat quantity significantly; And the quality of product has increased subsequently.Yet chronic GH uses has actual its availability and validity (people such as Chung, 1985 of weakening potentially with physiological limitation; Gopinath and Etherton, 1989).According to experiment, GH-releasing hormone (GHRH) is selected as another kind of physiology.For big species such as pig or ox, the upstream stimulator of GH, the use of GHRH is another kind of selectable strategy, this strategy not only can increase growth performance or milk production, and prior, can increase production efficiency from actual and metabolism angle (people such as Dubreuil, 1990; People such as Farmer, 1992).Yet the expensive and essential frequency of administration of recombinant peptide has limited the widespread use of this treatment at present.These main shortcomings can be by eliminating with a kind of gene therapy method of producing at the GHRH dystopy, but as long as its production long term maintenance.The inferior colliculus cerebral tissue of GHRH gene is specific expressed not to be that its activity is necessary, and this is because the exocrine GHRH of head can have biologic activity (people such as Faglia, 1992; Melmed, 1991).A kind of gene therapy method of sending GHRH is supported because of the following fact, i.e. gene, cDNA and natural and molecules several sudden changes existing fine sign in pig, ox and many other species, and to therapeutic efficiency determine simple and clear and definite.The skeletal muscle tissue is the ideal candidate of destination organization, has the long life-span and can be transduceed (people such as Bettan, 2000 by the cyclic DNA plasmid because intramuscularly is easy in industrial plants operation, myofiber; People such as Everett, 2000).Thereby, need not to use once more and transgenosis can be for years be expressed people such as (, 1992) Wolff effectively in the immunocompetence host.

Summary of the invention

[0015] in one embodiment of the invention, a kind of improvement or the enhancing method from the offspring's of jenny growth is arranged, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for wherein nucleotide sequence and the wherein introducing of carrier and improvement or the enhancing that expression causes the offspring to grow.In a specific embodiment, the cell of this jenny comprises diploid cell.In another specific embodiment, the cell of this jenny comprises muscle cell.In the specific embodiment of another one, this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.In further specific embodiment, this growth hormone releasing hormone is SEQ ID NO:1, SEQ ID NO:8 or its analogue separately.In the specific embodiment of another one, this promotor comprises synthetic myogenic promotor.In further specific embodiment, this 3 ' non-translational region comprises hGH 3 ' non-translational region.In another specific embodiment, this carrier by electroporation, through virus vector, combine with carrier or be incorporated into by parenteral route in the described cell of this jenny.In the specific embodiment of another one, this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.In further specific embodiment, this jenny is people, pig, cow, sheep, goat or chicken.In the specific embodiment of another one, this carrier is selected from plasmid, virus vector, liposome and cation lipid.In another specific embodiment, this carrier introduces in applied once that this is female.In the specific embodiment of another one, this takes place when being introduced in the 3rd phase in March of nourishing this offspring.In another specific embodiment, this method further comprises to this female step of using the part of secretagogue receptor.In another specific embodiment, this part dosage forms for oral administration.

[0016] in an extra embodiment of the present invention, the method of a kind of increase from level of growth hormone among the offspring of jenny arranged, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause level of growth hormone increase among the offspring.In a specific embodiment, the cell of this jenny comprises diploid cell.In another specific embodiment, the cell of this jenny comprises muscle cell.In the specific embodiment of another one, this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.In further specific embodiment, this growth hormone releasing hormone is SEQ ID NO:1, SEQID NO:8 or its analogue separately.In the specific embodiment of another one, this promotor comprises synthetic myogenic promotor.In further specific embodiment, this 3 ' non-translational region comprises hGH 3 ' non-translational region.In another specific embodiment, this carrier by electroporation, through virus vector, combine with carrier or be incorporated into by parenteral route in the cell of jenny.In the specific embodiment of another one, this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.In further specific embodiment, this jenny is people, pig, cow, sheep, goat or chicken.In the specific embodiment of another one, this carrier is selected from plasmid, virus vector, liposome and cation lipid.In another specific embodiment, this carrier introduces in applied once that this is female.In the specific embodiment of another one, this introducing is to take place when nourishing the 3rd phase in March of this offspring.In another specific embodiment, this method further comprises to this female step of using the part of secretagogue receptor.In another specific embodiment, this part dosage forms for oral administration.

[0017] in another embodiment of the invention, the method of a kind of increase from the offspring's of jenny lean mass arranged, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause lean mass increase among the offspring.In a specific embodiment, the cell of this jenny comprises diploid cell.In another specific embodiment, the cell of this jenny comprises muscle cell.In the specific embodiment of another one, this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.In further specific embodiment, this growth hormone releasing hormone is SEQ ID NO:1, SEQ ID NO:8 or its analogue separately.In the specific embodiment of another one, this promotor comprises synthetic myogenic promotor.In further specific embodiment, this 3 ' non-translational region comprises hGH 3 ' non-translational region.In another specific embodiment, this carrier by electroporation, through virus vector, combine with carrier or be incorporated into by parenteral route in the cell of jenny.In the specific embodiment of another one, this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.In further specific embodiment, this jenny is people, pig, cow, sheep, goat or chicken.In the specific embodiment of another one, this carrier is selected from plasmid, virus vector, liposome and cation lipid.In another specific embodiment, this carrier introduces in applied once that this is female.In the specific embodiment of another one, this introducing is to take place when nourishing the 3rd phase in March of this offspring.In another specific embodiment, this method further comprises to this female step of using the part of secretagogue receptor.In another specific embodiment, this part dosage forms for oral administration.

[0018] in another embodiment of the invention, the method of a kind of increase from the offspring's of jenny IGF-I level arranged, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause IGF-I level increase among the offspring.In a specific embodiment, the cell of this jenny comprises diploid cell.In another specific embodiment, the cell of this jenny comprises muscle cell.In the specific embodiment of another one, this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.In further specific embodiment, this growth hormone releasing hormone is SEQ ID NO:1, SEQID NO:8 or its analogue separately.In the specific embodiment of another one, this promotor comprises synthetic myogenic promotor.In further specific embodiment, this 3 ' non-translational region comprises hGH 3 ' non-translational region.In another specific embodiment, this carrier is by electroporation, through virus vector, combines with carrier or be incorporated into by parenteral route in the cell of jenny.In the specific embodiment of another one, this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.In further specific embodiment, this jenny is people, pig, cow, sheep, goat or chicken.In the specific embodiment of another one, this carrier is selected from plasmid, virus vector, liposome and cation lipid.In another specific embodiment, this carrier introduces in applied once that this is female.In the specific embodiment of another one, this introducing is to take place when nourishing the 3rd phase in March of this offspring.In another specific embodiment, this method further comprises to this female step of using the part of secretagogue receptor.In another specific embodiment, this part dosage forms for oral administration.

[0019] in an extra embodiment of the present invention, the method of a kind of increase from the offspring's of jenny feeding efficiency arranged, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause feeding efficiency increase among the offspring.In a specific embodiment, the cell of this jenny comprises diploid cell.In another specific embodiment, the cell of this jenny comprises muscle cell.In the specific embodiment of another one, this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.In further specific embodiment, this growth hormone releasing hormone is SEQ ID NO:1, SEQ ID NO:8 or its analogue separately.In the specific embodiment of another one, this promotor comprises synthetic myogenic promotor.In further specific embodiment, this 3 ' non-translational region comprises hGH 3 ' non-translational region.In another specific embodiment, this carrier by electroporation, through virus vector, combine with carrier or be incorporated into by parenteral route in the cell of jenny.In the specific embodiment of another one, this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.In further specific embodiment, this jenny is people, pig, cow, sheep, goat or chicken.In the specific embodiment of another one, this carrier is selected from plasmid, virus vector, liposome and cation lipid.In another specific embodiment, this carrier introduces in applied once that this is female.In the specific embodiment of another one, this introducing is to take place when nourishing the 3rd phase in March of this offspring.In another specific embodiment, this method further comprises to this female step of using the part of secretagogue receptor.In another specific embodiment, this part dosage forms for oral administration.

[0020] in another embodiment of the invention, the method of a kind of increase from the offspring's of jenny the speed of growth arranged, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause speed of growth increase among the offspring.In a specific embodiment, the cell of this jenny comprises diploid cell.In another specific embodiment, the cell of this jenny comprises muscle cell.In the specific embodiment of another one, this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.In further specific embodiment, this growth hormone releasing hormone is SEQ ID NO:1, SEQ ID NO:8 or its analogue separately.In the specific embodiment of another one, this promotor comprises synthetic myogenic promotor.In further specific embodiment, this 3 ' non-translational region comprises hGH 3 ' non-translational region.In another specific embodiment, this carrier by electroporation, through virus vector, combine with carrier or be incorporated into by parenteral route in the cell of jenny.In the specific embodiment of another one, this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.In further specific embodiment, this jenny is people, pig, cow, sheep, goat or chicken.In the specific embodiment of another one, this carrier is selected from plasmid, virus vector, liposome and cation lipid.In another specific embodiment, this carrier introduces in applied once that this is female.In the specific embodiment of another one, this introducing is to take place when nourishing the 3rd phase in March of this offspring.In another specific embodiment, this method further comprises to this female step of using the part of secretagogue receptor.In another specific embodiment, this part dosage forms for oral administration.

[0021] in an extra embodiment of the present invention, there is a kind of increase to produce cell is produced the ratio of cell to other hormone method from somatropin in the offspring's of jenny the pituitary body, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, condition of living in are expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause the ratio that somatropin production cell is produced cell to other hormone among the offspring to increase.In a specific embodiment, the cell of this jenny comprises diploid cell.In another specific embodiment, the cell of this jenny comprises muscle cell.In the specific embodiment of another one, this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.In further specific embodiment, this growth hormone releasing hormone is SEQID NO:1, SEQ ID NO:8 or its analogue separately.In the specific embodiment of another one, this promotor comprises synthetic myogenic promotor.In further specific embodiment, this 3 ' non-translational region comprises hGH 3 ' non-translational region.In another specific embodiment, this carrier by electroporation, through virus vector, combine with carrier or be incorporated into by parenteral route in the cell of jenny.In the specific embodiment of another one, this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.In further specific embodiment, this jenny is people, pig, cow, sheep, goat or chicken.In the specific embodiment of another one, this carrier is selected from plasmid, virus vector, liposome and cation lipid.In another specific embodiment, this carrier introduces in applied once that this is female.In the specific embodiment of another one, this introducing is to take place when nourishing the 3rd phase in March of this offspring.In another specific embodiment, this method further comprises to this female step of using the part of secretagogue receptor.In another specific embodiment, the dosage forms for oral administration of this part.In a specific embodiment, this hormone is produced cell and is selected from corticotroph, lactotroph and gonadotroph.

[0022] in an extra embodiment of the present invention, the method of a kind of delay from offspring's birth of jenny arranged, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for wherein nucleotide sequence and wherein the introducing of carrier and expression cause offspring's delay of being born.In a specific embodiment, the cell of this jenny comprises diploid cell.In another specific embodiment, the cell of this jenny comprises muscle cell.In the specific embodiment of another one, this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.In further specific embodiment, this growth hormone releasing hormone is SEQ ID NO:1, SEQ ID NO:8 or its analogue separately.In the specific embodiment of another one, this promotor comprises synthetic myogenic promotor.In further specific embodiment, this 3 ' non-translational region comprises hGH 3 ' non-translational region.In another specific embodiment, this carrier by electroporation, through virus vector, combine with carrier or be incorporated into by parenteral route in the cell of jenny.In the specific embodiment of another one, this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.In further specific embodiment, this jenny is people, pig, cow, sheep, goat or chicken.In the specific embodiment of another one, this carrier is selected from plasmid, virus vector, liposome and cation lipid.In another specific embodiment, this carrier introduces in applied once that this is female.In the specific embodiment of another one, this introducing is to take place when nourishing the 3rd phase in March of this offspring.In another specific embodiment, this method further comprises to this female step of using the part of secretagogue receptor.In another specific embodiment, this part dosage forms for oral administration.

[0023] in an extra embodiment of the present invention, the method that has a kind of milk that increases animal to produce, this method comprises that the carrier with significant quantity is incorporated into the intracellular step of this animal, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause animal milk production to increase.In a specific embodiment, the cell of this jenny comprises diploid cell.In another specific embodiment, the cell of this jenny comprises muscle cell.In the specific embodiment of another one, this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.In further specific embodiment, this growth hormone releasing hormone is SEQID NO:1, SEQ ID NO:8 or its analogue separately.In the specific embodiment of another one, this promotor comprises synthetic myogenic promotor.In further specific embodiment, this 3 ' non-translational region comprises hGH 3 ' non-translational region.In another specific embodiment, this carrier by electroporation, through virus vector, combine with carrier or be incorporated into by parenteral route in the cell of jenny.In the specific embodiment of another one, this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.In further specific embodiment, this jenny is people, pig, cow, sheep, goat or chicken.In the specific embodiment of another one, this carrier is selected from plasmid, virus vector, liposome and cation lipid.In another specific embodiment, this carrier introduces in applied once that this is female.In the specific embodiment of another one, this introducing is to take place when nourishing the 3rd phase in March of this offspring.In another specific embodiment, this method further comprises to this female step of using the part of secretagogue receptor.In another specific embodiment, this part dosage forms for oral administration.

[0024] any example by the preferred embodiment of reading following specification sheets and providing for disclosed purpose at present with reference to wherein subsidiary accompanying drawing part or the present invention, the present invention other and further purpose, feature and advantage will be apparent, and finally be easier to understand.

Accompanying drawing is described

[0025] Figure 1A has illustrated that to 1C the analogue of GHRH superactivity has increased GH secretogogue activity and stable.Figure 1A is the comparison of pig wild-type (1-40) OH aminoacid sequence and analogue HV-GHRH.Figure 1B represents the effect that different GHRH kinds discharge pig GH in pig prepituitary gland culture of former generation.Fig. 1 C has illustrated the change of the stability that betides HV-GHRH and wild-type pig GHRH in 6 hours incubation.

[0001] Fig. 2 A to 2E illustrated after the shot of superactivity analogue GHRH myogenic expression vector 2 the middle of the month GHRH, GH and the increase of IGF-I serum level.Fig. 2 A has described the construct of the 3 ' UTR that comprises SPc5-12 synthetic promoter and GH.As the model of mutein, use the HV-GHRH construct also to compare over against shining into row, and compare as bearing to contrast with the beta-galactosidase enzymes construct with the conduct of pig wild-type.Fig. 2 B has illustrated with the pig of pSP-GHRH injection and the relative level of the Serum GH RH of the contrast pig of using placebo injection.Fig. 2 C explanation increases the pig of correction back pSP-GHRH injection and the abswolute level of the Serum GH RH of contrast pig with weight/blood volume.Fig. 2 D is illustrated in the variation with GH level in the pig of pSP-HV-GHRH injection.Fig. 2 E is illustrated in and directly the pSP-GHRH construct is carried out after the intramuscularly level of IGF-I in the blood plasma.

[0027] Fig. 3 A is to the effect of 3C explanation myogenic GHRH expression vector to the pig growth.Fig. 3 A is illustrated in the variation of 2 middle of the month with weight in average in the pig of pSP-GHRH or pSP-HV-GHRH injection.Fig. 3 B represent with the pig of pSP-GHRH injection compared with the control feed change the situation of efficient.Fig. 3 C is back 45 days of injection, with the pig of pSP-HV-GHRH injection with the comparison of the contrast pig of placebo injection.

[0028] Fig. 4 illustrates the effect of the pSP-HV-GHRH of the different amounts of injection to the piggy of 10 ages in days.

[0029] Fig. 5 represents to inject the effect of the pSP-HV-GHRH of different amounts to the IGF-I level of the piggy of 10 ages in days.

[0030] Fig. 6 has illustrated the time course of pSP-HV-GHRH injection piggy.

[0031] Fig. 7 has illustrated the preferred embodiment of comparing injection-type electrode of the present invention with the another embodiment of external clamp electrode (caliper electrode).The top is the explanation to the external clamp electrode with 2 square flat board/1.5cm faces.The bottom is that the length to 18-26g in the array that is present in the 1cm diameter is the explanation of the 6-pin array instrument (solid pin) of 2cm pin.The explanation on the left side is a side-view and the explanation on the right side is a fish-eye view.

[0032] Fig. 8 has illustrated the birth weight of the piggy of contrast and experiment.

[0033] Fig. 9 has illustrated the weight of weanling experiment and contrast piggy.

[0034] Figure 10 represents to raise together with the animal of injection contrast and its littermate weight relatively of (cross-fostered).

[0035] Figure 11 illustrated from the weight of the piggy of the sow of handling with the GHRH-that raises together with of contrast sow and with the comparison of its littermate.

[0036] Figure 12 has illustrated comparison and has impinged upon increase total on the weight (being raised by the contrast sow).

[0037] Figure 13 represents to test and contrast the comparison of market weight.

[0038] Figure 14 has illustrated the offspring's in 3 week, 10 week and 24 week weight.

The muscle weight of every body weight when [0039] Figure 15 was illustrated in for 3 ages in week.

[0040] Figure 16 has illustrated the hypophysis weight of every offspring's gross weight.

[0041] Figure 17 RNA of being illustrated in GH, GHRH among the offspring and PRL analyzes, and illustrates GHRH and in utero as somatomedin hypophysis is being worked.

[0042] Figure 18 has illustrated the DAB dyeing of GH-secretory cell.

[0043] Figure 19 has illustrated the concentration of IGF-I among the offspring in 3 week, 10 week and 6 months.

Detailed Description Of The Invention

[0044] for a person skilled in the art, carry out in can disclosed herein the present invention that various to substitute and modify and do not deviate from scope and spirit of the present invention be conspicuous.

[0045] " (a) " or " (an) " used herein can refer to one or more in specification sheets.So be in the claim used, when with " by ... form " when being used in combination, " one (a) " or " one (an) " can refer to one or more than one.Refer at least the second or more as used herein " another ".

[0046] term " animal " used herein refers to any species in the animal kingdom.In preferred embodiments, it more specifically refers to animal (chicken, cow, fish), the farm-animals (pig, horse, cow, sheep, chicken) of the animal of people, wild state, the animal (bird, dog, cat, horse) that is used as pet, the animal (horse, cow, dog) that is used for work and production food or itself is animal (frog, chicken, fish, crab, lobster, shrimp, mussel, scallop, goat, boar, cow, lamb, pig, ostrich, emu, common eel) and other animals well known in the art of food.

[0047] term " significant quantity " used herein be defined as need be in the host amount of the composition of generation effect, the available several frontier points known in those skilled in the art of effect are wherein monitored.In a specific embodiment, these frontier points are surrogate markers things.

[0048] term " feed transformation efficient " used herein is defined as the amount of the foodstuff that the weight that obtains with this animal eaten with respect to this animal every day.Term used herein " efficient " or " feeding efficiency " can exchange with " feed transformation efficient ".

[0049] term " growth lacks " used herein is defined as normal few any healthy state, medical conditions or the disease of its growth fraction.This shortage can be distored result, and this distortion directly influences tethelin approach (as the GHRH-GH-IGF-I axle), remote effect tethelin approach or do not influence the tethelin approach.

[0050] term " tethelin " used herein be defined as relate to growth and serve as chemical messenger and in target cell the hormone of its effect of performance.

[0051] term " growth hormone releasing hormone " used herein is defined as and promotes or hormone that the stimulating growth hormone discharges.

[0052] term " analogue of growth hormone releasing hormone " used herein is defined as and contains amino acid mutation and/or disappearance (no synthetic dextrorotation or cyclic amino acid) but be not natural generation in the GHRH molecule in naturally occurring aminoacid sequence, and still keeps it to strengthen the protein of the synthetic and secreting function of tethelin.

[0053] term " secretagogue receptor " used herein (GHS-R) is defined as the acceptor of little synthetic compound, and this compound discharges relevant from pituitary body with tethelin directly or indirectly.

[0054] term " lean mass " used herein is defined as the body weight of animal owing to non-fat tissue such as muscle.

[0055] term " part of secretagogue receptor " used herein is defined as the compound of the agonist that serves as secretagogue receptor.This part can be synthetic or naturally occurring.This part can be peptide, protein, sugar, carbohydrate, lipid, nucleic acid or its combination.

[0056] term " myogenic " used herein specifically refers to muscle tissue.

[0057] term " newborn infant " used herein refers to be right after the animal after birth and all are with after ripening or growth phase.

[0058] term " offspring " used herein refers to the descendant of parental generation, and wherein this descendant is unborn fetus or newborn infant.

[0059] term " parenteral " used herein refers to except that the mechanism that through enteron aisle material is incorporated in the animal.In specific embodiment, that parenteral comprises is subcutaneous, in the intramuscular, intravenously, sheath, intraperitoneal or other.

[0060] term " medicine is acceptable " used herein refers to compound, and wherein using of this compound can be tolerated by the acceptor Mammals.

[0061] term " secretogogue " used herein refers to strengthen synthetic and the natural or synthetic molecule (is the secretogogue of GH as GHRH) of excretory of the molecule of regulating in the downstream.

[0062] term " somatotroph " used herein refers to produce the cell of tethelin.

[0063] term " treatment significant quantity " used herein refers to the amount of the compound used, wherein should amount on physiology significantly.If its existence of a kind of medicament cause receptor the physiology generation technique change on the physiology significantly.For example, in treatment growth shortage, the composition that increases growth is effective for treatment; Can reduce rate of loss or increase the composition of growing in consuming disease is to treat effectively.

[0064] term " carrier (vector) " used herein refers to any carrier that delivery of nucleic acids can be gone in cell or the organism.Example comprises plasmid, virus vector, liposome or cation lipid.In a specific embodiment, liposome and cation lipid are can be compound to increase the adjuvant (carrier (carrier)) of target cell to the picked-up of plasmid or virus vector with other carrier.In a preferred embodiment, this carrier comprises promotor, nucleotide sequence, preferably encode growth hormone releasing hormone or its analogue and 3 ' non-translational region.In a further preferred embodiment, this promotor, nucleotide sequence and 3 ' non-translational region operationally are connected to express in eukaryotic cell.

[0065] term " atrophy symptom " used herein is defined as symptom or the situation relevant with consumption or chronic wilt disease.

[0066] the application is on theme and in the U.S. Provisional Patent Application No.60/145 of submission on July 26th, 1999,624 and corresponding in the non-temporary patent application No.09/624 of the U.S. of submission on July 24th, 2000,268 is relevant, the two quoted as a reference herein.

[0067] in order to estimate the growth result of growth hormone releasing hormone (GHRH) gene therapy myogenic carrier, to injected the carrier of GHRH cDNA that 10mg contains (pSP-HV-GHRH) of wild-type (pSP-wt-GHRH) or sudden change the sow of last 3 months pregnancy of gestation.After this injection, carry out electroporation.With the sow of injection/electroporation not with comparing.Big at birth (p＜0.0014 is for contrast 1.27 ± 0.02kg) for average out to 1.65 ± 0.06kgHV-GHRH, p＜0.00002 and 1.46 ± 0.05kg wt-GHRH from the piggy of the sow of GHRH injection.Raise together with research.When wean, shine big from the piggy comparison of the sow of injecting.By the sow lactation of injection to raise together with contrast bigger than its littermate significantly.This advantage is kept, and in birth back 170 days, the offspring of the sow of injection is respectively 135.7kg and 129.3kg for HV-GHRH and wt-GHRH, and the contrast weighted average is 125.3kg.Piggy has been carried out the measurement of multi-biological chemistry.Gross protein has increased in from the piggy of injection sow, and the level of urea reduces in the time point blood of all checks, and these two constants have all illustrated the protein metabolism that improves.Creatinine concentration is normal, shows normal renal function.The level of glucose and Regular Insulin is normal.Thereby, carry out piggy that sow that gene therapy handles produces when keeping normal ortho states and stablize by plasmid DNA construction body with coding GHRH, show in back 170 days than the growth pattern of normal level increase and thinner being born at least.The modification of hypothalamic pituitary axis among the increase of the sow lactation that this increase is equally injected due to and the offspring.When the plasmid-mediated transfer of evidence explanation that should main experiment is used in the secondary effect of avoiding classical protein therapeutic institute associated, run through some animal character of enhancing from generation to generation.

[0068] in one embodiment of the invention, use a nucleotide sequence in the method for the invention, this sequence increases ratio, delay birth or the production of increase milk of growth, enhancing growth, increase feed transformation efficient, increase lean mass, increase IGF-I level, the speed of growth that increases, relative other hormones production cells of increase somatotroph in female offspring.In specific embodiment, this nucleotide sequence is growth hormone releasing hormone, tethelin, IGF-I, prolactin or its analogue.This is female can be mother, in the past never conceived or produce female, perhaps for substituting mother, as becoming pregnant by fetus transplant.

[0069] preferred embodiment of the present invention is used the growth hormone releasing hormone analog with aminoacid sequence SEQ IDNO:1 or SEQ ID NO:8 (wt GHRH).As used herein, term " wild-type " can be the endogenous form of the GHRH of any animal, or is the form of modifying slightly of this hormone such as pig GHRH.The technician knows that endogenous GHRH has 44 amino acid, and has an amide group at latter end, and its correct symbolic representation is (1-44) NH ₂-GHRH.In a specific embodiment, using only has 40 amino acid form of (lacking last 4 amino acid), and this form does not contain amide group yet, can be designated as (1-40) OH-GHRH.Be also referred to as wild-type as this used herein form, do not contain inner sudden change because it is compared with the sequence of wild-type, this has with described herein that introduced inner other forms (as HV) of suddenling change by rite-directed mutagenesis opposite.The technician knows in the form of 1-40 and the natural people of being present in of shorter form (for example 1-32 or 1-29) and other Mammalss (even in dissimilar GHRH secreting tumors), and these forms have and natural (1-44) NH ₂Suitable activity.In a preferred embodiment of the invention, use GHRH with the stability that increases than wild-type GHRH.

[0070] in another embodiment, the analogue of different types of GHRH or GHRH within the scope of the present invention.In one object of the present invention, during characteristic that known nucleic acid is used, do not carry out posttranslational modification by the residue of dna encoding.

[0071] Xia Mian kind within the scope of the present invention.U.S. Patent No. 4,223,019 discloses and has had aminoacid sequence NH ₂The pentapeptide of-Y-Z-E-G-J-COOH, wherein Y is selected from D-Methionin and D-arginine; Z and J are independently selected from tyrosine, tryptophane and phenylalanine; And E and G are independently selected from D-tyrosine, D-tryptophane and D-phenylalanine.U.S. Patent No. 4,223,020 discloses and has had following aminoacid sequence NH ₂The tetrapeptide of-Y-Z-E-G-COOH, wherein Y and G are independently selected from tyrosine, tryptophane and phenylalanine; And Z and E are independently selected from D-tyrosine, D-tryptophane and D-phenylalanine.U.S. Patent No. 4,223,021 discloses and has had following aminoacid sequence NH ₂The pentapeptide of-Y-Z-E-G-J-COOH, wherein Y and G are independently selected from tyrosine, tryptophane and phenylalanine; Z is selected from glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), oxyproline, Serine, Threonine, halfcystine and methionine(Met); E and J are independently selected from D-tyrosine, D-tryptophane and D-phenylalanine.U.S. Patent No. 4,224,316 disclose and have had following aminoacid sequence NH ₂The novel pentapeptide of-Y-Z-E-G-J-COOH, wherein Y and E are independently selected from D-tyrosine, D-tryptophane and D-phenylalanine; Z and G are independently selected from tyrosine, tryptophane and phenylalanine; And J is selected from glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), oxyproline, Serine, Threonine, halfcystine, methionine(Met), aspartic acid, L-glutamic acid, l-asparagine, glutamine, arginine and Methionin.U.S. Patent No. 4,226,857 disclose and have had following aminoacid sequence NH ₂The pentapeptide of-Y-Z-E-G-J-COOH, wherein Y and G are independently selected from tyrosine, tryptophane and phenylalanine; Z and J are independently selected from D-tyrosine, D-tryptophane and D-phenylalanine; And E is selected from glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), oxyproline, Serine, Threonine, halfcystine, methionine(Met), aspartic acid, L-glutamic acid, l-asparagine, glutamine and Histidine.U.S. Patent No. 4,228,155 disclose and have had following aminoacid sequence NH ₂The pentapeptide of-Y-Z-E-G-J-COOH, wherein Y is selected from tyrosine, D-tyrosine, tryptophane, D-tryptophane, phenylalanine and D-phenylalanine; Z and E are independently selected from D-tyrosine, D-tryptophane and D-phenylalanine; G is selected from Methionin and arginine; And J is selected from glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), oxyproline, Serine, Threonine, halfcystine and methionine(Met).U.S. Patent No. 4,228,156 disclose and have had following aminoacid sequence NH ₂The tripeptides of-Y-Z-E-COOH, wherein Y and Z are independently selected from D-tyrosine, D-tryptophane and D-phenylalanine; And E is selected from tyrosine, tryptophane and phenylalanine.U.S. Patent No. 4,228,158 disclose and have had following aminoacid sequence NH ₂The pentapeptide of-Y-Z-E-G-J-COOH, wherein Y and G are independently selected from tyrosine, tryptophane and phenylalanine; Z and E are independently selected from D-tyrosine, D-tryptophane and D-phenylalanine; And J is selected from natural amino acid and its D-form.U.S. Patent No. 4,833,166 disclose the synthetic peptide with molecular formula H-Asp-Pro-Val-Asn-Ile-Arg-Ala-Phe-Asp-Asp-Val-Leu-Y, and wherein Y is OH or NH ₂Or its non-toxic salts and have the synthetic peptide of molecular formula H-Val-Glu-Pro-Gly-Ser-Leu-Phe-Leu-Val-Pro-Leu-Pro-Leu-Le u-Pro-Val-His-Asp-Phe-Val-Gln-Gln-Phe-Ala-Gly-Ile-Y, wherein Y is OH or NH ₂Or its non-toxic salts.The hGHRH fragment of the 228-bp of 31 amino acid whose signal peptides of people such as Draghia-Akli (1997) use coding and complete mature peptide people GHRH (1-44) OH (Tyr1Leu44) that describes by people such as Mayo (1995) at first.People such as Guillemin (1982) have also determined the sequence of human pancreas's somatotropin releasing factor (hpGRF).

[0001] the extra embodiment of the present invention comprises: (1) improves the method for offspring's growth performance; (2) stimulate among the offspring to produce the method for tethelin than the level of the higher level relevant with normal growth; (3) strengthen the method that the offspring grows.All these methods comprise with plasmid vector during nourishing this offspring or before gestation time be incorporated into step among mother of this offspring, wherein this carrier comprise promotor, nucleotide sequence as the nucleotide sequence of coding SEQ ID NO:1 or SEQ ID NO:8 and operationally suitable distance be linked in sequence to carry out 3 ' non-translational region of functional expression.

[0001] in an extra particular, the method that has a kind of offspring of stimulation to produce tethelin with the level of the higher level more relevant than normal growth, this method is included in nourishes carrier from this offspring to mother of this offspring that introduce significant quantity during, this carrier comprise the nucleotide sequence of promotor, coding SEQ ID NO:1 or SEQ ID NO:8 and operationally suitable distance be linked in sequence to carry out 3 ' non-translational region of functional expression.The level of the higher level more relevant than normal growth comprise animal with the relevant shortage of growth or have with colony in the animal of the similar level of growth of other similar animals, comprise basic, the inherent growth of animal that those do not have the relevant shortage of growth.

[0074] in a preferred embodiment, a kind of method that strengthens growth of animal is arranged, this method comprises carrier from significant quantity to this animal that introduce, this carrier comprise the nucleotide sequence of promotor, coding SEQ ID NO:1 or SEQ ID NO:8 and operationally suitable distance be linked in sequence to carry out 3 ' non-translational region of functional expression.Its growth enhanced animal can have or not have growth and lack.

[0075] an object of the present invention is to increase the growth and/or the speed of growth of animal, it is preferably the offspring from mother.In a preferred embodiment, the growth of animal and/or the speed of growth have been subjected to secular influence, as greater than several weeks or greater than some months.In a specific embodiment, this reaches by the mother who growth hormone releasing hormone is applied to the offspring, preferably with the form of nucleic acid.In a preferred embodiment, this GHRH nucleic acid is as the episome of muscle cell and kept.In a specific embodiment, the increase of GHRH is produced the number of cell and is influenced pituitary body by increasing tethelin, and thereby has changed its cell lineage.In a specific embodiment, the ratio of somatotroph (tethelin production cell) is produced cell such as corticotroph, lactotroph and gonadotroph etc. with respect to other hormone in the hypophysis and is increased.In a specific embodiment, the increase of the tethelin relevant with the increase of tethelin production cell number is reflected in the increase of IGF-I level.In another specific embodiment, the increase of the increase of level of growth hormone and offspring's lean mass is relevant with the increase of the speed of growth.In another specific embodiment, the increase of lean mass is relevant with the increase of linear bone growth.In the specific embodiment of another one, offspring's feed changes efficient and increases.In another specific embodiment, offspring's birth postpones, and in a preferred embodiment, this is with the fetal growth speed improvement or increase relevant.

[0001] in a preferred embodiment, this promotor is a synthetic myogenic promotor, and hGH 3 ' non-translational region is positioned at 3 ' non-translational region.Yet this 3 ' non-translational region can be from any natural or synthetic gene.In a specific embodiment of the present invention, use the synthetic promotor, be called SPc5-12 (people such as Li, 1999) (SEQ ID NO:6), it contains from the nearside serum response element (SRE) of bone α-Ji Dongdanbai, multiple MEF-2 site, MEF-1 site and TEF-1 binding site, and surpasses the transcriptional capability of natural myogenic promotor widely.In a preferred embodiment, the used promotor of the present invention and can't help endogenous cell device or the factor and turn off significantly or reduce its activity.Other element comprises that trans-acting factor binding site and enhanser can use according to embodiment of the present invention.In another embodiment, use natural myogenic promotor, and the technician knows how to obtain such promoter sequence from database, this database comprises NCBI (National Center for Biotechnology Information) (NCBI) GenBank database or NCBI PubMed website.The technician knows that these Internets (world wide web) website can be used for obtaining sequence related to the present invention or pertinent literature.

[0077] in a specific embodiment, in nucleic acid carrier such as plasmid, uses hGH3 ' non-translational region (SEQ ID NO:7).

[0078] in specific embodiment, this carrier is selected from plasmid, virus vector, liposome or cation lipid.In further specific embodiment, this carrier is incorporated in myogenous cell or the muscle tissue.In further specific embodiment, this animal is people, pet animals, work with animal or edible animal.

[0079] except that this construct is incorporated into the particular in the animal by plasmid vector, also can use delivery system of nucleic acid transfection being gone into animal or its cell as known in the art.For example, can use method other non-viruses or virus.The technician recognizes that the goal systems of DNA or the non-viral form of RNA needs 4 compositions: 1) target DNA or RNA; 2) discern and be attached to cell surface receptor or antigenic part; 3) DNA bound fraction; With 4) make mixture to be transported to cracking section the kytoplasm from cell surface.Further, liposome can be used for the delivery treatments assortment of genes to reach identical effect with cation lipid.The potential virus vector comprises derived from the expression vector as viruses such as adenovirus (adenovirus), vaccinia virus (vaccinia virus), simplexvirus (herpes virus) and bovine papilloma viruses (bovine papilloma virus).In addition, can adopt the episome carrier.Other dna vectors and translocator system are known in this field.

[0080] those skilled in the art recognizes derived from various bacterial plasmids, retrovirus, adenovirus, bleb or from the expression vector of vaccinia virus and can be used for sending nucleotide sequence to Target organ, tissue or cell colony.Method known in those skilled in the art can be used for making up the recombinant vectors that will express coding growth hormone releasing hormone analog gene.Temporary transient expression is with sustainable 1 month of the carrier of non-replicating or longer, and if suitable reproduction element when being carrier system a part of even can be longer.

[0081] one object of the present invention is that the applied once of growth hormone releasing hormone is enough effective period for multiple pregnancies, and provides and strengthen the treatment of piggy to market weight performance, forms as the growth that increases and the health of change.

Nucleic acid

1. carrier

[0082] term " carrier " is used in reference to the vector nucleic acid molecule, can insert nucleotide sequence therein to be incorporated in the cell, and reproducible and this nucleotide sequence of this carrier can be expressed there.This nucleotide sequence can be " external source ", this means it for being external source to the cell of wherein introducing carrier, and perhaps this sequence and an intracellular sequence homology but be positioned in host's nucleic acid usually do not found on the position of this sequence.Carrier comprises plasmid, clay, virus (phage, animal virus and plant virus) and artificial chromosome (for example YACs).Those skilled in the art can train the carrier construction with the recombinant technology by standard well, and this recombinant technology is described in people such as Maniatis, and 1988 and people such as Ausubel, 1994, both are quoted as a reference herein.

[0083] term " expression vector " refers to contain the carrier of the nucleotide sequence of the gene product that can transcribe that is encoding to small part.In a specific embodiment, the GHRH that this nucleic acid sequence encoding is part or all of.In some cases, the RNA molecule is translated as protein, polypeptide or peptide then.Under other situation, these sequences are not translated, as in the production of antisense molecule or ribozyme.Expression vector can contain various " control sequences ", and this refers to transcribing and the necessary nucleotide sequence of possible translation of the encoding sequence that is operably connected in special host living beings.Except that the control sequence that domination is transcribed and translated, carrier and expression vector can contain the nucleotide sequence that other functions also is provided and describes hereinafter.

[0084] in a preferred embodiment, carrier of the present invention is to comprise synthetic myogenic (muscle specific) promotor, coding growth hormone releasing hormone or the nucleotide sequence of its analogue and the plasmid of 3 ' non-translational region.In another embodiment, this carrier is a virus vector, as adeno-associated virus (AAV), adenovirus or retrovirus.In another embodiment, can use bone α-Ji Dongdanbai promotor, myosin light chain promotor, cytomegalovirus promoter or SV40 promotor.In other other embodiments, end user's tethelin, Trobest, SV40 or bone α-Ji Dongdanbai 3 ' non-translational region in carrier.

A. promotor and enhanser

[0085] " promotor " is control sequence, and it is a zone of the initial sum speed of transcribing of the control on the nucleotide sequence.It can contain and regulates protein and molecule such as RNA polymerase and the combinative genetic elements of other transcription factors thereon.Phrase " operationally localized ", " being operably connected ", " under control " and " transcribing under the control " refer to that promotor is positioned at the correct position of function of relative nucleotide sequence and/or direction to control the initial and/or expression that this sequence is transcribed.Promotor can with or be not used in combination with " enhanser ", the trans-acting of the transcriptional activation of latter's reference and nucleotide sequence is regulated sequence.

[0086] promotor can be one of natural encoding sequence (naturally-coding sequences) that is positioned at coding section and/or exon upstream.Such promotor can be described as " endogenous ".Similarly, enhanser can be relevant with nucleotide sequence natively and is positioned at the sequence of this sequence downstream or upstream.Perhaps, the nucleic acid segment that makes coding can obtain some benefit under promotor control reorganization or allogenic, and this promotor refers to not be in its natural surroundings and the normal relevant promotor of nucleotide sequence.Reorganization or allogenic promotor also refer to not be in its natural surroundings and the normal relevant enhanser of nucleotide sequence.Such enhanser or enhanser can comprise the promotor of other genes or enhanser and from promotor or enhanser any other protokaryon, cellular segregation virus or eucaryon, and be not the promotor or the enhanser of " natural existence ", the different elements and/or the sudden change in the different transcriptional regulatory zone that promptly containing changes expresses.Except that the nucleotide sequence of production promotor synthetically or enhanser, this sequence also can comprise PCR with recombinant clone and/or nucleic acid amplification technologies ^TMProduce together with disclosed composition herein (referring to United States Patent (USP) 4,683,202, United States Patent (USP) 5,928,906, they are quoted as a reference separately herein).In addition, thinking also can be adopted the control sequence that guide sequence is transcribed and/or expressed in the organoid of non-nuclear such as plastosome, chloroplast(id) etc.

[0087] natively, adopt that can to instruct DNA section expression promoter and/or enhanser effectively at the selected cell type that is used for expressing, organoid and biology be important.The technician of those biology field generally knows that the purposes of the promotor, enhanser and the cell type combination that are used for protein expression, as referring to people such as Sambrook (1989), quotes as a reference herein.The promotor that is adopted can be composing type, tissue-specific, induction type and/or is used to instruct the DNA section high level expression introduced under proper condition, as favourable in the scale operation of recombinant protein and/or peptide.This promotor can be allogenic or endogenous.In a specific embodiment, this promotor is a synthetic myogenic promotor, as described by people such as Li (1999).

[0088] conventionally known to one of skill in the art to being determined as of the evaluation of tissue-specific promoter or element and activity characterization thereof.These regional examples comprise people LIMK2 gene (people such as Nomoto, 1999), somatotroph acceptor 2 genes (people such as Kraus, 1998), mouse epididymis vitamin A acid is in conjunction with gene (people such as Lareyre, 1999), people CD4 (people such as Zhao-Emonet, 1998), mouse α 2 (XI) collagen protein (people such as Tsumaki, 1998), D1A dopamine receptor gene (people such as Lee, 1997) insulin-like growth factor II (people such as Wu, 1997), human blood platelets endotheliocyte adhesion molecule-1 people such as (, 1996) Almendro.

B. start signal and internal ribosome binding site

[0089] specific start signal also is essential for the effective expression of encoding sequence.These signals comprise ATG initiator codon or the sequence of closing on.May need to provide the translation control signal of external source, comprise the ATG initiator codon.One of ordinary skill in the art is easy to determine this situation and essential signal is provided.As everyone knows, initiator codon must with the open reading-frame (ORF) " same frame (in frame) " of required encoding sequence to guarantee the translation of whole inset.The translation control signal of external source and initiator codon can be natural or synthetic.The efficient of expressing can strengthen by comprising suitable transcriptional enhancer element.

[0090] in certain embodiments of the invention, the purposes of internal ribosome entry site (IRES) element is used to produce polygene, or polycistron, information.The IRES element can be ignored 5 ' the methylate ribosome-scanning model of cap dependency translation and in the initial translation in inner site (Pelletier and Sonenberg, 1988).IRES element (Pelletier and Sonenberg from 2 members (polio and encephalomyocarditis) of picornavirus (picornavirus) family have been described, 1988) and from the IRES (Macejak and Sarnow, 1991) of Mammals information.The IRES element can be connected with allogenic open reading-frame (ORF).A plurality of open reading-frame (ORF)s can be transcribed together and be generated polycistronic message, and each free IRES separates.Utilize the advantage of IRES element, each open reading-frame (ORF) all can be near rrna effectively to translate.Polygene can be with single promotor/enhanser effective expression to be transcribed into single information (referring to United States Patent (USP) 5,925,565 and 5,935,819, quote as a reference) herein.

C. multiple clone site

[0091] carrier can comprise multiple clone site (MCS), and this site is the nucleic acid region that contains a plurality of restriction enzyme digestion sites, and wherein each all can be used in combination with digested vector with the standard recombinant technology.(referring to people such as Carbonelli, 1999, people such as Levenson, 1998 and Cocea, 1997, quote as a reference herein.) " digestion with restriction enzyme " refer to cut nucleic acid molecule with the enzyme catalysis in the specific position performance function of nucleic acid molecule only.Many restriction enzymes all have commodity.The purposes of such enzyme is extensively understood for those skilled in the art.Frequently, carrier is used in the restriction enzyme that cuts in the MCS and carries out linearizing or fragmentation so that exogenous array can connection carrier." connection " refers to form the process of phosphodiester bond between 2 nucleic acid molecule fragments, this fragment can adjacently or non-conterminous connect each other.The attach most importance to technician of technical group field of the technology that comprises restriction enzyme and ligation is known.

D. splice site

[0092] many eucaryotic RNA molecules of transcribing will carry out the RNA montage to remove intron from primary transcript.The carrier that contains genome eucaryon sequence can need donor and/or acceptor splice site to be used for the correct processing of the transcript of protein expression with assurance.(referring to people such as Chandler, 1997, quote as a reference herein.)

E. polyadenylation signal

[0093] in expression, generally polyadenylation signal will be comprised so that transcript correctly carries out polyadenylation.Do not think that the characteristic of polyadenylation signal is crucial for Successful Practice of the present invention and/or can adopts any such sequence.Preferred embodiment comprises SV40 polyadenylation signal and/or ox or human growth hormone polyadenylation signal, these signals be easily and/or known in all types of target cell performance good function.An element of also thinking deeply as expression cassette is the Transcription Termination site.These elements are suitable for strengthening information level and/or make the company from this box to other sequences read to minimize.

The starting point of f. duplicating

[0094] for amplification vector in the host, it can contain one or more replication orgin site (being commonly referred to " ori "), and this site is the initial specific nucleic acid sequence that duplicates at this place.Perhaps, if can adopt autonomously replicating sequence (ARS) when host cell is yeast.

G. selectivity and screening property mark

[0095] in certain embodiments of the invention, cell contains nucleic acid construct of the present invention, and this cell can be identified in external or the body by comprise mark in expression vector.Such mark can be given the appraisable change of cell and be easy to identify so that contain the cell of this expression vector.Normally, selected marker is to give the mark of the character that allows selection.Positive selectivity is labeled as the mark that allows its selection when it exists, and negative selected marker is for preventing the mark of its selection when it exists.An example of positive selected marker is the drug resistance mark.

[0096] comprise clone and the evaluation that the drug selectivity mark can help transformant usually, for example, the gene of giving Xin Meisu, tetracycline, Totomycin, DHFR, GPT, zeocin and histidinol resistance is useful selected marker.Except that the mark of giving permission distinguishes transformant based on implementation condition phenotype, also think deeply the mark of other types, comprise screening property mark such as GFP, its basis is colorimetric analysis.In addition, can use screening property enzyme such as herpes simplex virus thymidine kinase (tk) or E.C. 2.3.1.28 (CAT).Those skilled in the art also knows how to adopt amynologic label, may combine with facs analysis.Do not think that used mark is important, as long as it can be expressed simultaneously with the nucleic acid of encoding gene product.The further example of selectivity and screening property mark is conventionally known to one of skill in the art.

2. host cell

[0097] as used herein, term " cell ", " clone " and " cell culture " are used interchangeably.All these terms also comprise its descendant, and this can be any or all successive generations.Can understand all descendants because have a mind to or sudden change and may be inequality unintentionally.Under the situation of expressing heterologous nucleotide sequence, chief cell contracts " refer to protokaryon or eukaryotic cell, but and it comprise any can replicating vector and/or express inverting biological body by the heterologous gene of vector encoded.Host cell can and be used as the acceptor of carrier.Host cell can be by " transfection " or " conversion ", and this refers to that exogenous nucleic acid shifts or introduce the process of host cell.Cell transformed comprises cell and the descendant thereof that former pickup is tested.

[0098] host cell can be derived from prokaryotic organism or eukaryote, and this depends on required result is replicating vector or the nucleotide sequence of expressing part or all of vector encoded.Many clones and culture can be used as host cell, and can pass through American Type Culture Collection (ATCC) acquisition, and this preservation be is as the tissue (www.atcc.org) of the archives of live body culture and genetic stocks.Suitable host can be determined based on the main chain and the required result of carrier by those skilled in the art.For example, plasmid or clay can be incorporated in the prokaryotic organism host cell to duplicate many carriers.Comprise DH5 α, JM109 and KC8 as host cell with the bacterial cell that carries out carrier and duplicate and/or express, and many commodity host bacteriums such as SURE  Competent Cells and SOLOPACK  Gold Cells (STRATAGENE , LaJolla).In addition, bacterial cell such as intestinal bacteria (E.coli) LE392 can be used as the host cell of phage virus.

[0099] duplicates as carrier and/or the eukaryotic host cell example of expressing comprises HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos and PC12.Many host cells from various cell types and organism can be used and be conventionally known to one of skill in the art.Similarly, virus vector can be used in combination with eucaryon or prokaryotic host cell, and particularly those allow carriers to duplicate or express.

[0100] some carriers can adopt and allow its control sequence of duplicating and/or expressing in protokaryon and eukaryotic cell.Those skilled in the art further can understand the condition of all above-mentioned host cell incubations to keep this host and to allow carrier to duplicate.Understand equally with knowing and allow the scale operation carrier and by the technology and the condition of the nucleic acid of the vector encoded polypeptide relevant, protein or peptide with it.

3. expression system

[0101] many at least partly or entirely expression systems of above-mentioned composition that comprise is arranged.Can be used for the present invention to produce nucleotide sequence or its relevant polypeptide, protein or peptide based on prokaryotic organism and/or Eukaryotic system.Many such systems are commodity and extensively available.

[0102] insect cell/baculovirus (baculovirus) system can produce the segmental high-level protein expression of heterologous nucleic acids, as in U.S. Patent No. 5,871,986,4,879, described in 236, both are quoted as a reference, and this system can buy, for example be from INVITROGEN herein ^MAXBAC ^2.0 with from CLONTECH ^BACPACK ^TMBACULOVIRUS EXPRESSION SYSTEM.

[0100] example of other expression systems comprises STRATAGENE ^COMPLETECONTROL  induction type mammalian expression system, this system comprises that the synthetic moulting hormone induces acceptor, or its pET expression system, promptly a kind of escherichia expression system.Another example of this inducible expression system can derive from INVITROGEN ^, it contains T-REX ^TM(expression that tsiklomitsin is regulated) system, promptly a kind of induction type mammalian expression system that uses total length CMV promotor.INVITROGEN ^The yeast expression system of a kind of Pichia of being called methanolicaExpression System also is provided, and this system design is a high level production recombinant protein in methylotrophy yeast Pichia methanolica.How those skilled in the art knows that expression vector such as expression construct are to produce nucleotide sequence polypeptide, protein or the peptide relevant with it.

Sudden change

[0104] in use, sudden change realizes by the sudden change program of various standards.Sudden change is the process that changes in the amount of biology and structure whereby.Sudden change can relate to modifies term single gene, a collection of gene or whole chromosomal nucleotide sequence.Variation in the term single gene can be the result of point mutation, and this point mutation comprises in the dna sequence dna removes, adds or substitute single nucleotide base, and perhaps this variation can be the result of the change of the insertion that comprises a large amount of Nucleotide or deletion.

[0105] sudden change can be used as the result of the incident of moving as the mistake in the dna replication dna fidelity or transposable genetic element (transposon) and takes place naturally in genome.They also can be after being exposed to chemistry or physics mutagens and being induced.Such sudden change inductor comprises ionizing rays, ultraviolet ray and various chemical medicaments such as alkylating agent and polynuclear aromatics, and all these are (generally being to follow after some metabolism biologicals transform) and nucleic acid interaction directly or indirectly.When affected DNA duplicates or repairs, can cause the modification of base sequence by such environment medicament inductive DNA infringement, and thereby cause suddenling change.Sudden change can be that fixed point is carried out by using special target-seeking method also.

Rite-directed mutagenesis

[0106] the strong instrument (people such as Wells, 1996, people such as Braisted, 1996) of the analysis of carrying out protein-ligand interaction and manipulation has been represented in the sudden change of the locus specificity of structure guiding.This technology provides preparation and check to sequence variants by introduce one or more nucleotide sequence in selected DNA.

[0107] the specific oligonucleotides sequence of the dna sequence dna of the required sudden change of locus specificity sudden change use coding and the unmodified Nucleotide of enough number adjacency.In this approach, provide primer sequence with enough sizes and complicacy with across the both sides of disappearance contact form stable duplex.The primer that length is about 17 to 25 Nucleotide is for preferred, this primer 5 to 10 residues of respectively having an appointment in the contact both sides of reformed sequence.

[0108] the general phage vector that exists with strand and double chain form that adopts of this technology.Useful carrier comprises the carrier as the M13 phage in rite-directed mutagenesis.These phage vectors have commodity selling, and its purposes generally is known for those skilled in the art.Double-stranded plasmid also is used for rite-directed mutagenesis routinely, and it has saved goal gene is transferred to step the plasmid from phage.

[0109] usually, at first will obtain the carrier of strand, perhaps with two chains fusions of double-stranded carrier, this carrier comprises coding desired protein or genic dna sequence dna in its sequence.Oligonucleolide primers with required mutant nucleotide sequence and the annealing of single stranded DNA prepared product that to synthesize preparation then, the degree of consideration mispairing when selecting hybridization conditions.The product of hybridization is handled to finish synthesizing of chain with sudden change with archaeal dna polymerase such as Escherichia coli polymerase I (Klenow fragment).Thereby, formed heteroduplex, wherein chain encoding did not have the sequence of sudden change originally, and the second chain has required sudden change.Then this heteroduplex carrier is used to transform appropriate host cell such as Bacillus coli cells, and selects those to comprise the clone of recombinant vectors with mutant nucleotide sequence arrangement.

[0110] preferably can obtain the functional importance of given residue of protein and the comprehensive information of information content, wherein 19 all amino acid replacements all be checked by saturation mutation.The shortcoming of this method is that the logistic work of many residues saturation mutation is (people such as Warren, 1996, people such as Brown, 1996 gloomy; People such as Zeng, 1996; Burton and Barbas, 1994; People such as Yelton, 1995; People such as Jackson, 1995; People such as Short, 1995; People such as Wong, 1996; People such as Hilton, 1996).Must the hundreds of even thousands of locus specificities sudden changes of possibility of research.Yet the technology of improvement makes that the generation and the rapid screening of sudden change are simpler.Also referring to United States Patent (USP) 5,798,208 and 5,830,650 descriptions to " walkthrough (walk-through) " sudden change.

[0111] method of other rite-directed mutagenesises is disclosed in United States Patent (USP) 5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377 and 5,789,166.

Dosage and preparation

[0112] composition (activeconstituents of the present invention; Herein for the nucleotide sequence that comprises promotor, coding SEQ ID NO:1 or SEQ ID NO:8 and operationally in suitable distance order continuously to carry out 3 ' non-translational region of functional expression) can be produced and use by any method various growth deficiency state are had an effect, method wherein contacts the intravital medicament action site generation of activeconstituents and animal.Said composition of the present invention is defined as the carrier of the nucleotide sequence that contains the compound of the present invention of encoding, and it is an aminoacid sequence analogue described herein.Said composition is to use to generate the treatment significant quantity of this compound with enough amounts.Those skilled in the art recognizes term administering " and " introducing " be used interchangeably.They as independent treatment effective constituent or with the treatment combination of active principles, can use by any ordinary method that can be used in combination with medicine.Activeconstituents separately or use in the damping fluid as PBS but can be with based on selected route of administration and standard medication practice and the pharmaceutical carrier of selection is used in a preferred embodiment.Such pharmaceutical composition can be used for the purpose of treatment in people and animal doctor's the clinical medicine or diagnosis.For example, they are used for the treatment to the growth phase related disorders, as the pituitary dwarf disease that is caused unusually by tethelin production.In addition, they also can be used for stimulating the growth of storing up animal that is used for meat production or strengthen the production that its feed changes the production of efficient, enhancing milk and stimulates eggs.

[0113] dosage of using will be the treatment significant quantity of activeconstituents, and will depend on known factor certainly and change, this factor is for as the type of the treatment of the nature and extent of age of the pharmacokinetic characteristic of given activity composition and the pattern of using thereof and approach, type of animal, receptor, receptor's sex, receptor's reproductive status, receptor's health, receptor's weight, symptom, coexistence, the frequency and the required effect of treatment.The suitable dosage of the carrier that the present invention will use will depend on the parameter of indivedual subjects and other and some change.Skilled practitioner can be determined suitable dosage based on the activity to growth hormone release of known tethelin cyclical level relevant with normal growth and carrier.As known in the art, must change dosage to female or mother's treatment to produce bigger animal between individuality, this dosage depends on the degree of essential tethelin increase level.

[0114] thereby, according to the method that the invention provides a kind of offspring's of increasing growth, this method comprises that the female or mother to this offspring uses a certain amount of analogue of the present invention, and this amount enough makes tethelin be increased to level than the higher level relevant with normal growth.Normal level of growth hormone has sizable variation between individuality, and for any given individuality, the level of round-robin tethelin has sizable variation in one day process.

[0015] also provide a kind of by using a certain amount of GHRH analogue of the present invention and increase the method for animal growth rate, the enough stimulating growth hormones of amount wherein are with than the level production higher with the normal growth related levels.

Gene therapy is used

[0116] in suitable part, gene therapy vector can be formulated as the preparation of solid, semisolid, liquid or gas form with approach well known in the art according to route of administration separately.Method well known in the art can be used for preventing the release and the absorption of composition, arrives Target organ or guarantees that the timing of composition discharges up to it.Can adopt the acceptable form of medicine, this form does not make composition of the present invention invalid.In pharmaceutical dosage forms, said composition can be used separately or suitably unite with the other drug active compound and be used in combination.

[0117] therefore, pharmaceutical composition of the present invention each site that can send and arrive the animal health via various approach is to realize that special effect is (referring to as people such as Rosenfeld (1991); People such as Rosenfeld (1991a); People such as Jaffe, 1992).Although person of skill in the art will appreciate that more than a kind of approach and can use, comparable other the approach of particular approach provides more direct and more effective reaction.Part or systemic delivery can be used preparation or splash into body cavity, suck or be blown into aerosol-applied by comprising, perhaps by comprise intramuscular, intravenously, peritonaeum, parenteral introducing and topical application subcutaneous, intracutaneous realize.

[0118] those skilled in the art recognizes that different delivering methods can be used for vector administration in cell.Example comprises: (1) utilizes the method for physical means, as electroporation (electricity), particle gun (power of physics) or use a large amount of liquid (pressure); (2) wherein this carrier and other entities such as liposome or translocator molecule composite methods.

[0119] therefore, the invention provides the method that therapeutic gene is transferred to the host, this method comprises with any aforesaid route of administration or as well known to those skilled in the art and be suitable for other route of administration of special applications, uses carrier of the present invention, preferably as the part of composition.Carrier can be monitored (as alleviating of some symptoms relevant with the special disease of being treated) according to result of treatment to host's effective gene transfer according to the present invention, perhaps the evidence of expressing in the host by gene or this gene of transfer is further monitored (as using polymerase chain reaction and order-checking, Northern or Southern are hybridized or are transcribed mensuration to survey nucleic acid in host cell, perhaps use immunoblotting assay, antibody-mediated detection, research of mRNA or protein transformation period or the mensuration that particularly points out are to survey by level and affected protein of function or the polypeptide nucleic acid encoding that shifts or that cause owing to such transfer).

[0120] these methods described herein are not equipped with entirely, and the further method that is suitable for application-specific is apparent for general technician.In addition, the significant quantity of said composition can be further by estimating with the known composition analogy of bringing into play required effect.

[0121] in addition, it is not to use with other pharmaceutical composition that actual dosage and timetable can be dependent on said composition, or depends on pharmacokinetics, drug accumulation (disposition) and metabolic interindividual variation and change.Similarly, this amount can be dependent on employed special cells system (as the number based on the carrier acceptor that is present in cell surface, or the ability of transgenosis with the special carrier that duplicates of carrying out that is adopted) and changes to some extent in external application in this clone.In addition, the amount of the carrier that every cell will add changes according to the characteristic of the length that is inserted into the therapeutic gene in the carrier and stability and this sequence probably, this amount is the special parameter that need determine according to experiment, and can change owing to the extrinsic factor of method of the present invention (as with synthetic cost related).Those skilled in the art is easy to do any essential adjustment according to pressing for of Special Circumstances.

[0122] the following examples are that mode with example provides, and are not meaning by any way and limit the scope of the invention.

Embodiment 1

GHRH superactivity analogue increase the GH secretogogue active and stable

[0123] GHRH has relative short about 12 minutes transformation period in the recycle system of people people such as (, 1984) Frohman and pig.Prolong its biological half time and/or improve the active GHRH analogue of its GH secretogogue by adopting, realize strengthening the GH secretion.The GHRH mutant generates by rite-directed mutagenesis.Gly15 is replaced by Ala15 to increase alpha-helix conformation and amphipathic structure to reduce by the cutting of trypsin-like enzyme people such as (, 1991) Su.Have Ala15 alternate GHRH analogue and show the avidity doubly high people such as (, 1991) Campbell the 4-5 of GHRH acceptor.For with reducing in the more stable a little form of molecule with free COOH-end because the forfeiture of the biologic activity that the oxidation of Met causes people such as (, 1989) Kubiak, carried out alternative to Leu27 and Ash28 of Met27 and Ser28.Thereby, form the triamino acid that is expressed as GHRH-15/27/28 and substituted mutant.DPP IV is main Serum GH RH degrading enzyme (people such as Walter, 1980; People such as Martin, 1993).Worse pepx substrate also is replaced by Ile2 Ala2 (GHRH-TI) or Val2 (GHRH-TV) then by adopting GHRH15/27/28, or by changing Tyr1 and Ala2 into His1 and Val2[GHRH-HV (Figure 1A); H1V2A15L27N28] and produce.

Embodiment 2

DNA construct

[0124] in a specific embodiment, uses the plasmid of SEQ IDNO:9 (pSPc5-12-HV-GHRH) in the present invention.In another specific embodiment, used a plasmid vector, wherein this plasmid comprise pVC0289 main chain (SEQ ID NO:10), promotor as SEQ ID NO:6, GHRH cDNA such as pig HV-GHRH (mutant HV-GHRH cDNA) and 3 ' UTR as from people GH (SEQ ID NO:7).

[0125] for the biological efficiency of the pig GHRH cDNA sequence of checking sudden change, plasmid vector is reassembled as it can instructs the skeletal muscle expression of specific gene by the synthetic muscle promotor of new description, this promotor is SPc5-12, it contains from the nearside serum response element (SRE) of bone α-Ji Dongdanbai, multiple MEF-2 site, multiple MEF-1 site and TEF-1 binding site (people such as Li, 1999).After people GH cDNA 3 ' non-translational region, the pig GHRH fragment of 228-bp is incorporated in the myogenic GHRH expression vector by means commonly known in the art 31 amino acid whose signal peptides of pig GHRH fragment coding wherein and complete mature peptide pig GHRH (Tyr1-Gly40) and or GHRH mutant.(people such as Draghia-Akli, 1997) comprise the Sac I/BamH I fragment (people such as Li, 1999) of the 360bp of SPc5-12 synthetic promoter to plasmid pSPc5-12 in the Sac of pSK-GHRH main chain I/BamH I site.

[0126] site II external abruptly-changing system (Altered Sites II in vitro the MutagenesisSystem) (Promega of the pig GHRH cDNAs of wild-type and sudden change by changing with test kit; Madison WI) carries out rite-directed mutagenesis to people GHRH cDNA and obtains.People GHRH cDNA is gone on the corresponding site of pALTERPromega carrier as BamH I-Hind III fragment subclone, and suddenly change according to manufacturer's specification sheets.Pig wild-type cDNA obtains by changing human amino acid 34 and 38 with primer SEQ ID NO:2:5 '-AGGCAGCAGGGAGAGAGGAACCAAGAGCAAGGAGCATAATGACTGC-AG-3 ' from people cDNA.Pig HV sudden change is to make with primer SEQ ID NO:3:5 '-ACCCTCAGGATGCGGCGGCACGTAGATGCCATCTTCACCAAC-3 '.Pig 15Ala sudden change is to make with primer SEQ ID NO:4:5 '-CGGAAGGTGCTGGCCCAGCTGTCCGCC-3 '.Pig 27Leu28Asn sudden change is to make with primer SEQ ID NO:5:5 '-CTGCTCCAGGACATCCTGAACAGGCAGCAGGGAGAG-3 '.After sudden change, the clone of gained is as a result checked order confirming its exactness, and subsequently by well known to a person skilled in the art that the method subclone goes into the BamH I/HindIII site of the pSK-GHRH that describes in the present embodiment.

Embodiment 3

Cell cultures and transfection

[0127] experiment is equally successfully carried out in pig pituitary frontal lobe culture and former generation chicken sarcoplast culture.Yet, illustrate the data that pig pituitary frontal lobe culture generates.In former generation,, chicken sarcoplast culture obtained as described below.With Embryo Gallus domesticus tissue results, dissect and remove skin and cartilage and mechanically dissociate.Make cell suspending liquid pass through a kind of cheese cloth and lens wiping paper, then with 1 * 10 ⁸To 2 * 10 ⁸The density of/100mm plastic culture dish is carried out flat board and is cultivated.To still be present in the cell colony of suspension with 2 * 10 ⁶To 3 * 10 ⁶The density of the 100mm plastic culture dish that individual cell/collagen protein covers is carried out dull and stereotyped cultivate and at 5% CO ₂Carry out incubation in 37 ℃ in the environment.Before transfection, make cell with 1.5 * 10 then ⁶The density of/100mm flat board minimum minimum medium (Minimal Essential Medium) (MEM) middle plateform cultivate, the horse serum (Heat Inactivated Horse Serum) that has replenished 10% heat inactivation in this substratum (HIHS), 5% Embryo Gallus domesticus extract (CEE) (Gibco BRL; Grand Island, NY) and gentamicin.Further details are referring to people such as Draghia-Akli, and 1997 and people such as Bergsma, 1986.Pig pituitary frontal lobe culture obtains (people such as Tanner, 1990) substantially as described.Briefly, dissociate under the pituitary tissue enzyme condition, and cultivate time enough at the plastic ware upper flat plate and adhere to allowing.Then before experiment with this cell rinsing and be exposed in the incubation substratum.Details are referring to people such as Tanner (1990).

[0100] specification sheets according to the manufacturer comes transfectional cell with lipofection amine (lipofectamine) with the dull and stereotyped 4 μ g plasmids of every 100mm.After the transfection, the MEM that substratum is become the CEE that contains 2% HIHS and 2% is to allow cytodifferentiation.At differentiation back 72 hours results substratum and cell.The efficient of transfection is estimated as 10% by contrasting the dull and stereotyped sweet enzyme histochemistry of beta galactose.In the day before yesterday of results, with cell washed twice and substratum become MEM, 0.1% bovine serum albumin in Han Keshi balanced salt solution (HBSS).Conditioned medium is by adding 1% trifluoroacetic acid and the 1mM phenylmethylsulfonyl fluoride of 0.25 volume, freezing in-80 ℃, freeze-drying, at C-18 Sep-Columns (Peninsulalaboratories, Belmont, CA) go up purifying, again freeze-drying and be used for radioimmunoassay or be resuspended in the conditioned medium of former generation pig pituitary frontal lobe culture.

Embodiment 4

It is active and stable that GHRH superactivity analogue increases the GH secretogogue

[0129] becomes Skeletal Muscle Cell (skeletal myoblast) as embodiment 3, to carry out transfection, and the GHRH part of purifying the substratum cell of cultivating from condition is measured tethelin release in pig pituitary frontal lobe cell culture with each construct.Shown in Figure 1B, after 24 hours, collect and show GHRH kind (GH15/27/28 for modification with the quantitative substratum of pig specificity GH-radioimmunoassay; GHRH-TI; GHRH-TV) add up to 20% to 50% than wild-type pig GHRH suitable the increasing of GH excretory.Have only 1 to be that GHRH-HV has real increase in the secretogogue activity in 4 mutants, wherein pig GH level is increased to 1600ng/ml (Figure 1B) from baseline value 200ng/ml.

Embodiment 5

The blood plasma incubation of HV-GHRH molecule

[0100] from the contrast pig, collects the porcine blood plasma of concentrating and be stored in-80 ℃.The HV-GHRH of chemosynthesis is by the synthetic preparation of peptide.It is also centrifugal that porcine blood plasma is thawed, and places 37 ℃ to allow balance.The GHRH mutant is dissolved in the final concentration that reaches 100 μ g/ml in the plasma sample.Be right after after adding the GHRH mutant and after 15,30,60,120 and 240 minutes, get the blood plasma of 1ml and carry out acidifying with the 1M TFA of 1ml.Acidifying blood plasma is purifying on C18 sterile S EP-Pak post, freeze-drying is also used Walters 600 multisystem delivery systems, Walters intelligence sample processor, 717 types and Walters optical spectrum monitor device (spectromonitor) 490 (Walters Associates, MilliporeCorp., Milford MA) analyzes by HPLC.This detection is carried out at 214nm.Percentage ratio at the peptide of these time points degraded is measured by the peak method of masurement of integrating (integratedpeak measurements).

[0131] stability of wild-type GHRH and its analogue GHRH-HV incubation by the GHRH peptide in porcine blood plasma then, and solid phase extractions and HPLC analyze and check subsequently.Shown in Fig. 1 C, 95% wild-type GHRH (1-44) NH ₂Incubation was degraded in 60 minutes in blood plasma.On the contrary, the incubation of GHRH-HV in porcine blood plasma be presented at incubation during 4 to 6 hours at least 75% polypeptide be subjected to protection to the enzyme cutting.Thereby under identical condition, the major portion of GHRH-HV is still complete, and wild-type GHRH has then degraded fully, shows the quite high increase (Fig. 1 C) of GHRH-HV to the plasma proteins enzyme stability.

Embodiment 6

Zooscopy

[0132] in GHRH research, 3 groups have been used, every group of 5 3-4 week crossbreeding sow in age (barrow) (Yorkshire, Landrace, Hampshire and Duroc).This animal can be unrestrictedly near its body weight meals of water and 6% (24% protein pig feed, Producers Cooperative Association, Bryan, stable breeding TX) and separately.Every other day in the morning 8:30 animal is weighed, and add feed subsequently.This animal is kept according to NIH guide, USDA and Animal Welfare Act guide.

Embodiment 7

Give pig intramuscularly plasmid DNA

[0133] (CA) preparation pSPc5-12-HV-GHRH, pSPc5-12-wt-GHRH and pSPc5-12bgal are diluted to 1mg/ml in PBS (pH7) for Qiagen Inc., Chatsworth will not have endotoxic plasmid.Make animal equally distribute seance.With isoflurane (isoflurane) (concentration 2-6% be used to induce and 1-3% be used to keep) anesthesia pig.By operative procedure the jugular vein conduit is implanted in the animal to get blood on the 3rd, 7,14,21,28,45 and 65 after injection.When anesthesia, the 10mg plasmid is injected directly in the semitendinosus muscle of pig.Injected back 2 minutes, and the muscle of injection was placed between the cover clamp and to be 200V/cm with the suitableeest condition carry out electroporation people such as (, 1998) Aihara with 4 pulses of 60 milliseconds.In injection back 65 days, put to death animal and the muscle of internal and injection is collected, weigh, freezing and be stored in-80 ℃ in liquid nitrogen.Carcass is weighed and analyze with neutron activation.Measured the fat at back.

Embodiment 8

The intramuscular injection of pSP-HV-GHRH increased the blood plasma level of pig GHRH, GH and IGF-I in 2 months

[0100] having the pSP-HV-GHRH carrier of suitable protease resistant promote that GHRH is long-time expresses and stimulates the ability of GH and IGF-I secretion level to be determined.PSP-HV-GHRH and as the wild-type construct pSP-wt-GHRH of wild-type contrast with as the sweet expression of enzymes carrier of synthetic myogenic promotor intestinal bacteria beta galactose of placebo, the synoptic diagram of pSP-β gal is represented in Fig. 2 A.The boar of 3 castratings in age in week is anaesthetized, and the jugular vein conduit is inserted to collect blood sample under the situation that does not make the animal uneasiness.Plasmid expression vector DNA (DNA of the pSP-HV-GHRH of 10mg, pSP-wt-GHRH or pSP-β gal) is injected directly into semitendinosus muscle, then muscle is carried out electroporation (referring to embodiment 7).

Embodiment 9

Pig GHRH, GH and IGF-I measure

[0135] pig GHRH measures system with allogenic people (PeninsulaLaboratories, Belmont CA) measures.The sensitivity of this mensuration is the 1pg/ pipe.Pig GHRH in the blood plasma measures with specific double antibody program RIA (The PennsylvaniaState University).The sensitivity of this mensuration is the 4ng/ pipe.Pig IGF-I measures with allogenic people that (Diagnostic System Lab., Webster TX) measures.Data are analyzed with Microsoft Excel statistical study bag.The value of Xian Shiing is mean number ± s.e.m in the drawings.Specific p value obtains by comparing with the Studentst check.The level of p＜0.05 as statistically significant is set.In semitendinosus intramuscular injection in the pig body of pSP-HV-GHRH, the GHRH level increases (Fig. 2 B) back 7 days of injection, and on 14th be control level 150% (652.4 ± 77pg/ml is to 419.6 ± 13pg/ml).The pSP-HV-GHRH expression activity has reached maintenance level on 60th, this level is higher 2 ~ 3 times than the control value level of having injected placebo approximately.(1426.49 ± 10.47ng is to 266.84 ± 25.45ng) high 3 times (Fig. 2 C), and wherein this absolute magnitude has carried out proofreading and correct (blood volume account for TBW 8%) to the body weight of increase between 0 day and 60 days than the control value of having injected placebo by the absolute magnitude of the pig excretory Serum GH RH of pSP-HV-GHRH injection.The animal of wild-type pSP-GHRH injection is injected at the semitendinosus intramuscular, only began to show the suitable increase of GHRH level in back 45 days in injection, but in the back increase (779.36ng) that had 2 times on the 60th of injection, its level enough causes biological effect.

[0136] young animal has the high-level GH that reduces gradually with the age.To after the initial injection 7 and 14 days in 24 hours during in the blood sample got every 15 minutes carried out the mensuration of pGH level, and this level is extrapolated to total variation of pGH content.The pig (Fig. 2 D) of pSP-HV-GHRH injection is at back 7 days (δ variation HV=+1.52 of injection, wt=-0.73 to the contrast=-3.2ng/ml) and the injection back 14 days (6 variation HV=+1.09, wt=-4.42 to the contrast=-6.88ng/ml) show that its GH content significantly increases.

[0137] another of the whole body GH level of Zeng Jiaing is designated as the increase of IGF-I level.Porcine blood serum IGF-I level began to increase (Fig. 2 E) in about 3 days after injection in the pig of pSP-HV-GHRH injection.On 21st, the serum I GF-I level of these animals on average increased about 3 times, and this level is maintained to (p＜0.03) on the 60th.As a comparison, only in its circulation IGF-I level, has 40% increase (p=0.39) with the pig of wild-type pSP-GHRH expression vector injection, shown in Fig. 2 E.

Embodiment 10

Myogenic GHRH expression vector strengthens the growth of pig

[0100] growth that is secreted into the young boar that the pig GH of systemic circulation will castrate behind intramuscularly myogenic pSP-GHRH expression vector has increased by 65 days.Health form to measure in body, carried out in 30th and 65 after the injection (light densitometry, K40) or after death carrying out (organ, carcass, body fat, directly dissect and carry out the neutron activation chamber subsequently).The animal average specific placebo of wild-type pSP-GHRH injection weighs 21.5% (37.125kg is to 29.375kg), and the pig weightening finish 37.8% (41.775kg of pSP-HV-GHRH injection; P=0.014), as shown in Fig. 3 A.When comparing, changing efficient with the feed of the pig of GHRH construct injection has also improved 20% (per kilogram weight has increased for being 0.267kg food/sky at pSP-HV-GHRH, be 0.274kg in pSP-wt-GHRH, in the pig of pSP-β gal injection, being 0.334kg) (Fig. 3 B).Pass through light densitometry, the health composition research that carry out K40 potassium chamber and neutron activation chamber is presented at the proportional increase of all body compositions in the animal of GHRH injection, and does not have internal organs enlargement (organomegaly), body fat correlation proportion and relevant pathological sign.The photo of pig after 45 days of the contrast pig of placebo injection and pSP-HV-GHRH injection is shown in Fig. 3 C.

[0139] the metabolism overview the pig of the pSP-HV-GHRH shown in Table I injection means that the remarkable reduction of pSP-GHRH and pSP-HV-GHRH serum urea level separately (is 9 ± 0.9mg/dl in the contrast, in the pig of injection is 8.3 ± 1mg/dl and 6.875 ± 0.5mg/dl) (p=0.006), shows the amino acid metabolism that reduces.Serum level of glucose is that similar (in the contrast pig is 99.2 ± 4.8mg/dl between the pig of contrast and plasmid GHRH injection, in the pig of pSP-HV-GHRH injection is 104.8 ± 6.9mg/dl, and is 97.5 ± 8mg/dl) (p＜0.27) in the pig of wild-type pSP-GHRH injection.Do not find other metabotic change.

The pig of Table I: GHRH injection and the metabolism overview of contrast (value is mg/ml).

Glucose urea creatinine gross protein

Contrast 99.2 ± 4.8 9 ± 0.9 0.82 ± 0.06 4.6 ± 0.22

pSP-wt-GHRH??97.5±8?????8.3±1??????0.83±0.056??4.76±0.35

pSP-HV-GHRH??104.8±6.9??6.875±0.5??0.78±0.04???4.88±0.23

Embodiment 11

The experiment of different levels pSP-HV-GHRH

[0140] in order further to study the effect of pSP-HV-GHRH, the group of 2 piggys was carried out the injection (3mg, 1mg, 100 micrograms) of pSP-HV-GHRH in back 10 days with new injection-type six needle-array electrodes (six needle-array electrode) in birth to the piggy growth.These electrodes are checked in advance, and higher 10 times than clamp electrode efficiency well known in the art.Therefore, pin electrode is preferred in the method for the present invention.As shown in Figure 4, the group of injecting with 100 microgram plasmids has shown best growth curve, has significant significant difference behind 50 ages in days compared with the control.Developing antibody and showing significantly reduced growth pattern with an animal in the group of 3mg injection.

[0141] similarly, the groups of 2 piggys is injected with specified pSP-HV-GHRH dosage back 10 days of birth.The IGF-I value began to increase in injection in back 10 days, and contrasted high 10.62 times at back 35 days IGF-I average specifics with the pig of 100 microgram plasmids injection of injection.With the pig comparison of 1mg injection according to 7.94 times of mean heights, and with the pig of 3mg injection than 1.16 times of control value mean heights.

[0142] thereby, in a specific embodiment, injected pSP-HV-GHRH than low dosage.In a specific embodiment, used the plasmid of about 100 micrograms (.1 milligram).In another specific embodiment, injected about 200-300 microgram.In the another one embodiment, used the 50-100 microgram.

Embodiment 12

The age age ratio of pSP-HV-GHRH

[0143], the groups of 2 piggys is begun to inject with the pSP-HV-GHRH of 2mg after birth for the age optimization of the piggy that is used in pSP-HV-GHRH injection.As shown in Figure 6, shown best growth curve in the group of back 14 days of birth injection, this curve all has significant significant difference with comparing on each time point.An animal in the group of injection on the 21st has been developed antibody and has shown significantly reduced growth pattern.If handle and too early to have insulin resistance (promptly＜about 10-14 age in days).In a specific embodiment, when this treatment is minimum in natural GH and IGF-I level the most effective (being about life 10-14 day), and may not reach the target of imagination during for normal height when the GHRH level.In a specific embodiment, suppose the immunosurveillance system when gestation time reduces, compare with not conceived animal, then the reduction of the antibody number of in the animal of pregnancy, producing for the GHRH that modifies.

Embodiment 13

Specific embodiment

[0144] in a word, the suitableeest time point that is used to inject is back 14 days of birth (40 per day comparisons are according to weighing 8 pounds (p＜0.04) after injection).The preferred dose that is used for injecting is 2-5ml volume 100 microgram plasmids (40 per day comparisons are according to weighing 6 pounds (p＜0.02) after injection).Hormone normally and with weight increases relevant (IGF-I, IGF-BP3, Regular Insulin, urea, glucose, gross protein, creatinine) with the biological chemistry constant in the offspring of sow 1 (time course) and sow 3 (dose curve), and does not have deleterious side effect.The health composition research of previous experiments shows that HV-GHRH has determined consistent the increasing (health is formed similar but bigger to contrast) of all body portion, and wt-GHRH has determined the increase of lean mass and the reduction of fat.

[0145] increase of supposing tethelin can cause the increase of body temperature, and in a preferred embodiment, sow is to inject under about 62 about conditions of 80 in temperature.

Embodiment 14

Before the first nest son, give conceived sow with Ghrh myogenic vector injection

[0146], the carrier that the sow of pregnancy contained GHRH at last 3 months of gestation with 10mg is injected in order to measure the growth effect of GHRH myogenic carrier.In this certain embodiments, sow (～800 pounds) is injected with in its conceived for the first time gestation 90 days of the pSP-HV-GHRH carrier of 10mg.Delivering method can be any as known in the art any one.In a specific embodiment, except use was used for the clamp electrode of electroporation, this plasmid was sent as embodiment 7 (Fig. 7).This electrode has 6 pin 22g, and these pin length are on 2cm and the round plastic support that is positioned at diameter 1cm.

[0147] table 2 has illustrated the piggy weight (kg) in time of the sow production of injecting with pSP-HV-GHRH (p2) by electroporation gestation 90 days.Table 3 has illustrated the weight (kg) of the control animal of sow (p3) production of not injecting at the phase same date.The health of the piggy of the sow of table 4 expression pSP-HV-GHRH injection and the sow of not injecting is formed data (fatty %/body weight (BW)/d mean number).On behalf of fat, this table the relative proportion of body weight is shown that also the fat of the piggy per unit weight of the sow of injecting has reduced 18.5%.Before the acquisition health is formed data, pig p1/2 and p2/6 are put to death.Biochemical data of piggy and conceived illustrated similar (referring to the embodiment 15) second time of this sow.P value highly significant all on all time points.These tables are presented at piggy that sow that its Gestation period injects with pSP-HV-GHRH produces significantly, and to compare the piggy that produces according to sow significantly heavier.Do not limiting the scope of the invention and do not giving under the situation that boundary of the present invention and scope use restraint, the GHRH that the application's conjecture is injected into muscle cell is secreted and is passed through placenta.To the hypertrophy of hypophysis and the result of proliferative effect, the number that discharges the pituicyte of GH has increase as GHRH.

Embodiment 15

The second nest son of the sow of injection

[0148] table 5 has illustrated the second nest son's of sow weight data, and this sow is injected with pSP-HV-GHRH when it is conceived for the first time.

Table 5: the piggy health is formed over time

1 day 5/,4/2,000 5/,8/2,000 5/1,1/2,000 5/1,6/2,000 5/,18/,200 5/2,3/2,000 7/13/2000 May of April 27

Sow 2 the 1st day the 5th day the 7th day the 11st day the 14th day the 19th day the 21st day the 26th day the 77th day

Pig 1 2.097 3.26 4.22 5.627 6.505 8.4 9.1 10.75 36.32

Pig 2 2.264 3.512 4.46 5.882 6.799 8.7 9.4 11.25 37.228

Pig 3 1.758 2.78 3.68 4.817 5.7 7.5 8.25 10.25 35.866

Pig 4 1.895 2.843 3.62 4.733 5.714 7.1 7.6 8.9 32.234

Pig 5 2.397 3.458 4.24 5.704 6.692 8.85 9.6 11.35 39.498

Pig 7 2.457 3.599 4.68 6.132 7.05 8.9 9.65 11.55 37.682

Pig 8 1.907 2.882 3.58 4.767 5.593 6.95 7.55 9.65 36.32

Pig 9 2.381 3.52 4.23 5.635 6.45 8.25 8.9 10.65 34.504

Pig 10 2.473 3.655 4.57 5.935 6.87 8.6 9.25 10.7 39.952

Average 2.181 3.2787 4.14222 5.47022 6.37478 8.13889 8.81111 10.56111 36.62267

STDEV?????0.2733???0.3509??0.41817???0.54711???0.55986????0.75778????0.81616???0.85322????2.3808

SE????????0.1933???0.2481??0.29569???0.38686???0.39588????0.53583????0.57711???0.60332????1.68348

Increase by 0 1.0977 1.96122 3.28922 4.19378 5.95789 6.63011 8.38011 34.44167

Amount to (kg) 19.629 29.509 37.28 49.232 57.373 73.25 79.3 95.05 329.604

Pound 43.183 64.919 82.016 108.3104 126.2206 161.15 174.46 209.11 725.1288

Increased by 0.32231 0.44729 in average day

[0149] at the second nest son's pregnancy duration or do not carry out GHRH afterwards and use.Second nest is young with regard to (piggy of other sows of raising under similar environment weight in average at birth is 1.71kg greatly when birth; And these piggys average out to 2.181kg at birth).On 21st, all weight of piggy that are characterized as the nest son of this kind and mean level (ML) add up to～130 pounds (～59kg), and in the past with the piggy of the sow of pSP-HV-GHRH injection add up to 174 pounds (～79kg).This advantage is maintained, and compares average big 11-15 pound (5.5-6kg)/pig with optimal cases in this kind known in the field in back 77 days this weight of birth.In birth back 169 days, the animal average specific of injection contrasted big 22 pounds (10kg), p＜0.0007.

[0150] sow is only anaesthetized in injection/electroporation program, and they have been used the TELAZOL of 2.2mg/kg dosage ^(mixture of ring thiophene ethamine oxyhydroxide and azoles fluorine nitrogen oxyhydroxide).For piggy, estimating combination that health has used ketamine/xylazine HCl when forming to anaesthetize, piggy must be (DEXA) quiet lying on the back about 15 minutes on the machine of double X-ray spectrodensitometry instrument (Dual X-ray Densitometry) at that time.Particularly, use ketamine 20mg/kg+ xylazine 1mg/kg (regular xylazine dosage is 2mg/kg).In another specific embodiment, use other narcotic well known in the art, as ketamine 15mg/kg+ vetrnquil 0.4mg/kg.In the specific embodiment of another one, need not piggy anaesthetized can take a blood sample, injection etc.

[0151] supposes that other animal of pig and some is responsive and can then use coromegine sometimes by the main change of its temperature regulation process after anesthesia dead (hypothermia or hyperpyrexia, the latter is more frequent) to dissimilar narcotic usually.Coromegine is an anticholinergic agents, it uses before anesthesia usually and thinks that it can promote the drying of secretory product and reduces essential narcotic amount, prevents in this program that heart rate is disorderly and increase the level of comfort of animal in anesthesia recovery, has also reduced the frequency of disadvantageous unusual hot outbreak.In specific embodiment, carry out pre-treatment with 0.05mg/kg subq (subcutaneous) coromegine.Other similar medicines well known in the art can be used as atropinic alternative.

[0152] piggy having been carried out multiple biological chemistry measures.Table 6 provides the data that relate to these measurements to 12.Regular Insulin experiment (table 6) is measured at 5-25-00.The mean number of the control group that all are checked previously is 6.8 μ U/ml, and the experiment piggy mean number be 4.785 μ U/ml, no significance,statistical (p=0.07).

Table 6: the insulin concentration of piggy

The 25th day

Pig 1 4.3827

Pig 2 4.131

Pig 3 4.8176

Pig 4 5.7899

Pig 5 4.4267

Pig 7 4.3076

Pig 8 4.1648

Pig 9 6.0921

Pig 10 4.9527

Average 4.78501

STDEV??????????0.71397

SE?????????????0.23799

[0153] IGF-I is determined at 5-25-00 and carries out (referring to table 7).The mean number of experimental group is 145.509ng/ml, and the mean number of all control groups of checking previously is 53.08ng/ml.Therefore, p value highly significant (p＜0.0001).Suppose that GH has stimulated production and the release of IGF-I, then this IGF-I measures indication and the use so usually in the art that the GHRH level increases.

Table 7: the IGF-I concentration of piggy

The 1st day the 10th day the 18th day the 25th day

Pig 1 290.46 118.63 185.01 356.02

Pig 2 265.7 115.62 117.99 172.28

Pig 3 109.27 77.389 200.75 109.99

Pig 4 94.689 36.746 93.795 65.113

Pig 5 155.98 95.946 138.24 179.3

Pig 7 171.41 19.463 213.29 226.43

Pig 8 178.3 101.55 98.478 165.88

Pig 9 104.86 78.872 84.7 77.214

Pig 10 262.4 131.36 206.23 138.99

Average 181.4521 86.17511 148.7203 165.6908

STDEV????74.91415????37.61337????52.67175????87.96496

SE???????24.97138????12.53779????17.55725????29.32165

[0154] for table 8, IGF-BP3 (IGF-conjugated protein 3) immunoradiometric assay(IRMA) (IRMA) is checked at 5-25-00.IRMA adopted two sites immunoradiometric assay(IRMA) (referring to Miles LEM, Lipschitz DA, Bieber CP and Cook JD: the immunoradiometric assay(IRMA) by two sites is to the measurement of serum ferritin.Analyt Biochem 61:209-224,1974) IRMA is non-competitive assay, therein the analyte that will measure " by clamping (sandwiched) " between two antibody.First antibody is fixed in the inwall of test tube.Another antibody has carried out radio-labeling to survey.Be present in analyte in unknown, standard and sample contrast by two kinds of antibodies to form " sandwich (sandwich) " mixture.Unconjugated material is removed by decant and washing to test tube.Measurement in the table 8 comprises correction factor x50.Table 8 has illustrated that the mean number of experimental group is 238.88, and the mean number of the control group that all are checked previously is 205.44ng/ml.The significance,statistical that p＜0.048 is arranged.

Table 8: the IGF-BP3 concentration of piggy

The 1st day the 10th day the 18th day the 25th day the 1st day the 10th day the 18th day the 25th day

Pig 1 7.9841 3.917 7.1657 3.5957 399.205 195.85 358.285 179.785

Pig 2 7.5463 3.4327 3.3382 4.4706 377.315 171.635 166.91 223.53

Pig 3 3.4187 4.9039 6.7961 6.3021 170.935 245.195 339.805 315.105

Pig 4 5.6354 4.2184 3.8551 1.9101 281.77 210.92 192.755 95.505

Pig 5 4.282 4.5592 5.2783 3.8224 214.1 227.96 263.915 191.12

Pig 7 3.7328 4.4454 2.9426 4.8232 186.64 222.27 147.13 241.16

Pig 8 5.4265 3.3285 4.1714 7.1258 271.325 166.425 208.57 356.29

Pig 9 3.7912 5.6354 3.9117 6.7643 189.56 281.77 195.585 338.215

Pig 10 4.7668 5.6099 5.24 3.8474 238.34 280.495 262 192.37

Average 5.17598 4.45004 4.74434 4.74018 258.7989 222.5022 237.2172 237.0089

STDEV???1.652????0.83658??1.48489??1.70536??82.6??????41.8289???74.24472??85.2679

SE??????0.55067??0.27886??0.49496??0.56845??27.53333??13.94297??24.74824??28.42263

[0155] table 9 has illustrated the concentration (g/dl) of gross protein.The mean number of experimental group is 5.3g/dl, and the mean number of the control group that all are checked previously is 4.02g/dl.The very high significance,statistical that p＜0.0001 is arranged.

Table 9: the total protein concentration of piggy

The 1st day the 10th day the 18th day the 25th day

Pig 1 5.7 5.9G.H. 5.5

Pig 2 5.3 5.6 5.5 5

Pig 3 5.2 5.3 5.3 5.4

Pig 4 5.3 5.5 4.9 5.4

Pig 5 5.8 5.3 5 5.4

Pig 7 5.6 5.4 5.3 5.2

Pig 8 4.5 5G.H. 4

Pig 9 5.3 5.1 5.3 5.2

Pig 10 6.3 5 5.2 5.5

Average 5.44444 5.34444 5.21429 5.17778

STDEV??0.49526??0.29627??0.20354??0.47111

SE?????0.16509??0.09876??0.06795??0.15704

[0156] table 10 has illustrated the concentration (mg/dl) of creatinine.The mean number of experimental group is 0.936mg/dl, and the mean number of the control group that all are checked previously is 0.982mg/dl.No significance,statistical (p＜0.34), this is the indication of normal renal function.

Table 10: the creatinine concentration of piggy

The 1st day the 10th day the 18th day the 25th day

Pig 1 0.75 0.96G.H. 1.14

Pig 2 0.73 1.03 0.98 1.46

Pig 3 0.69 0.92 0.95 1.1

Pig 4 0.65 0.94 1.18 1.18

Pig 5 0.64 0.8 0.91 0.92

Pig 7 0.72 0.93 1.02 1.12

Pig 8 0.68 0.9 0.83 1.2

Pig 9 0.68 0.87 1 1.07

Pig 10 0.74 1.02 1.02 1.03

Average 0.69778 0.93 0.98625 1.13556

STDEV??0.0393???0.07124??0.10113??0.14783

SE?????0.0131???0.02375??0.03371??0.04928

[0157] table 11 has illustrated BUN (blood urea level) (mg/dl).The mean number of experimental group is 3.88mg/dl, and the mean number of the control group that all are checked previously is 8.119mg/dl.The significant significance,statistical that p＜0.0012 is arranged.

Table 11: the BUN concentration of piggy

The 1st day the 10th day the 18th day the 25th day

Pig 14354

Pig 24336

Pig 36657

Pig 45345

Pig 53233

Pig 73333

Pig 82357

Pig 93344

Pig 10 3334

Average 3.66667 3.22222 3.88889 4.77778

STDEV???1.22474????1.09291???0.92796????1.56347

SE??????0.40825????0.3643????0.30932????0.52116

[0158] concentration (mg/dl) of table 12 expression glucose.The mean number of experimental group is 123.23mg/dl, and the mean number of the control group that all are checked previously is 122.8mg/dl.No significance,statistical (p＜0.67), term G.H. represents total hemocytolysis; In these embodiments the biological chemistry constant determined it is impossible.

Table 12: the glucose concn of piggy

The 1st day the 10th day the 18th day the 25th day

Pig 1 117 115G.H. 115

Pig 2 112 137 130 119

Pig 3 133 138 143 115

Pig 4 125 127 132 90

Pig 5 115 123 133 120

Pig 7 114 120 123 115

Pig 8 126 123G.H. 116

Pig 9 118 129 124 119

Pig 10 142 134 136 112

Average 122.4444 127.3333 131.5714 113.4444

STDEV??9.98888????7.88987????6.90066????9.15302

SE?????3.32963????2.62996????2.30022????3.05101

[0159] illustrated as these tables, IGF, IGF-BP3 have increased (as the result that the GH axle is stimulated), and urea and gross protein reduce and increase (signal that this is the protein metabolism of improvement) respectively, yet Regular Insulin and glucose are kept normally.The normal level of Regular Insulin and glucose is an advantage of the present invention, because classical GH treatment has produced " diabetes " sample situation with hyperglycemia.Normal in this experiment creatinine is the parameter that is used to measure renal function, in animal sometimes under unsuitable metabolism condition renal function weakened.

[0160] thereby, in a specific embodiment, by having shown than normal level or not with the growth of the animal that sow the produced increase of the DNA injection of any type of GHRH of coding at first therein with the piggy that produces in the repeatedly pregnancy after the sow pregnancy of pSP-HV-GHRH injection.The pregnancy of pig continues about 114 days, and the time of nursing of allowing allows no more than 2 pregnancy/years.

[0161] in a specific embodiment, it is relevant with the increase of the about 25-50% of GH production cell that the nucleotide sequence of coding GHRH is applied to female or mother.

[0162] in another embodiment, the sow of non-pregnancy is injected before pregnancy.

[0163] in another embodiment, replace using of pSP-HV-GHRH carrier of the present invention, can use other growth hormone releasing hormone analogs known in the field.For example, use the GHRH of wild-type.This experiment is carried out to be similar to instruction provided herein.

[0164] in another embodiment, the hypophysis from piggy is variation that collect and that measure hypophysis content when putting to death piggy.That is, piggy reach market weight (～100kg) time with its execution and collect hypophysis.This mensuration comprises the hypophysis relative content (relative proportion of the cell of secretion tethelin, prolactin, follicle stimulating hormone (FSH) etc.) of dissimilar hormone secretion cells.

Embodiment 16

Extra experiment

[0165] in a specific embodiment, with more sow according to appointment 20 use with embodiment 14 and 15 in the same or analogous processing that provides inject.Multiple plasmid amount is tested, and as from 100 micrograms to 10 milligram, and each handles the group of using 5 pigs.Goner and the offspring of the sow of not injecting compare.In a specific embodiment, these experiments are carried out in the farm, so these data can the Documentary Records stdn.

Embodiment 17

The optimization experiment

[0166] in order when for the first time conceived, to determine the suitableeest frequency injection, used conceived rat.The gestation of rat continues about 21 days.Conceived rat begins to inject 5 days and 18 days of gestation, and its offspring different time point after birth is tested.Specific experiment comprises that weight, health are formed and the hypophysis relative content of dissimilar hormone secretion cells (relative proportion of the cell of secretion tethelin, prolactin, FSH etc.).

Embodiment 18

Increase the method that milk is produced

[0167] in one embodiment of the invention, a kind of method (being also referred to as lactation) that milk is produced that increases is arranged, this method comprises the step of under the following conditions carrier of significant quantity being introduced zooblast, the nucleotide sequence of growth hormone releasing hormone of wherein encoding expressed and wherein this carrier comprise promotor, the nucleotide sequence of this growth hormone releasing hormone of encoding and being operably connected so that this nucleotide sequence carries out 3 ' non-translational region of functional expression, and wherein the introducing and the expression of this carrier cause the increase that milk is produced in the animal.In a specific embodiment, this animal behaviour, cow, pig, goat or sheep.

[0168] carrier that will comprise GHRH by the method for describing herein is incorporated into the production that increases animal milk in the animal.In a specific embodiment, this animal is female or mother or conceived female.In further specific embodiment, this offspring female or mother grows very fast in 2 initial approximately week owing to the increase of female or mother's milk production.As discussing herein, the increase of milk production is to take place when the nucleic acid shot animal of the GHRH that will encode.

[0169] technician knows how to measure the increase that milk is produced, as in U.S. Patent No. 5,061,690; 5,134,120 and 5,292,721 or people such as Peel, J.Nutr., 1981, the method among the 111:1662.

[0170] milk sample is when farrowing (colostrum) and 13 days of lactation and 20 days are artificial expresses.Used the intramuscularly (except that collecting colostrum) of 40IU pitocin and as quickly as possible two glands of every sow have been milked up to there not being more milk.To deposit in 2 bottles with sanitas such as potassium bichromate from the sample thorough mixing and the aliquots containig of two glands.Bottle is freezing up to analyzing.Milk fat, dry-matter and protein are determined according to the standard program such as A.O.A.C. (1980) program of this area.In a specific embodiment, milk lactose semi-automatic (27 type industrial analysis instruments, YellowSprings Instrument Co., Inc., Yellow Springs, Ohio) enzyme program (schedule of operation OP-025, Monsanto Co., St.Louis Mo.) analyzes.In a specific embodiment, the output of every sow milk was passed through on 13 and 20 to determine with a hour interval weighing pig before and after the nurture of describing as people (1971) such as people such as Lewis (1978) and Mahan.Note and prevent from this moment or consider to urinate and drain and lose.In a specific embodiment, initial 2 nurture phases are used for making sow and the young calculating that adapts to and be not included in milk production every day of nest.Milk production multiply by 4 by the output that will obtain and calculates in subsequently 6 hours.

Embodiment 19

Other embodiment

[0171] in another embodiment of the invention, the part of secretagogue receptor (GHS-R) obtains similar result to sending of GHRH nucleic acid.The technician knows many different GHS-R ligand structure types well known in the art, and they all play a role by GHS-R.Example comprise MK-0677 from Merck (Whitehouse Station, NJ), GHRP-6 (summary referring to Bowers, 1998) and endogenous ligands ghrelin (people such as Kojima, 1999; Dieguez and Casanueva, 2000).Other comprise hexarelin (Europeptides), L-692,943 (Merck ﹠amp; Co.; Whitehouse Station, NJ), NN703 (Novo Nordisk; Bagsvaerd, Denmark) or any compound that serves as the antagonist of GHS-R acceptor, they are all known (referring to as people such as Pong (1996) by the technician; People (1997) such as people such as Howard (1996) or Smith).

[0172] technician knows that GHS-R is in the upstream of GHRH and increase the release of GHRH from pituitary body.In a specific embodiment, GHS-R part dosage forms for oral administration (as by being added in feed and the tap water), this will amplify the effect that GHRH causes GH to discharge from pituitary body.In the present embodiment, GHRH delivery of nucleic acids of the present invention can more be strengthened.Under the situation that does not limit the scope of the invention, it is that extra GHRH increases pit-1 (a kind of relate to prepituitary gland GH produces cell, the developmental transcription factor of somatotroph in embryo's generating process) and expresses that the present inventor proposes mechanism of action likely.The activation of GHS-R also increases the expression of pit-1.The expression of pit-1 is also increased by cAMP, and the GHS-R part increases the amount of the cAMP that produces in response to GHRH.Therefore, pig has the somatotroph concentration of increase at birth probably.Therefore, pig produces more GH.Therefore, in a specific embodiment, sending with at least a GHS-R is ligand united of GHRH nucleic acid of the present invention used.The GHS-R part is used with the medicine acceptable composition.

[0173] all patents mentioned in specification sheets and publication all are the indications of the level of various equivalent modifications of the present invention.All patents and publication are all quoted as a reference herein, as specific or quote each independent publication individually as a reference.

Embodiment 20

Use the multiple action of GHRH to sow and offspring

[0174] in one object of the present invention, the GHRH that dystopy is produced in the animal of pregnancy for example passes to the offspring by placenta and strengthen the production of GH in the descendant, and its offspring shows the health composition of the growth and the change of increase then.The sow of being injected simultaneously, significantly produces more milk.

[0175], the sow of 6 pregnancies is injected with 10mg plasmid DNA pSP-HV-GHRH (n=4) or pSP-wt-GHRH (n=2) gestation 95 days for estimating that GHRH myogenic vector injection large mammals sends effect to the sow lactation to the effect of offspring's growth and GHRH.Recently, in rodents and large mammals, utilize electroporation technology to express and obtained significant progress (people such as Bettan, 2000 to take in the body that strengthens plasmid muscle being used for different position gene; People such as Draghia-Akli, 1999; People such as Mir, 1999).In this case, carry out electroporation with the 6-needle-array electrode after the plasmid injection, and condition is as describing with people such as (, 1999) Draghia-Akli herein.With 6 sows that are complementary with comparing.This animal produced in 24 hours in succession.132 piggys in research subsequently, have been analyzed altogether.

[0176] the known processing that 2 weeks carried out in order to the reorganization GHRH of drug administration by injection before childbirth has increased the weight of pig and has improved surviving rate people such as (, 1992) Etienne of pig when 13 days and wean just.In this case, significantly big at birth (average out to 1.65 ± 0.06kg HV-GHRH, p＜0.00002 and 1.46 ± 0.05kg wt-GHRH, p＜0.0014, and of the piggy of the sow of GHRH injection to contrasting 1.27 ± 0.02kg) (Fig. 8).

[0177] piggy was weaned on 21st, and analyzed the killing-out weight amount in back 170 days in birth.The piggy of the sow of injection is average weight gain 18% (Fig. 9) when wean just.Half of each nest son and the sow (from the piggy of contrast sow) of contrast sow (from the piggy of the sow of injecting) or injection are raised together with.What is interesting is, with the animal contrast of raising together with of injection significantly than its littermate big (reaching 12.2%), p＜0.02 (Figure 10).The milk production that the sow of the change of weight indication injection significantly increases in the control animal that the animal of being handled by GHRH is raised together with.Yet, tend to (Figure 11) from the sow of GHRH-processing and the piggy of raising together with by the contrast sow than its littermate little (reaching 5.8%), but it is remarkable to be worth statistics, and the offspring of the animal that this explanation GHRH handles has the growth of endogenous variation and increase at its hypothalamic pituitary axis.Compare total increase as shown in figure 12 with contrast (raising) with the contrast sow.

[0178] this advantage can be maintained to market weight; For HV-GHRH and wt-GHRH difference average out to 135.7 ± 1.89kg and 129.3 ± 2.17kg, is 125.3 ± 1.74kg (Figure 13) and contrast weighted average this weight on the 170th.This weight differential puts at any time that all statistics is remarkable, and the p value is 0.05 and 10 ^-5Between.

[0179] carried out multi-biological chemical measurement (table 13a and 13b).As the anabolic signal that increases, gross protein and albumin concentration (g/dl) show increase in experimental group.Gross protein has increased by 8%, and albumin has increased by 7.5%, and at the time point of test fine difference (at postnatal 50 and 170 days) (table 13a and 13b) is arranged.

Table 13a

The 50th day	Total protein	White protein
The 50th day	Total protein	White protein	Contrast	????5.209+/-.379	????3.207+/-.411
?WT-GHRH	????5.617+/-.298	????3.639+/-.301	Contrast	????5.209+/-.379	????3.207+/-.411
?WT-GHRH	????5.617+/-.298	????3.639+/-.301	The P value	????p＜4.3037E-05	????p＜4.83477E-05
?HV-GHRH	????5.533+/-0.291	????3.415+/-0.291	The P value	????p＜4.3037E-05	????p＜4.83477E-05
?HV-GHRH	????5.533+/-0.291	????3.415+/-0.291	The P value	????p＜1.52284E-05	????p＜0.003470198

Table 13b

The 170th day	Total protein	White protein
The 170th day	Total protein	White protein	Contrast	????7.07+/-0.56	????3.82+/-0.39
??WT	????7.68+/-0.31	????4.07+/-0.38	Contrast	????7.07+/-0.56	????3.82+/-0.39
??WT	????7.68+/-0.31	????4.07+/-0.38	The P value	????p＜4.045E-06	????p＜0.04199035
??HV	????7.33+/-0.29	????4.01+/-0.20	The P value	????p＜4.045E-06	????p＜0.04199035
??HV	????7.33+/-0.29	????4.01+/-0.20	The P value	????p＜0.00609905	????p＜0.00423639

[0180] normally (0.936mg/dl relative comparison 0.982mg/dl, p＜.34), this is the indication of normal renal function to creatinine concentration (mg/dl).

[0181] glucose concn is at the time point all normal (table 14a and 14b) of all checks.

Table 14a

The 50th day	Glucose
The 50th day	Glucose	Contrast	????99.36+/-12.03
????WT-GHRH	????98.5+/-10.11	Contrast	????99.36+/-12.03
????WT-GHRH	????98.5+/-10.11	The P value	????p＜0.76483343
????HV-GHRH	????98.41+/-10.63	The P value	????p＜0.76483343
????HV-GHRH	????98.41+/-10.63	The P value	????p＜0.67921581

Table 14b

The 170th day	Regular Insulin	Glucose
The 170th day	Regular Insulin	Glucose	Contrast	????14.79+/-9.23	????78.68+/-19.01
??WT	????10.16+/-2.13	????81.14+/-8.90	Contrast	????14.79+/-9.23	????78.68+/-19.01
??WT	????10.16+/-2.13	????81.14+/-8.90	The P value	????p＜0.00548803	????p＜0.49606217
??HV	????15.55+/-11.64	????81.11+/-10.52	The P value	????p＜0.00548803	????p＜0.49606217
??HV	????15.55+/-11.64	????81.11+/-10.52	The P value	????p＜0.76677483	????p＜0.44978079

[0182] insulin level is normal.The normal level of Regular Insulin and glucose is an advantage, because classical GH treatment produces situation people such as (, 1990) Pursel of " diabetes " sample of hyperglycemia.

[0183] among the offspring of the sow that is being subject to processing of the surviving rate in whole research significantly higher (table 15).Sickness rate significantly reduces in the group of handling.

Table 15

The classification of pig	Sum # pig	The pig of # death	% death	Pathology		Clinical note
The classification of pig	Sum # pig	The pig of # death	% death	Pathology		Clinical note	Contrast	63	?7	?11.11	Sudden death	1
Prolapsus	1										Sudden death	1
Prolapsus	1		Lame	1	Back leg
Enteritis			Lame	1	Back leg
Enteritis		1	7/26 prolapsus-10/10 enteritis
Arthroncus		1	7/26 prolapsus-10/10 enteritis
Arthroncus		2	Tenderfooted Hermiths	8/30 abscess
The ulcer of bleeding		2	Tenderfooted Hermiths	8/30 abscess	1	Consumption-anaemia
The ulcer of bleeding	WT-GHRH	18	?1	?5.56	1	Consumption-anaemia	Sudden death	1
HV-GHRH	WT-GHRH	18	?1	?5.56	42	?2	Sudden death	1		?4.76	Sudden death	1
	Lame	1	8/21 hinders leg fights								Sudden death	1

[0184] different with the injection of the somatotropin (rpST) of recombinating with pig, this injection can cause the cavity of hemorrhagic ulcer, liver and kidney to form or even the death of sow (people such as Smith, 1991), the GHRH gene therapy can well be tolerated, and does not see side effect in animal.Also notice among the offspring of the animal that being grown in of this increase is subject to processing to obtain, and do not have the GHRH plasmid there.Shift by transgenosis, sarcoplast before the plasmid of the specific hGH of containing of tissue/fiber type that regulates or liposome-mediated intravenous injection is used for host in livestock and GH-defective and carries out sending of GH and stably manufactured (people such as Dahler, 1994; People such as Pursel, 1990; Barr and Leiden, 1991).Yet these technology have significant disadvantage is excluded in outside large-scale operation and/or the edible animal it: 1) possible toxicity or the immune response relevant with liposome delivery; 2) in transfection sarcoplast method, need isolated operation widely; And/or 3) important side effect or inefficient danger in transgenosis (people such as Mililer, 1989; People such as Dhawan, 1991).Compare with these technology, the plasmid DNA injection is simple and effective, and does not have the complication that is relevant to delivery system or overexpression.

[0185] data presentation enhanced biological efficiency provided herein realizes that in the offspring of the large mammals of GHRH plasmid injection this usefulness increases GH production and excretory physiological level, reduces mortality ratio and sickness rate.The sow that is subject to processing shows significantly higher milk production.How the offspring piggy is treated side effect and has normal biological chemistry overview without successive, does not also have relevant pathology or internal organs enlargement.The remarkable enhancing of growth show the ectopic expression of myogenic GHRH carrier will substitute probably classical therapeutic modality also can physiology on more suitable mode irritate the GH axle.Show that in pig high stability and the secreting active HV-GHRH molecule of GH also can be used for other Mammalss, because the serum protein enzyme of degraded GHRH is similar in most of animals.

[0186] Xia Mian paragraph has been described the materials and methods of present embodiment.

[0187] DNA construct.Plasmid pSPc5-12 comprises the Sac I/BamH I fragment (people such as Draghia-Akli, 1997) of the SPc5-12 synthetic promoter of the 360bp that is positioned at pSK-GHRH main chain SacI/BamH I site.Wild-type pig GHRH is by becoming Arg to people GHRHcDNA (1-40) OH at position 34:Ser, and 38:Arg becomes Glu and carries out rite-directed mutagenesis and obtain; The pig HV-GHRH DNA of sudden change is by becoming His to people GHRH cDNA (1-40) OH at position 1:Tyr, 2:Ala becomes Val, 15:Gly becomes Ala, 27:Met becomes Leu, 28:Ser becomes Asn, 34:Ser becomes Arg, 38:Arg becomes Glu and carries out rite-directed mutagenesis and obtain (the change external abruptly-changing system of site II (AlteredSites II in vitro Mutagenesis System), Promega, Madison, and be cloned into the BamH I/Hind III site of pSP-GHRH WI).After this GHRH cDNA is 3 ' non-translational region of human growth hormone, and generates pSPc5-12-wt-GHRH and pSPc5-12-HV-GHRH.Control plasmid contains e.coli under the control of identical synthetic promoter to produce pSP-bgal.

[0188] Zooscopy.Weight is used for these GHRH research for the young sow of PIC strain 22 first nests of about 365kg.This animal was taken the research department, farm on 87th in gestation, and placed the corral of independent farrowing separately, and they unrestrictedly can last till the end of 25 day lactation near water and food there.Experiment starts from March, and the first nest son is in April birth and analyze mid-October always.This farmstead has been equipped cooling system, and this makes it possible to can make in hot weather top temperature to hang down 2-5 ℃ than the temperature of outside.The average maximum in July, August and September is respectively 40.6 ℃, 41.6 ℃ and 36.6 ℃.This animal is kept according to NIH guide, USDA and Animal Welfare Act guide.

[0189] The intramuscularly of plasmid DNA in the pig.To not have endotoxic plasmid prepared product pSPc5-12-HV-GHRH and pSPc5-12-wt-GHRH (Qiagen Inc., Chatsworth, CA, the U.S.) and in PBS pH=7.4, be diluted to 1mg/ml.Each sow is distributed seance.4 sows are injected with pSPc5-12-HV-GHRH, 2 sows inject with pSPc5-12-wt-GHRH and 6 sows with comparing.Gestation 95 days, animal is carried out slight anesthesia with the telazol of 2.2mg/kg.Be injected directly in the left side semitendinosus muscle of pig amounting to the 10mg plasmid.Injected back 2 minutes, with the muscle diameter of injection is that 1cm, 22 specifications and length are that the injection-type electrode of the 6-pin array of 2cm carries out electroporation, adopt following condition: alternating-electric field between 6 pulses, pin, 200V/cm, 60 milliseconds/pulse, (people such as Draghia-Akli, 1999 as described; Aihara and Miyazai, 1998).

[0190] Raise together with research.Be right after after birth each nest son is divided into 2 groups.Half that makes each nest son is still by its mother's nurture, and second half of this nest son then raised together with (raise together with as the contrast piggy and the animal of HV-or wt-injection, piggy and control animal that HV or wt are born are raised together with) with different groups.Per week record weight.

[0191] Meals.When just having weaned on 21st, (MN) feeding young pigs is 60 days for Cargill, Minneapolis with the Nutrena 18% Medicated Pig Starter with 1.012% Methionin.Subsequently, raised pig 45 days with having 1.4% Methionin Custom Mix PigStarter, 24% protein, raised pig 45 days with having 1.4% Methionin Custom Mix22.7% protein, then with having 1.2% Methionin and 20% proteinic Custom Mix (Cargill, Minneapolis MN) keeps the research of pig to be left.

[0192] Biological chemistry.Back 50 days of birth with collected serum on the 170th, and by laboratory independently (Antech Diagnostics, Irvine CA) analyzes.

[0193] Pig IGF-I RIA.The IGF-I of pig by allogenic people IGF-I measure (Diagnostic System Lab., Webster, TX).

[0194] Pork insulin RIA.The Regular Insulin of pig is measured (Linco Research Inc. by allogenic people; St.Charles, Missouri).The sensitivity of measuring is 2 little U/ml.

[0195] Health is formed data.Per week 2 times is measured weight with identical calibration ratio (be accredited as and have ± accuracy of .2kg and 0.3% variation factor) in whole research process.

[0196] Statistics.Data are analyzed with Microsoft Excel statistical study bag.The value of Xian Shiing is mean number ± s.e.m in the drawings.Specific p value obtains by comparing with the Studentst check.The level of p＜.05 as statistically significant is set.

Embodiment 21

Multiple action to the rat of handling with GHRH

[0197] secretion of tethelin (GH) is stimulated by natural GH secretogogue growth hormone releasing hormone (GHRH), and (SS) is suppressed by somatostatin (stomatostatin), and both are hypothalamic hormone people such as (, 1995) Thorner.The GH pulse is to reduce or cancel relevant GHRH excretory result with the somatostatin excretory.When in addition, it seems that the mechanism of impulse generator controlled by the GH-reverse feedback.In addition, have recognized that initial separation is the important instrumentality of GH secretion and energy homeostasis from the new peptide ghrelin of rat stomach.Ghrelin is that GH-discharges activity and depends on GHRH people such as (, 2001) Hataya in the endogenous ligands of secretagogue receptor and its body.In the healthy adult Mammals, GH discharges with the pulsation mode of the uniqueness of altitude mixture control, and this took place 4-8 time in 24 hours and to its biologic activity utmost importance (people such as Argente, 1996).Excretory paroxysm pattern relates in the suitableeest induce (Veldurs, 1998) of periphery level to physiological role.The expression of GH isotype, processing and/or release and the relative proportion between them are in (Araburo waits the people, 2000) under the different control in the g and D process.

[0198] adjusting of somatotroph and differentiation also depend in the paracrine process of hypophysis inside itself and relate to somatomedin and several neuropeptide, as vasoactive intestinal peptide (people such as Rawlings, 1995), angiotonin 2, endothelin (people such as Tomic, 1999) and activin (people such as Billesbup, 1990).GH and insulin-like growth factor I (IGF-I) approach effectively and the expression of being regulated for the suitableeest linear growth and carbohydrate, protein and lipometabolic stable state, and be crucial (Murray and Shalet, 2000) for positive nitrogen balance is provided.GHRH, GH, ghrelin, prolactin (PRL) and IGF-I under physiology and pathological condition, play a significant role in the adjusting to body fluid and cell immune response (people such as Geffner, 1997; People such as Hattori, 2001).

[0199] inferior colliculus cerebral tissue of GHRH gene is specific expressed is not active necessary, and this is because the exocrine GHRH of head can have biologic activity (people such as Faglia, 1992; Melmed, 1991).The active pathologic GHRH of GH stimulates (no matter its source, from the transgenic models to the pancreatic neoplasm) can cause propagation, hyperplasia and adenoma (people such as Asa, 1992 of adenopituicyte; People such as Sano, 1988).Yet, dock the long-term effect that GHRH that the offspring of subject animal continues handles and do still unknown.

[0200] front has shown that the ectopic expression of the pig GHRH of the new serum protein enzyme resistance that the expression plasmid by synthetic muscle specific promotor control instructs can cause high GH and IGF-I level people such as (, 1999) Lopez-Calderon after electroporation is sent in intramuscularly and body in the pig body.The experiment purpose of describing in the present embodiment is for estimating GHRH, and it is sent to strengthen growth and change health among the offspring with the animal of handling last 3 middle of the month in gestation by the plasmid DNA gene therapy and forms.

[0201] in a specific embodiment, the GHRH that dystopy is produced in the animal of pregnancy passes to the offspring by placenta, in the descendant, determined the hyperplasia of hypophysis and strengthened long-time GH production that this will show the health composition of the growth and the change of increase then.In order to estimate of the growth effect of GHRH myogenic vector injection Mammals, the rat of pregnancy is injected in gestation plasmid DNA pSP-HV-GHRH or pSP-β gal with 30 μ g on the 16th offspring.After this injection, carry out electroporation to strengthen the plasmid picked-up.

[0202] all animals produced in pregnant 20-22 day.Average offspring's number similar ((T) of processing, n=10.8 young baby/nest of nest son between each group; Contrast (C) n=11.75 young baby/nest).Make the cub number equalization between each mother turn to 10 cub/mothers.In back 2 week of birth, the nest son's of treatment group weight in average has increased 9%:T=31.47 ± 0.52g for C=28.86 ± 0.75g, p＜0.014.

[0203] when wean just, weight significantly increases in the T offspring: T female (TF) average out to 51.97 ± 0.83g is for contrast female (CF) 47.07 ± 4.4g, p＜0.043, and the male average out to 60.89 ± 1.02g that handles is for contrast male (CM) 49.85 ± 4.9g, p＜0.001 (Figure 14).This advantage was maintained to for 10 ages in week, and weight differential becomes not remarkable in 24 weeks.

[0204] two sex all has the hypertrophy of muscle when 3 ages in week, and has significant difference (Figure 15) on gastrocnemius muscle (G) and tibialis anterior muscle (TA) muscle/weight.TF keeps the hypertrophy of muscle during whole research, and the male sign that after 10 ages in week, does not show the muscle hypertrophy.This variation may be owing to the male variation of sex steroid when ripe, and it has weakened GH that physiology the increases effect to skeletal muscle.

[0205] at after death first minute pituitary body of dissecting and weigh.Main in IF hypophysis weight the ratio of TBW reached after birth 12 week increased (Figure 16) significantly.The increase most probable of this hypophysis weight is owing to the somatotroph hyperplasia, because known GHRH can stimulate GH synthetic and secretion and somatotroph had specific hypertrophy effect (people such as Morel, 1999 from prepituitary gland; People such as Murray, 2000).This has obtained the support of hormone (Figure 17) and histology (Figure 18) evidence.Northern engram analysis from the hypophysis of the animal of injection is shown the remarkable increase of GH and PRL mRNA level, and in conjunction with the minimizing of endogenous rat GHRH mRNA level.Utilize histological techniques, specific Chinese People's Anti-Japanese Military and Political College's murine antibody has been illustrated the increase of somatotroph number.

[0206] indication of whole body GHRH of Zeng Jiaing and GH level is the increase of serum I GF-I concentration.Serum rat IGF-I reaches in 24 week significantly higher after the rat offspring birth of pSP-HV-GHRH injection, all has p＜0.05 (Figure 19) at the time point of all checks.

[0207] organ (lung, the heart, liver, kidney, stomach, intestines, suprarenal gland, sexual gland, brain) is collected and weighed.In any animal, do not observe related pathologies.In the non-virus technology of transgenosis, it is simple, cheap and safe directly plasmid DNA being injected into muscle in vivo, but this methodological application has been subjected to the restriction of the low relatively expression level of the DNA expression vector of transfer.In a specific embodiment, in order to obtain the adjusting that gene therapy is formed growth and health, must utilize the method for innovation, wherein directly processing of target animal, but owing to the processing to mother of pregnancy has the enhanced biological property.As described herein, another significant improvement of plasmid vector is the gene of using the more stable GHRH analogue HV-GHRH of coding people such as (, 1999) Draghia-Akli.Electricity gene (electrogene) treatment is shifted and is made gene to shift effectively in required organ or tissue and to express, and long expression can be provided after single administration.This method can be represented a kind of novel method that does not need virogene or the efficient nucleic acid of particulate to shift.

[0208] for big species such as pig or ox, GH upstream stimulator, the use of GHRH is a kind of selectable strategy, this strategy not only can increase growth performance or milk production, and the more important thing is that seeing from actual and metabolic viewpoint to increase production efficiency people such as (, 1990) Dubreuil.Yet the expensive and essential frequency of administration of recombinant peptide has limited the widespread use of this processing at present.These main shortcomings can be eliminated with the dystopy production of instructing GHRH by using the nucleic acid transfer method, particularly when its production long term maintenance.

[0209] thereby, after the injection of single electroporation is grown up in the tibialis anterior muscle muscle of conceived rat, the enhanced growth of animal has taken place in the offspring at the plasmid of growth hormone releasing hormone (GHRH) cDNA that will express sudden change.Newborn rat (F1) is significantly bigger at birth.Weight and health composition research have shown between two sexes the difference with the age longitudinally.Hormone consistent with growth pattern with biochemical measurement.F1 has the GH content of bigger pituitary body, somatotroph hyperplasia and increase.F1 blood plasma IGF-I level significantly raises.In a word, these new discovery explanations GHRH is used in based on strengthen some animal character after the gene therapy of plasmid between the generation.

[0210] Xia Mian paragraph has been described the experiment of carrying out in the present embodiment.

[0211] DNA construct.Plasmid pSPc5-12 comprises the Sac I/BamH I fragment (people such as Draghia-Akli, 1997) of the 360bp SPc5-12 synthetic promoter (people such as Li, 1999) that is positioned at pSK-GHRH main chain SacI/BamH I site.The pig GHRHcDNA of sudden change obtain by people GHRH cDNA being carried out rite-directed mutagenesis (change the external abruptly-changing system of site II (Altered Sites II in vitro Mutagenesis System), Promega, Madison, WI).Encode 228-bp fragment (the exon 2 part of pig GHRH of sudden change of pig GHRH (1-40) OH of 31 amino acid whose signal peptides and sudden change, whole parts of exon 3s and exon 4) be characterised in that following amino acid replacement: Gly15 becomes Ala, and Met27 becomes Leu and Ser28 and becomes Asn and the Tyr1 transformation to His and Ala2 to Val.BamH I/Hind III site with this fragment cloning pSK-GHRH.HGH pA is from 3 ' non-translational region of people GH gene and polyadenylic acid signal.Plasmid in bacillus coli DH 5 alpha, grow (Gibco BRL, Carlsbad, CA).To not have endotoxic plasmid (Qiagen Inc., Chatsworth, the CA U.S.) prepared product and be diluted to 1mg/ml among the pH7.4 at PBS.

[O212] The intramuscularly of plasmid and electroporation.At Baylor College of Medicine, Houston preserves in the zooscopy chamber of TX and brings up with the conceived adult Wistar female rats that clocks.Animal is kept under the envrionment conditions of 10 hours illumination/14 hour dark according to NIH guide, USDA and Animal Welfare Act guide, and these rules have obtained the approval of Instutional Animal Care and Use Committee.To test and repeat 2 times.16 days of gestation, animal (n=20 group) weighed and the combination of the vetrnquil of the xylazine of the ketamine of 42.8mg/ml, 8.2mg/ml and the 0.7mg/ml dosage i.m. with 0.5-0.7ml/kg is used and anaesthetizes.(Becton-Dickinson, Franklin Lakes NJ) injects to use the pSP-HV-GHRH of the 30mg among the 100ml PBS to use the 0.3cc insulin syringe flesh muscle before the left tibia of rat.Control animal is injected with PBS.For two groups, after this injection as described people such as (, 1999) Draghia-Akli carry out the clamp electroporation.Briefly, in injection back 2 minutes, with the rat leg place 2 length be between 1cm, 26 specifications and the pin for the pin electrode of 1cm (Genetronics, San Diego, CA) between and electricimpulse is applied to this zone.Pulse in that a direction has been used 3 60-ms in the voltage of 100V/cm makes field inversion then, and uses other 3 pulses in opposite direction.(Genetronics, San Diego CA) generate by T-820 ElectroSquare Porator in this pulse.

[0213] Offspring's research.The rat of all injections produced in pregnant 20-22 day.60 offsprings in 240 offsprings in first research and second research are analyzed from being born to 5 monthly ages (birth,

birth

2,3,6,8,12,16,22 weeks of back).Body weight is carried out record with identical gauged balance at these time points.At the end of experiment, studied the health composition after death.Collect blood, centrifugal and before analysis, be stored in-80 ℃ in 0 ℃ immediately.With organ (from injection with the heart, liver,spleen,kidney, hypophysis, brain, suprarenal gland, skeletal muscle--tibialis anterior muscle (TA), gastrocnemius muscle (G), soleus muscle (S) and musculus extensor digitorum longus pedis (EDL), carcass, the fat of control animal) removal, weighing on the analytical balance and quick-frozen in liquid nitrogen.Measure and write down shin bone length.

[0214] The Northern engram analysis of hypophysis.With hypophysis quick-frozen and homogenate in solution D, extracting then.The total RNA of 20mg is handled, separates size and transfer on the nylon membrane on 1.5% agarose-formaldehyde gel with DNase I.Hybridize by causing at random by the GHRH cDNA probe of 32P-mark with specific for this film.

[0215] Rat IGF-I radioimmunoassay.(Diagnostic System Laboratories, Webster Texas) measures rat IGF-I by specific radioimmunoassay.The sensitivity of this mensuration is 0.8ng/ml; The variation of measuring between interior and mensuration is respectively 2.4% and 4.1%.

[0216] Statistics.The value of Xian Shiing is mean number ± s.e.m in the drawings.Specific p value obtains by analyzing to compare with Students t check or ANOVA.The level of p＜0.05 as statistically significant is set.

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Publication

Aihara，H.?&?Miyazaki，J.Nat.Biotechnol.16，867-870(1998).

Albanese，A.and?R.Stanhope.1997.GH?treatment?induces?sustained?catch-up?growth?inchildren?with?intrauterine?growth?retardation：7-year?results.Horm.Res.48：173-177.

Allen，D.B.，A.C.Rundle，D.A.Graves，and?S.L.?Blethen.1997.Risk?of?leukemia?in?childrentreated?with?human?growth?hormone：review?and?reanalysis.?J.Pediatr.?131：S32-S36

Aramburo，C.，Luna，M.，Carranza，M.，Reyes，M.，Martinez-Coria，H.，Scanes，C.G.(2000)Growth?hormone?size?variants：changes?in?the?pituitary?during?development?of?thechicken.Proc.Soc.Exp.Biol.Med.223，67-74

Argente，J.，Pozo，J.，Chowen，J.A.(1996)The?growth?hormone?axis：control?and?effects.Hormone?Research?45?Suppl?1，9-11

Asa，S.L.，Kovacs，K.，Stefaneanu，L.，Horvath，E.，Billestrup，N.，Gonzalez-Manchon，C.，Vale，W.(1992)Pituitary?adenomas?in?mice?transgenic?for?growth?hormone-releasinghormone.Endocrinology?131，2083-2089

Azcona，C.，A.Albanese，P.Bareille，and?R.Stanhope.1998.Growth?hormone?treatment?ingrowth?hormone-sufficient?and?-insufficient?children?with?intrauterine?growthretardation/Russell-Silver?syndrome.Horm.Res.50：22-27.

Barr，E.，Leiden，J.M.(1991)Systemic?delivery?of?recombinant?proteins?by?geneticallymodified?myoblasts.Science?254，1507-1509

Bartke，A.1998.Growth?hormone?and?aging.Endocrine?8：103-108.

Benfield，M.R.and?E.C.Kohaut.1997.Growth?hormone?is?safe?in?children?after?renaltransplantation.J.Pediatr.131：S28-S31

Bercu，B.B.，R.F.Walker.1997.Growth?hormone?secretagogues?in?children?with?alteredgrowth.Acta?Paediatrica?86：102-106.

Bergsma，D.J.，Grichnik，J.M.，Gossett，L.M.?&?Schwartz，R.J.Mol.Cell.Biol.6，2462-2475(1986).

Bettan，M.，Emmanuel，F.，Darteil，R.，Caillaud，J.M.，Soubrier，F.，Delaere，P.，Branelec，D.，Mahfoudi，A.，Duverger，N.，Scherman，D.(2000)High-level?protein?secretion?intoblood?circulation?after?electric?pulse-mediated?gene?transfer?into?skeletal?muscle.Mol.Ther.2，204-210

Billestrup，N.，Gonzalez-Manchon，C.，Potter，E.，Vale，W.(1990)Inhibition?of?somatotrophgrowth?and?growth?hormone?biosynthesis?by?activin?in?vitro.Mol.Endocrinol.4，356-362

Blethen，S.L.and?A.C.Rundle.1996.Slipped?capital?femoral?epiphysis?in?children?treatedwith?growth?hormone.A?summary?of?the?National?Cooperative?Growth?Studyexperience.Horm.Res.46：113-116.

Bowers，C.Y.1998.Growth?hormone-releasing?peptide(GHRP).Cell?Mol?Life?Sci.54(12)：1316-29.

Campbell，R.M.，Y.Lee，J.Rivier，E.P.Heimer，A.M.Felix，and?T.F.Mowles.1991.GRFanalogs?and?fragments：correlation?between?receptor?binding，activity?and?structure.Peptides?12：569-574.

Chung，C.S.，Etherton，T.D.，Wiggins，J.P.(1985)?Stimulation?of?swine?growth?by?porcinegrowth?hormone.J.Anim?Sci.60，118-130

Corpas，E.，S.M.Harman，and?M.R.Blackman.1993.Human?growth?hormone?and?humanaging.Endocrine?Reviews?14：20-39.

Corpas，E.，S.M.?Harman，M.A.Pineyro，R.?Roberson，and?M.R.?Blackman.?1993.Continuous?subcutaneous?infusions?of?growth?hormone(GH)?releasing?hormone?1-44for?14?days?increase?GH?and?insulin-like?growth?factor-I?levels?in?old?men.?Journalof?Clinical?Endocrinology?&?Metabolism?76：134-138.

Dahler，A.，Wade，R.P.，Muscat，G.E.，Waters，M.J.(1994)?Expression?vectors?encodinghuman?growth?hormone(hGH)?controlled?by?human?muscle-specific?promoters：prospects?for?regulated?production?of?hGH??delivered?by?myoblast?transfer?orintravenous?injection.?Gene?145，305-310

Davis，H.L.，Whalen，R.G.?&?Demeneix，B.A.Hum.Gene?Ther.4，151-159(1993).

D′Costa，A.P.，R.L.Ingram，J.E.Lenham，and?W.E.Sonntag.1993.?The?regulation?andmechanisms?of?action?of?growth?hormone?and?insulin-like?growth?factor?1?duringnormal?aging.J.?Reprod.?Fert.-Supp.?46：87-98.

Dhawan，J.，Pan，L.C.，Pavlath，G.K.，Travis，M.A.，Lanctot，A.M.，Blau，H.M.(1991)Systemic?delivery?of?human?growth?hormone?by?injection?of?genetically?engineeredmyoblasts.Science?254，1509-1512

Dieguez?C，Casanueva?FF.2000.?Ghrelin：a?step?forward?in?the?understanding?of?somatotrophcell?function?and?growth?regulation.?Eur?J?Endocrinol.?142(5)：413-7.

Draghia-Akli，R.，Li，X.G.，Schwartz，R.J.，et?al.Nat.Biotechnol.15，1285-1289(1997).

Draghia-Akli，R.Fiorotto?ML，Hill?LA，Malone?PB，Deaver?DR，Schwartz?RJ?NatBiotechnol，17(12)，1179-83(1999).

Dubreuil，P.，Petitclerc，D.，Pelletier，G.，Gaudreau，P.，Farmer，C.，Mowles，TF，Brazeau，P.(1990)?Effect?of?dose?and?frequency?of?administration?of?a?potent?analog?of?humangrowth?hormone-releasing?factor?on?hormone?secretion?and?growth?in?pigs.?Journal?ofAnimal?Science?68，1254-1268

Eicher，E.M.and?W.G.Beamer.1976.?Inherited?ateliotic?dwarfism?in?mice.Characteristicsof?the?mutation，little，on?chromosome?6.?J.Hered?67：87-91.

Erling，A.1999.?Methodological?considerations?in?the?assessment?of?health-related?quality?oflife?in?children.?Acta?Paediatrica?Scandin.-Supp.?428：106-107.0803-5326.

Esch，F.S.，P.Bohlen，N.C.Ling，P.E.Brazeau，W.B.Wehrenberg，M.O.Thorner，M.J.Cronin，and?R.Guillemin.?1982.?Characterization?of?a?40?residue?peptide?from?ahuman?pancreatic?tumor?with?growth?hormone?releasing?activity.?Biochemical?&Biophysical?Research?Communications?109：152-158.

Etherton，T.D.，Wiggins，J.P.，Chung，C.S.，Evock，C.M.，Rebhun，J.F.，Walton，P.E.(1986)?Stimulation?of?pig?growth?performance?by?porcine?growth?hormone?andgrowth?hormone-releasing?factor.?Journal?of?Animal?Science?63，1389-1399

Etienne，M.，Bonneau，M.，Kann，G.，Deletang，F.(1992)?Effects?of?administration?of?growthhormone-releasing?factor?to?sows?during?late?gestation?on?growth?hormone?secretion，reproductive?traits，and?performance?of?progeny?from?birth?to?100?kilograms?liveweight.?Journal?of?Animal?Science?70，2212-2220

Everett，R.S.，Gerrard，D.E.，Grant，A.L.(2000)?Factors?affecting?production?of?luciferaseand?epitope-tagged?IGF-I?in?porcine?muscle?after?DNA?injection.J?Endocrinol.166，255-263

Farmer，C.，Petitclerc，D.，Pelletier，G.，Brazeau，P.(1992)?Lactation?performance?of?sowsinjected?with?growth?hormone-releasing?factor?during?gestation?and(or)?lactation.Journal?of?Animal?Science?70，2636-2642

Faglia，G.，Arosio，M.，Bazzoni，N.(1992)Ectopic?acromegaly.[Review].Endocrinology?&Metabolism?Clinics?of?North?America?21，575-595

Frohman，M.A.，T.R.Downs，P.Chomczynski，and?L.A.?Frohman.1989.?Cloning?andcharacterization?of?mouse?growth?hormone-releasing?hormone(GRH)complementaryDNA：increased?GRH?messenger?RNA?levels?in?the?growth?hormone-deficient?lit/litmouse.?Mol.Endocrinol.?3：1529-1536.

Frohman，L.A.，J.L.Thominet，C.B.Webb，M.L.Vance，H.Uderman，J.Rivier，W.Vale，andM.O.Thorner.1984.?Metabolic?clearance?and?plasma?disappearance?rates?of?humanpancreatic?tumor?growth?hormone?releasing?factor?in?man.?J.Clin.Invest.?73：1304-1311.

Geffner，M.(1997)?Effects?of?growth?hormone?and?insulin-like?growth?factor?I.ActaPaediatr.Suppl?423，76-79

Gesundheit，N.and?J.K.Alexander.1995.?Endocrine?Therapy?with?Recombinant?Hormonesand?Growth?Factors.?In?Molecular?Endocrinology：Basic?Concepts?and?ClinicalCorrelations.B.D.?Weintraub，editor.?Raven?Press，Ltd.，New?York.491-507.

Gopinath，R.，Etherton，T.D.(1989)?Effects?of?porcine?growth?hormone?on?glucosemetabolism?of?pigs：I.Acute?and?chronic?effects?on?plasma?glucose?and?insulin?status.J.Anim?Sci.67，682-688

Hataya，Y.，Akamizu，T.，Takaya，K.，Kanamoto，N.，Ariyasu，H.，Saijo，M.，Moriyama，K.，Shimatsu，A.，Kojima，M.，Kangawa，K.，Nakao，K.(2001)A?low?dose?of?ghrelinstimulates?growth?hormone(GH)?release?synergistically?with?GH-releasing?hormonein?humans.J?Clin.Endocrinol.Metab?86，4552

Hattori，N.，Saito，T.，Yagyu，T.，Jiang，B.H.，Kitagawa，K.，Inagaki，C.(2001)GH，GHreceptor，GH?secretagogue?receptor，and?ghrelin?expression?in?human?T?cells，B?cells，and?neutrophils.J?Clin.Endocrinol.Metab?86，4284-4291

Heptulla，R.A.，S.D.Boulware，S.Caprio，D.Silver，R.S.Sherwin，and?W.V.Tamborlane.1997.?Decreased?insulin?sensitivity?and?compensatory?hyperinsulinemia?afterhormone?treatment?in?children?with?short?stature.?J.Clin.Endocrinol.Metab.?82：3234-3238.

Horvath，T.L.，Diano，S.，Sotonyi，P.，Heiman，M.，Tschop，M.(2001)Minireview：ghrelinand?the?regulation?of?energy?balance-a?hyPothalamic?perspective.?Endocrinology?142，4163-4169

Howard，A.D.，Feighner，S.D.，Cully，D.F.Arena，J?P.，Liberator，P.A.，Rosenblum，C.I.，Hamelin，M.，Hreniuk，D.L.，Palyha，O.C.，Anderson，J.，Paress，P.S.，Diaz，C.，Chou，M.，Liu，K.K.，McKee，K.K.，Pong，S.S.，Chaung，L.，Elbrecht，A.，Dashkevicz，M.，Heavens，R.，Rigby，M.，Sirinathsinghji，D.，Dean，D.C.，Melillo，D.G.，Patchett，A.A.，Nargund，R.，Griffin，P.R.，DeMartino，J.A.，Gupta，S.K.，Schaeffer，J.M.，SmithR.G.，Van?der?Ploeg，L.H.T.(1996)?Receptor?in?pituitary?and?hypothalamus?thatfunctions?growth?hormone?release.?Science?273：974-977.

Iranmanesh，A.，G.Lizarralde，and?J.D.Veldhuis.1991.?Age?and?relative?adiposity?arespecific?negative?determinants?of?the?frequency?and?amplitude?of?growth?hormone(GH)secretory?bursts?and?the?half-life?of?endogenous?GH?in?healthy?men.?Journal?ofClinical?Endocrinology?&?Metabolism?73：1081-1088.

Jacobs，P.A.，P.R.Betts，A.E.Cockwell，J.A.Crolla，M.J.Mackenzie，D.O.Robinson，andS.A.Youings.1990.?A?cytogenetic?and?molecular?reappraisal?of?a?series?of?patientswith?Turner′s?syndrome.?Ann.Hum.Genet.?54：209-223.

Jaffe，H.A.，C.Danel，G.Longenecker，M.Metzger，Y.Setoguchi，M.A.Rosenfeld，T.W.Gant，S.S.Thorgeirsson，L.D.Stratford-Perricaudet，M.Perricaudet，A.Pavirani，J.-P.Lecocq?and?R.G.Crystal.1992.?Adenovirus-mediated?in?vivo?gene?transfer?andexpression?in?normal?rat?liver.?Nat?Genet?1(5)：372-8.

Key，L.L.J.and?A.J.Gross.?1996.?Response?to?growth?hormone?in?children?withchondrodysplasia.?J.Pediatr?128：S14-S17

Klindt，J.，Yen，J.T.，Buonomo，F.C.，Roberts，A.J.，Wise，T.(1998)Growth，bodycomposition，and?endocrine?responses?to?chronic?administration?of?insulin-like?growthfactor?I?and(or)?porcine?growth?hormone?in?pigs.J.Anim?Sci.76，2368-2381

Kojima?M，Hosoda?H，Date?Y，Nakazato?M，Matsuo?H，Kangawa?K.1999.Ghrelin?is?agrowth-hormone-releasing?acylated?peptide?from?stomach.?Nature?402(6762)：656-60.

Kubiak，T.M.，C.R.Kelly，and?F.Krabill.1989.?In?vitro?metabolic?degradation?of?a?bovinegrowth?hormone-releasing?factor?analog?Leu27-bGRF(1-29)NH2?in?bovine?andporcine?plasma.?Correlation?with?plasma?dipeptidylpeptidase?activity.?DrugMetabolism?&?Disposition?17：393-397.

Li，X.，Eastman，E.M.，Schwartz，R.J.，&?Draghia-Akli，R.Nat.Biotechnol.17.3，241-245(1999).

Lopez-Calderon，A.，Soto，L.，Villanua，M.A.，Vidarte，L.，Martin，A.I.(1999)The?effect?ofcyclosporine?administration?on?growth?hormone?release?and?serum?concentrations?ofinsulin-like?growth?factor-I?in?male?rats.Life?Sci.64，1473-1483

Martin，R.A.，D.L.?Cleary，D.M.Guido，H.A.Zurcher-Neely，and?T.M.Kubiak.1993.Dipeptidyl?peptidase?IV(DPP-IV)?from?pig?kidney?cleaves?analogs?of?bovine?growthhormone-releasing?factor?(bGRF)?modified?at?position?2?with?Ser，Thr?or?Val.Extended?DPP-IV?substrate?specificity？Biochimica?et?Biophysica?Acta?1164：252-260.

Melmed，S.(1991)?Extrapituitary?Acromegaly.[Review].?Endocrinology?&?MetabolismClinics?of?North?America?20，507-518

Miller，K.F.，Bolt，D.J.，Pursel，V.G.，Hammer，R.E.，Pinkert，C.A.，Palmiter，R.D.，Brinster，R.L.(1989)?Expression?of?human?or?bovine?growth?hormone?gene?with?amouse?metallothionein-1?promoter?in?transgenic?swine?alters?the?secretion?of?porcinegrowth?hormone?and?insulin-like?growth?factor-I.J.Endocrinol.?120，481-488

Mir，L.M.，Bureau，M.F.，Gehl，J.，Rangara，R.，Rouy，D.，Caillaud，J.M.，Delaere，P.，Branellec，D.，Schwartz，B.，Scherman，D.(1999)?High-efficiency?gene?transfer?intoskeletal?muscle?mediate?(by?electric?pulses.?Proc.Natl.Acad.Sci.U.S.A?96，4262-4267

Monti，L.D.，P.Brambilla，A.Caumo，F.Magni，S.Omati，G.Nizzoli，B.di?Natale，M.Galli-Kienle，C.Cobelli，G.Chiumello，and?G.Pozza.1997.?Glucose?turnover?and?insulinclearance?after?growth?hormone?treatment?in?girls??with?Tumer′s?syndrome.Metabolism?46：1482-1488.

Morel，G.，Gallego，R.，Boulanger，L.，Pintos，E.，Garcia-Caballero，T.，Gaudreau，P.(1999)Restricted?presence?of?the?growth?hormone-releasing?hormone?receptor?tosomatotropes?in?rat?and?human?pituitaries.Neuroendocrinology?70，128-136

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Watkins，S.L.1996.?Bone?disease?in?patients?receiving?growth?hormone.?Kidney?Int.Suppl.53：S126-7：S126-S127

Wolff，J.A.，Ludtke，J.J.，Acsadi，G.，Williams，P.，Jani，A.(1992)?Long-term?persistence?ofplasmid?DNA?and?foreign?gene?expression?in?mouse?muscle.?Human?MolecularGenetics?1，363-369

[0217] invention of those skilled in the art's easy to understand this patent is very suitable for realizing its purpose and obtains described and inherent target and advantage.The tethelin of Miao Shuing, growth hormone releasing hormone, analogue, plasmid, carrier, pharmaceutical composition, treatment, method, program and technology are the representative of preferred embodiment at present and are intended to demonstrate but not to the restriction of scope herein.Change wherein and other purposes can take place for those skilled in the art, and these are included in the spirit of the present invention or are defined by the scope of unsettled claim.

Sequence table

<110>Schwartz，Robert?A.

Draghia-Akli，Ruxandra

Smith，Roy?G.

Carpenter，Robert?H.

Kern，Douglas?R.

＜120〉the jenny administration of nucleic acid sequence is grown to strengthen the offspring

<130>HO-P02021WO0/10021476/OTA?00-91

<140>TBA

<141>2001-12-12

<150>60/255,021

<151>2000-12-12

<160>11

<170>PatentIn?version?3.1

<210>1

<211>40

<212>PRT

＜213〉pig

<400>1

Tyr?Ala?Asp?Ala?Ile?Phe?Thr?Asn?Ser?Tyr?Arg?Lys?Val?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

Leu?Ser?Ala?Arg?Lys?Leu?Leu?Gln?Asp?Ile?Met?Ser?Arg?Gln?Gln?Gly

20??????????????????25??????????????????30

Glu?Arg?Asn?Gln?Glu?Gln?Gly?Ala

35??????????????????40

<210>2

<211>48

<212>DNA

＜213〉synthetic

<400>2

aggcagcagg?gagagaggaa?ccaagagcaa?ggagcataat?gactgcag???????????????????48

<210>3

<211>42

<212>DNA

＜213〉synthetic

<400>3

accctcagga?tgcggcggca?cgtagatgcc?atcttcacca?ac?????????????????????????42

<210>4

<211>27

<212>DNA

＜213〉synthetic

<400>4

cggaaggtgc?tggcccagct?gtccgcc??????????????????????????????????????????27

<210>5

<211>36

<212>DNA

＜2l3〉synthetic

<400>5

ctgctccagg?acatcctgaa?caggcagcag?ggagag????????????????????????????????36

<210>6

<211>358

<212>DNA

＜213〉synthetic

<400>6

gagctccacc?gcggtggcgg?ccgtccgccc?tcggcaccat?cctcacgaca?cccaaatatg?????60

gcgacgggtg?aggaatggtg?gggagttatt?tttagagcgg?tgaggaaggt?gggcaggcag????120

caggtgttgg?cgctctaaaa?ataactcccg?ggagttattt?ttagagcgga?ggaatggtgg????180

acacccaaat?atggcgacgg?ttcctcaccc?gtcgccatat?ttgggtgtcc?gccctcggcc????240

ggggccgcat?tcctgggggc?cgggcggtgc?tcccgcccgc?ctcgataaaa?ggctccgggg????300

ccggcggcgg?cccacgagct?acccggagga?gcgggaggcg?ccaagctcta?gaactagt??????358

<210>7

<211>623

<212>DNA

＜213〉people

<400>7

gggtggcatc?cctgtgaccc?ctccccagtg?cctctcctgg?ccctggaagt?tgccactcca?????60

gtgcccacca?gccttgtcct?aataaaatta?agttgcatca?ttttgtctga?ctaggtgtcc????120

ttctataata?ttatggggtg?gaggggggtg?gtatggagca?aggggcaagt?tgggaagaca????180

acctgtaggg?cctgcggggt?ctattgggaa?ccaagctgga?gtgcagtggc?acaatcttgg????240

ctcactgcaa?tctccgcctc?ctgggttcaa?gcgattctcc?tgcctcagcc?tcccgagttg????300

ttgggattcc?aggcatgcat?gaccaggctc?agctaatttt?tgtttttttg?gtagagacgg????360

ggtttcacca?tattggccag?gctggtctcc?aactcctaat?ctcaggtgat?ctacccacct????420

tggcctccca?aattgctggg?attacaggcg?tgaaccactg?ctcccttccc?tgtccttctg????480

attttaaaat?aactatacca?gcaggaggac?gtccagacac?agcataggct?acctggccat????540

gcccaaccgg?tgggacattt?gagttgcttg?cttggcactg?tcctctcatg?cgttgggtcc????600

actcagtaga?tgcctgttga?att?????????????????????????????????????????????623

<210>8

<211>40

<212>PRT

＜213〉synthetic

<400>8

His?Val?Asp?Ala?Ile?Phe?Thr?Asn?Ser?Tyr?Arg?Lys?Val?Leu?Ala?Gln

1???????????????5???????????????????10??????????????????15

Leu?Ser?Ala?Arg?Lys?Leu?Leu?Gln?Asp?Ile?Leu?Asn?Arg?Gln?Gln?Gly

20??????????????????25??????????????????30

Glu?Arg?Asn?Gln?Glu?Gln?Gly?Ala

35??????????????????40

<210>9

<211>3534

<212>DNA

＜213〉synthetic

<400>9

gttgtaaaac?gacggccagt?gaattgtaat?acgactcact?atagggcgaa?ttggagctcc?????60

accgcggtgg?cggccgtccg?ccctcggcac?catcctcacg?acacccaaat?atggcgacgg????120

gtgaggaatg?gtggggagtt?atttttagag?cggtgaggaa?ggtgggcagg?cagcaggtgt????180

tggcgctcta?aaaataactc?ccgggagtta?tttttagagc?ggaggaatgg?tggacaccca????240

aatatggcga?cggttcctca?cccgtcgcca?tatttgggtg?tccgccctcg?gccggggccg????300

cattcctggg?ggccgggcgg?tgctcccgcc?cgcctcgata?aaaggctccg?gggccggcgg????360

cggcccacga?gctacccgga?ggagcgggag?gcgccaagct?ctagaactag?tggatcccaa????420

ggcccaactc?cccgaaccac?tcagggtcct?gtggacagct?cacctagctg?ccatggtgct????480

ctgggtgttc?ttctttgtga?tcctcaccct?cagcaacagc?tcccactgct?ccccacctcc????540

ccctttgacc?ctcaggatgc?ggcggcacgt?agatgccatc?ttcaccaaca?gctaccggaa????600

ggtgctggcc?cagctgtccg?cccgcaagct?gctccaggac?atcctgaaca?ggcagcaggg????660

agagaggaac?caagagcaag?gagcataatg?actgcaggaa?ttcgatatca?agcttatcgg????720

ggtggcatcc?ctgtgacccc?tccccagtgc?ctctcctggc?cctggaagtt?gccactccag????780

tgcccaccag?ccttgtccta?ataaaattaa?gttgcatcat?tttgtctgac?taggtgtcct????840

tctataatat?tatggggtgg?aggggggtgg?tatggagcaa?ggggcaagtt?gggaagacaa????900

cctgtagggc?ctgcggggtc?tattgggaac?caagctggag?tgcagtggca?caatcttggc????960

tcactgcaat?ctccgcctcc?tgggttcaag?cgattctcct?gcctcagcct?cccgagttgt????1020

tgggattcca?ggcatgcatg?accaggctca?gctaattttt?gtttttttgg?tagagacggg????1080

gtttcaccat?attggccagg?ctggtctcca?actcctaatc?tcaggtgatc?tacccacctt????1140

ggcctcccaa?attgctggga?ttacaggcgt?gaaccactgc?tcccttccct?gtccttctga????1200

ttttaaaata?actataccag?caggaggacg?tccagacaca?gcataggcta?cctggccatg????1260

cccaaccggt?gggacatttg?agttgcttgc?ttggcactgt?cctctcatgc?gttgggtcca????1320

ctcagtagat?gcctgttgaa?ttcgataccg?tcgacctcga?gggggggccc?ggtaccagct????1380

tttgttccct?ttagtgaggg?ttaatttcga?gcttggcgta?atcatggtca?tagctgtttc????1440

ctgtgtgaaa?ttgttatccg?ctcacaattc?cacacaacat?acgagccgga?agcataaagt????1500

gtaaagcctg?gggtgcctaa?tgagtgagct?aactcacatt?aattgcgttg?cgctcactgc????1560

ccgctttcca?gtcgggaaac?ctgtcgtgcc?agctgcatta?atgaatcggc?caacgcgcgg????1620

ggagaggcgg?tttgcgtatt?gggcgctctt?ccgcttcctc?gctcactgac?tcgctgcgct????1680

cggtcgttcg?gctgcggcga?gcggtatcag?ctcactcaaa?ggcggtaata?cggttatcca????1740

cagaatcagg?ggataacgca?ggaaagaaca?tgtgagcaaa?aggccagcaa?aaggccagga????1800

accgtaaaaa?ggccgcgttg?ctggcgtttt?tccataggct?ccgcccccct?gacgagcatc????1860

acaaaaatcg?acgctcaagt?cagaggtggc?gaaacccgac?aggactataa?agataccagg????1920

cgtttccccc?tggaagctcc?ctcgtgcgct?ctcctgttcc?gaccctgccg?cttaccggat????1980

acctgtccgc?ctttctccct?tcgggaagcg?tggcgctttc?tcatagctca?cgctgtaggt????2040

atctcagttc?ggtgtaggtc?gttcgctcca?agctgggctg?tgtgcacgaa?ccccccgttc????2100

agcccgaccg?ctgcgcctta?tccggtaact?atcgtcttga?gtccaacccg?gtaagacacg????2160

acttatcgcc?actggcagca?gccactggta?acaggattag?cagagcgagg?tatgtaggcg????2220

gtgctacaga?gttcttgaag?tggtggccta?actacggcta?cactagaaga?acagtatttg????2280

gtatctgcgc?tctgctgaag?ccagttacct?tcggaaaaag?agttggtagc?tcttgatccg????2340

gcaaacaaac?caccgctggt?agcggtggtt?tttttgtttg?caagcagcag?attacgcgca????2400

gaaaaaaagg?atctcaagaa?gatcctttga?tcttttctac?ggggtctgac?gctcagaaga????2460

actcgtcaag?aaggcgatag?aaggcgatgc?gctgcgaatc?gggagcggcg?ataccgtaaa????2520

gcacgaggaa?gcggtcagcc?cattcgccgc?caagctcttc?agcaatatca?cgggtagcca????2580

acgctatgtc?ctgatagcgg?tccgccacac?ccagccggcc?acagtcgatg?aatccagaaa????2640

agcggccatt?ttccaccatg?atattcggca?agcaggcatc?gccatgggtc?acgacgagat????2700

cctcgccgtc?gggcatgcgc?gccttgagcc?tggcgaacag?ttcggctggc?gcgagcccct????2760

gatgctcttc?gtccagatca?tcctgatcga?caagaccggc?ttccatccga?gtacgtgctc????2820

gctcgatgcg?atgtttcgct?tggtggtcga?atgggcaggt?agccggatca?agcgtatgca????2880

gccgccgcat?tgcatcagcc?atgatggata?ctttctcggc?aggagcaagg?tgagatgaca????2940

ggagatcctg?ccccggcact?tcgcccaata?gcagccagtc?ccttcccgct?tcagtgacaa????3000

cgtcgagcac?agctgcgcaa?ggaacgcccg?tcgtggccag?ccacgatagc?cgcgctgcct????3060

cgtcctgcag?ttcattcagg?gcaccggaca?ggtcggtctt?gacaaaaaga?accgggcgcc????3120

cctgcgctga?cagccggaac?acggcggcat?cagagcagcc?gattgtctgt?tgtgcccagt????3180

catagccgaa?tagcctctcc?acccaagcgg?ccggagaacc?tgcgtgcaat?ccatcttgtt????3240

caatcatgcg?aaacgatcct?catcctgtct?cttgatcaga?tcttgatccc?ctgcgccatc????3300

agatccttgg?cggcaagaaa?gccatccagt?ttactttgca?gggcttccca?accttaccag????3360

agggcgcccc?agctggcaat?tccggttcgc?ttgctgtcca?taaaaccgcc?cagtctagca????3420

actgttggga?agggcgatcg?gtgcgggcct?cttcgctatt?acgccagctg?gcgaaagggg????3480

gatgtgctgc?aaggcgatta?agttgggtaa?cgccagggtt?ttcccagtca?cgac??????????3534

<210>10

<211>2192

<212>DNA

＜213〉synthetic

<400>10

cgataccgtc?gacctcgagg?gggggcccgg?taccagcttt?tgttcccttt?agtgagggtt??????60

aatttcgagc?ttggcgtaat?catggtcata?gctgtttcct?gtgtgaaatt?gttatccgct?????120

cacaattcca?cacaacatac?gagccggaag?cataaagtgt?aaagcctggg?gtgcctaatg?????180

agtgagctaa?ctcacattaa?ttgcgttgcg?ctcactgccc?gctttccagt?cgggaaacct?????240

gtcgtgccag?ctgcattaat?gaatcggcca?acgcgcgggg?agaggcggtt?tgcgtattgg?????300

gcgctcttcc?gcttcctcgc?tcactgactc?gctgcgctcg?gtcgttcggc?tgcggcgagc?????360

ggtatcagct?cactcaaagg?cggtaatacg?gttatccaca?gaatcagggg?ataacgcagg?????420

aaagaacatg?tgagcaaaag?gccagcaaaa?ggccaggaac?cgtaaaaagg?ccgcgttgct?????480

ggcgtttttc?cataggctcc?gcccccctga?cgagcatcac?aaaaatcgac?gctcaagtca?????540

gaggtggcga?aacccgacag?gactataaag?ataccaggcg?tttccccctg?gaagctccct?????600

cgtgcgctct?cctgttccga?ccctgccgct?taccggatac?ctgtccgcct?ttctcccttc?????660

gggaagcgtg?gcgctttctc?atagctcacg?ctgtaggtat?ctcagttcgg?tgtaggtcgt?????720

tcgctccaag?ctgggctgtg?tgcacgaacc?ccccgttcag?cccgaccgct?gcgccttatc?????780

cggtaactat?cgtcttgagt?ccaacccggt?aagacacgac?ttatcgccac?tggcagcagc?????840

cactggtaac?aggattagca?gagcgaggta?tgtaggcggt?gctacagagt?tcttgaagtg?????900

gtggcctaac?tacggctaca?ctagaagaac?agtatttggt?atctgcgctc?tgctgaagcc?????960

agttaccttc?ggaaaaagag?ttggtagctc?ttgatccggc?aaacaaacca?ccgctggtag????1020

cggtggtttt?tttgtttgca?agcagcagat?tacgcgcaga?aaaaaaggat?ctcaagaaga????1080

tcctttgatc?ttttctacgg?ggtctgacgc?tcagaagaac?tcgtcaagaa?ggcgatagaa????1140

ggcgatgcgc?tgcgaatcgg?gagcggcgat?accgtaaagc?acgaggaagc?ggtcagccca????1200

ttcgccgcca?agctcttcag?caatatcacg?ggtagccaac?gctatgtcct?gatagcggtc????1260

cgccacaccc?agccggccac?agtcgatgaa?tccagaaaag?cggccatttt?ccaccatgat????1320

attcggcaag?caggcatcgc?catgggtcac?gacgagatcc?tcgccgtcgg?gcatgcgcgc????1380

cttgagcctg?gcgaacagtt?cggctggcgc?gagcccctga?tgctcttcgt?ccagatcatc????1440

ctgatcgaca?agaccggctt?ccatccgagt?acgtgctcgc?tcgatgcgat?gtttcgcttg????1500

gtggtcgaat?gggcaggtag?ccggatcaag?cgtatgcagc?cgccgcattg?catcagccat????1560

gatggatact?ttctcggcag?gagcaaggtg?agatgacagg?agatcctgcc?ccggcacttc????1620

gcccaatagc?agccagtccc?ttcccgcttc?agtgacaacg?tcgagcacag?ctgcgcaagg????1680

aacgcccgtc?gtggccagcc?acgatagccg?cgctgcctcg?tcctgcagtt?cattcagggc????1740

accggacagg?tcggtcttga?caaaaagaac?cgggcgcccc?tgcgctgaca?gccggaacac????1800

ggcggcatca?gagcagccga?ttgtctgttg?tgcccagtca?tagccgaata?gcctctccac????1860

ccaagcggcc?ggagaacctg?cgtgcaatcc?atcttgttca?atcatgcgaa?acgatcctca????1920

tcctgtctct?tgatcagatc?ttgatcccct?gcgccatcag?atccttggcg?gcaagaaagc????1980

catccagttt?actttgcagg?gcttcccaac?cttaccagag?ggcgccccag?ctggcaattc????2040

cggttcgctt?gctgtccata?aaaccgccca?gtctagcaac?tgttgggaag?ggcgatcggt????2100

gcgggcctct?tcgctattac?gccagctggc?gaaaggggga?tgtgctgcaa?ggcgattaag????2160

ttgggtaacg?ccagggtttt?cccagtcacg?ac??????????????????????????????????2192

<210>11

<211>308

<212>DNA

＜213〉pig

<400>11

ggatcccaag?gcccaactcc?ccgaaccact?cagggtcctg?tggacagctc?acctagctgc??????60

catggtgctc?tgggtgttct?tctttgtgat?cctcaccctc?agcaacagct?cccactgctc?????120

cccacctccc?cctttgaccc?tcaggatgcg?gcggcacgta?gatgccatct?tcaccaacag?????180

ctaccggaag?gtgctggccc?agctgtccgc?ccgcaagctg?ctccaggaca?tcctgaacag?????240

gcagcaggga?gagaggaacc?aagagcaagg?agcataatga?ctgcaggaat?tcgatatcaa?????300

gcttatcg??????????????????????????????????????????????????????308

25100478????-?1?-

Claims

1. improve or strengthen the method for jenny offspring growth, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of this jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and the wherein introducing of this carrier and improvement or the enhancing that expression causes this offspring's growth.

2. the process of claim 1 wherein that the cell of this jenny comprises diploid cell.

3. the process of claim 1 wherein that the cell of this jenny comprises the myocyte.

4. the process of claim 1 wherein this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.

5. the method for claim 4, wherein this growth hormone releasing hormone is SEQ IDNO:1, SEQ ID NO:8 or its analogue separately.

6. the process of claim 1 wherein that this promotor comprises synthetic myogenic promotor.

7. the process of claim 1 wherein that this 3 ' non-translational region comprises hGH 3 ' non-translational region.

8. the process of claim 1 wherein this carrier by electroporation, through virus vector, combine, be incorporated in this cell of this jenny with carrier (carrier) by parenteral route or its combination.

9. the process of claim 1 wherein that this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.

10. the process of claim 1 wherein that this jenny is people, pig, cow, sheep, goat or chicken.

11. the process of claim 1 wherein that this carrier is plasmid, virus vector, liposome, cation lipid or its combination.

12. the process of claim 1 wherein this carrier in single administration, introduce this female in.

13. the process of claim 1 wherein that this takes place when being introduced in the 3rd phase in March of nourishing this offspring.

14. the method for claim 1, it further comprises to this female step of using the part of secretagogue receptor.

15. the method for claim 14, wherein this part dosage forms for oral administration.

16. increase the method for jenny offspring body inner growth hormone level, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of this jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of this carrier cause offspring's body inner growth hormone level to increase.

17. the method for claim 16, wherein the cell of this jenny comprises diploid cell.

18. the method for claim 16, wherein the cell of this jenny comprises the myocyte.

19. the method for claim 16, wherein this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.

20. the method for claim 19, wherein this growth hormone releasing hormone is SEQ IDNO:1, SEQ ID NO:8 or its analogue separately.

21. the method for claim 16, wherein this promotor comprises synthetic myogenic promotor.

22. the method for claim 16, wherein this 3 ' non-translational region comprises hGH 3 ' non-translational region.

23. the method for claim 16, wherein this carrier by electroporation, through virus vector, combine with carrier, be incorporated in this cell of this jenny by parenteral route or its combination.

24. the method for claim 16, wherein this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.

25. the method for claim 16, wherein this jenny is people, pig, cow, sheep, goat or chicken.

26. the method for claim 16, wherein this carrier is plasmid, virus vector, liposome, cation lipid or its combination.

27. the method for claim 16, wherein this carrier in single administration, introduce this female in.

28. the method for claim 16 takes place when wherein this is introduced in the 3rd phase in March of nourishing this offspring.

29. the method for claim 16, it further comprises to this female step of using the part of secretagogue receptor.

30. the method for claim 29, wherein this part dosage forms for oral administration.

31. increase the method for jenny offspring's lean mass, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of this jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause offspring's lean mass to increase.

32. the method for claim 31, wherein the cell of this jenny comprises diploid cell.

33. the method for claim 31, wherein the cell of this jenny comprises the myocyte.

34. the method for claim 31, wherein this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.

35. the method for claim 34, wherein this growth hormone releasing hormone is SEQ IDNO:1, SEQ ID NO:8 or its analogue separately.

36. the method for claim 31, wherein this promotor comprises synthetic myogenic promotor.

37. the method for claim 31, wherein this 3 ' non-translational region comprises hGH 3 ' non-translational region.

38. the method for claim 31, wherein this carrier by electroporation, through virus vector, combine with carrier, be incorporated in this cell of this jenny by parenteral route or its combination.

39. the method for claim 31, wherein this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.

40. the method for claim 31, wherein this jenny is people, pig, cow, sheep, goat or chicken.

41. the method for claim 31, wherein this carrier is plasmid, virus vector, liposome, cation lipid or its combination.

42. the method for claim 31, wherein this carrier in single administration, introduce this female in.

43. the method for claim 31 takes place when wherein this is introduced in the 3rd phase in March of nourishing this offspring.

44. the method for claim 31, it further comprises to this female step of using the part of secretagogue receptor.

45. the method for claim 44, wherein this part dosage forms for oral administration.

46. increase the method for jenny offspring's IGF-I level, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of this jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause offspring IGF-I level to increase.

47. the method for claim 46, wherein the cell of this jenny comprises diploid cell.

48. the method for claim 46, wherein the cell of this jenny comprises the myocyte.

49. the method for claim 46, wherein this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.

50. the method for claim 49, wherein this growth hormone releasing hormone is SEQ IDNO:1, SEQ ID NO:8 or its analogue separately.

51. the method for claim 46, wherein this promotor comprises synthetic myogenic promotor.

52. the method for claim 46, wherein this 3 ' non-translational region comprises hGH 3 ' non-translational region.

53. the method for claim 46, wherein this carrier by electroporation, through virus vector, combine with carrier, be incorporated in this cell of this jenny by parenteral route or its combination.

54. the method for claim 46, wherein this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.

55. the method for claim 46, wherein this jenny is people, pig, cow, sheep, goat or chicken.

56. the method for claim 46, wherein this carrier is plasmid, virus vector, liposome, cation lipid or its combination.

57. the method for claim 46, wherein this carrier in single administration, introduce this female in.

58. the method for claim 46 takes place when wherein this is introduced in the 3rd phase in March of nourishing this offspring.

59. the method for claim 46, it further comprises to this female step of using the part of secretagogue receptor.

60. the method for claim 59, wherein this part dosage forms for oral administration.

61. increase the method for jenny offspring feeding efficiency, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of this jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause offspring's feeding efficiency to increase.

62. the method for claim 61, wherein the cell of this jenny comprises diploid cell.

63. the method for claim 61, wherein the cell of this jenny comprises the myocyte.

64. the method for claim 61, wherein this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.

65. the method for claim 64, wherein this growth hormone releasing hormone is SEQ IDNO:1, SEQ ID NO:8 or its analogue separately.

66. the method for claim 61, wherein this promotor comprises synthetic myogenic promotor.

67. the method for claim 61, wherein this 3 ' non-translational region comprises hGH 3 ' non-translational region.

68. the method for claim 61, wherein this carrier by electroporation, through virus vector, combine with carrier, be incorporated in this cell of this jenny by parenteral route or its combination.

69. the method for claim 61, wherein this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.

70. the method for claim 61, wherein this jenny is people, pig, cow, sheep, goat or chicken.

71. the method for claim 61, wherein this carrier is plasmid, virus vector, liposome, cation lipid or its combination.

72. the method for claim 61, wherein this carrier in single administration, introduce this female in.

73. the method for claim 61 takes place when wherein this is introduced in the 3rd phase in March of nourishing this offspring.

74. the method for claim 61, it further comprises to this female step of using the part of secretagogue receptor.

75. the method for claim 74, wherein this part dosage forms for oral administration.

76. increase the method for the jenny offspring speed of growth, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of this jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause offspring's speed of growth to increase.

77. the method for claim 76, wherein the cell of this jenny comprises diploid cell.

78. the method for claim 76, wherein the cell of this jenny comprises the myocyte.

79. the method for claim 76, wherein this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.

80. the method for claim 79, wherein this growth hormone releasing hormone is SEQ IDNO:1, SEQ ID NO:8 or its analogue separately.

81. the method for claim 76, wherein this promotor comprises synthetic myogenic promotor.

82. the method for claim 76, wherein this 3 ' non-translational region comprises hGH 3 ' non-translational region.

83. the method for claim 76, wherein this carrier by electroporation, through virus vector, combine with carrier, be incorporated in this cell of this jenny by parenteral route or its combination.

84. the method for claim 76, wherein this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.

85. the method for claim 76, wherein this jenny is people, pig, cow, sheep, goat or chicken.

86. the method for claim 76, wherein this carrier is plasmid, virus vector, liposome, cation lipid or its combination.

87. the method for claim 76, wherein this carrier in single administration, introduce this female in.

88. the method for claim 76 takes place when wherein this is introduced in the 3rd phase in March of nourishing this offspring.

89. the method for claim 76, it further comprises to this female step of using the part of secretagogue receptor.

90. the method for claim 89, wherein this part dosage forms for oral administration.

91. increase somatotroph in jenny offspring's the pituitary body relatively other hormone produce the method for the ratio of cell, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of this jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, condition of living in express for nucleotide sequence wherein and wherein the introducing of carrier cause relative other hormones of offspring's somatotroph to produce the ratio increase of cells with expression.

92. the method for claim 91, wherein the cell of this jenny comprises diploid cell.

93. the method for claim 91, wherein the cell of this jenny comprises the myocyte.

94. the method for claim 91, wherein this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.

95. the method for claim 94, wherein this growth hormone releasing hormone is SEQ IDNO:1, SEQ ID NO:8 or its analogue separately.

96. the method for claim 91, wherein this promotor comprises synthetic myogenic promotor.

97. the method for claim 91, wherein this 3 ' non-translational region comprises hGH 3 ' non-translational region.

98. the method for claim 91, wherein this carrier by electroporation, through virus vector, combine with carrier, be incorporated in this cell of this jenny by parenteral route or its combination.

99. the method for claim 91, wherein this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.

100. the method for claim 91, wherein this jenny is people, pig, cow, sheep, goat or chicken.

101. the method for claim 91, wherein this carrier is plasmid, virus vector, liposome, cation lipid or its combination.

102. the method for claim 91, wherein this carrier in single administration, introduce this female in.

103. the method for claim 91 takes place when wherein this is introduced in the 3rd phase in March of nourishing this offspring.

104. the method for claim 91, it further comprises to this female step of using the part of secretagogue receptor.

105. the method for claim 104, wherein this part dosage forms for oral administration.

106. the method for claim 91, wherein said hormone is produced cell and is selected from corticotroph, lactotroph and gonadotroph.

107. postpone the method for jenny offspring birth, this method be included in nourish before this offspring or during the carrier of significant quantity is incorporated into the intracellular step of this jenny, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing of carrier and expression cause offspring's delay of being born.

108. the method for claim 107, wherein the cell of this jenny comprises diploid cell.

109. the method for claim 107, wherein the cell of this jenny comprises the myocyte.

110. the method for claim 107, wherein this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.

111. the method for claim 110, wherein this growth hormone releasing hormone is SEQ IDNO:1, SEQ ID NO:8 or its analogue separately.

112. the method for claim 107, wherein this promotor comprises synthetic myogenic promotor.

113. the method for claim 107, wherein this 3 ' non-translational region comprises hGH 3 ' non-translational region.

114. the method for claim 107, wherein this carrier by electroporation, through virus vector, combine with carrier, be incorporated in this cell of this jenny by parenteral route or its combination.

115. the method for claim 107, wherein this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.

116. the method for claim 107, wherein this jenny is people, pig, cow, sheep, goat or chicken.

117. the method for claim 107, wherein this carrier is plasmid, virus vector, liposome, cation lipid or its combination.

118. the method for claim 107, wherein this carrier in single administration, introduce this female in.

119. the method for claim 107 takes place when wherein this is introduced in the 3rd phase in March of nourishing this offspring.

120. the method for claim 107, this method further comprise to this female step of using the part of secretagogue receptor.

121. the method for claim 120, wherein this part dosage forms for oral administration.

122. increase the method that animal milk is produced, comprise that the carrier with significant quantity is incorporated into the intracellular step of this animal, wherein this carrier comprises promotor, nucleotide sequence and 3 ' non-translational region, and condition of living in is expressed for nucleotide sequence wherein and wherein the introducing and the expression of carrier cause animal milk production to increase.

123. the method for claim 122, wherein the cell of this jenny comprises diploid cell.

124. the method for claim 122, wherein the cell of this jenny comprises muscle cell.

125. the method for claim 122, wherein this nucleic acid sequence encoding growth hormone releasing hormone or its analogue.

126. the method for claim 125, wherein this growth hormone releasing hormone is SEQ IDNO:1, SEQ ID NO:8 or its analogue separately.

127. the method for claim 122, wherein this promotor comprises synthetic myogenic promotor.

128. the method for claim 122, wherein this 3 ' non-translational region comprises hGH 3 ' non-translational region.

129. the method for claim 122, wherein this carrier by electroporation, through virus vector, combine with carrier, be incorporated in this cell of this jenny by parenteral route or its combination.

130. the method for claim 122, wherein this jenny is that animal is used in people, pet animals, farm-animals, edible animal or work.

131. the method for claim 122, wherein this jenny is people, pig, cow, sheep, goat or chicken.

132. the method for claim 122, wherein this carrier is plasmid, virus vector, liposome, cation lipid or its combination.

133. the method for claim 122, wherein this carrier in single administration, introduce this female in.

134. the method for claim 122 takes place when wherein this is introduced in the 3rd phase in March of nourishing this offspring.

135. the method for claim 122, it further comprises to this female step of using the part of secretagogue receptor.

136. the method for claim 135, wherein this part dosage forms for oral administration.