KR100358754B1 - The production method of transgenic porcine producing human erythropoietin and the transgenic porcine - Google Patents

Abstract

The present invention relates to the production of transgenic pigs (TRANSGENIC PORCINE) capable of producing a large amount of human hematopoietic promoter (EPO: erythropoietin) in pig mammary gland using genetic engineering techniques, which are genetic engineering methods. Injects and transforms the hematopoietic promoter (EPO) gene present in human kidneys into the fertilized egg of pigs to produce pigs with human hematopoietic promoters (EPO) genes (Saromyi), It is an invention having the characteristics that can contribute to the advancement of human health as well as to contribute to the development of the national economy and national competitiveness through the export of expensive medicines by enabling the secretion of human hematopoietic accelerators in large quantities.

The production of transgenic pigs for the production of human hematopoietic promoter (EPO) of the present invention is characterized in that the gene sequence is configured as shown in FIG.

Description

TECHNICAL METHOD OF TRANSGENIC PORCINE PRODUCING HUMAN ERYTHROPOIETIN AND THE TRANSGENIC PORCINE}

The present invention is a genetic engineering method, that is, a genetic recombination technique that takes a part of a gene (DNA; deoxyribonucleic acid) and attaches it to another DNA molecule to make a gene. The present invention relates to the production of transgenic pigs (males) capable of producing a large amount of hematopoietic promoters (EPO :: erythropoietin), and more specifically, the injection of hematopoietic promoter (EPO) genes present in human kidneys into pig embryos, By transforming, it produces pigs with human hematopoietic promoter (EPO) gene (also known as 'Saromei') and secretes a large amount of human hematopoietic promoter (EPO), which is an expensive medicine, through its milk. By doing so, it can contribute to the progress of human health and can contribute greatly to the establishment of the national economy and national competitiveness base through the export of expensive medicines. Invention.

In general, the lifespan of human erythrocytes is about 120 days, and about 120 minutes of the total erythrocytes are destroyed in the reticuloendothelial system every day, while a relatively constant number of erythrocytes are generated daily to maintain a constant concentration of erythrocytes (Guyton, Textbook of Medical Physiology, pp 56-60, WBSaunders Co., Philadelphia (1976).

Erythrocytes are produced by the maturation and differentiation of erythrocytes in the bone marrow and hematopoietic promoters (EPO) act on less differentiated cells to induce differentiation into erythrocytes. (Guyton, same as above).

Stem promoters (EPO) is to be ₅₉ Fe is inserted a lot of red cells that are newly formed when injecting the plasma of anemic animals to normal animals in the 1950s, it was found by being observed (Borsook, et, al., Blood, 9, 734 ( 1954), glycoproteins produced mainly in the kidneys of adults in the absence of oxygen in the atmosphere or increased cell numbers in bleeding or anemia play an important role in regulating erythropoiesis and maintaining erythrocyte counts. (Carnot, et al., Comot.Rend. 143, 384 (1906); Kranz. SB, Blood 77, 419 (1991); Goldwasser, E. et al., In Peptide Growth Factors and their Receptors I, Sporn, MB and AB Roberts, eds., Springer-Verlag, Berlin, p. 747 (1990)).

As is well known to those skilled in the art, natural hematopoietic promoters (EPO), hormones that regulate red blood cell production, begin to be secreted from the liver of the fetus and begin to migrate to the kidney between 120 and 140 days of gestation and are completely at 40 days postpartum. The migration is completed and in adults it is mainly produced in the kidneys, but about 10% of the total amount is known to be produced in the liver, in addition to the small amount is known to be produced in the macrophage of the bone marrow (bone marrow).

Hematopoietic promoters (EPO) are maintained at about 15 to 30 mU or 0.01 mM per 1 ml of normal human blood (Garcia, JF, Lab. Clin. Med. 99, 624-635 (1982)) and diseases such as aplastic anemia In patients with this condition, hematopoietic promoters (EPO) are produced at higher concentrations than normal people, and their blood or urine may be used for the production of hematopoietic promoters (EPO) (White, et al., Rec. Prog. Horm. Res. 16, 219 (1960); Espada, et al., Biochem. Med. 3, 475 (1970); Fisher, Pharmacol. Rev. 24, 459 (1972).

Hematopoietic promoter (EPO) is a glycoprotein hormone that has a molecular weight of about 30 Kd, has an N-linked sugar chain at amino acids 24, 38 and 83, and an O-linked sugar chain at the amino acid 126. (PSEBM 216, 358-369 (1997)), but conventionally used recombinant hematopoietic promoters (EPO) produced in animal cells, but the secretion amount is too small and the physiological activity is not the same as the natural type, and low problems Have.

Hematopoietic promoters (EPO) are preferably prepared from human-derived raw materials due to promising therapeutics or antigenic problems for the clinical treatment of anemia, especially renal anemia, and therefore a large amount of hematopoietic promoters (EPO) as described above. It can be obtained by using blood or urine of a patient suffering from diseases such as aplastic anemia that secrete thyroid. However, the amount is extremely limited overall.

Hematopoietic promoters (EPO) can also be recovered from animal plasma, but hematopoietic promoters (EPO) isolated from sheep plasma provide somewhat satisfactory titers and stable water-soluble agents, but can also act as antigens for the human body. Implies

Overseas, Cuban biotechnology company used human hematopoietic promoter (hEPO) cDNA to produce transgenic animals expressing hematopoietic promoter (hEPO) in rabbit's mammary gland tissue, and Kuopioeogkr of Finland Although there have been studies on the production of hemopoietic promoter (hEPO) cDNA transgenic animals in the mammary gland, the production of transgenic animals in pigs has not been reported so far. In Korea, as in Korean Patent Publication No. 93-5917, human hematopoiesis has been reported. Cloning (cloning) of the promoter (EPO) gene has been developed to express it in animal cells or insect cells.However, in this method, the hematopoietic promoter (EPO) expression level is weak or glycosylation does not occur precisely and is rapidly degraded in vivo. Korean Patent Application No. 94-12082 discloses a modified recombinant human hematopoietic factor (rhEPO) gene. The expression vector was developed. The animal cell line COS-7 (ATCC CRL 1651, African Green Monkey Kidney Cell) transformed with the expression vector was able to produce a large amount of recombinant human hematopoietic factor (rhEPO). Due to the hassle of transforming, there was an unsuitable problem in mass production.

In addition, a permanent cell line transformed with a recombinant human erythropoietic factor (rhEPO) by a mass expression vector can be recombined with Korean Patent Registration No. 0184778, which can efficiently and stably produce recombinant human erythropoietic factor (rhEPO) in animal cells. There is a method for producing human erythropoietic factor (rhEPO), but is different from the present invention for producing human erythropoietin (rhEPO) in pig milk.

The present invention was devised to solve the conventional problems as described above, and a main object of the present invention is to produce a transgenic animal usefully producing a hematopoietic promoter (EPO), which is a human hematopoietic factor.

Another object of the present invention is to produce a large amount of hematopoietic promoter (EPO), a human hematopoietic factor through the milk of a transgenic animal, thereby promoting human health welfare, converting livestock into a high value-added industry The purpose is to sleep.

Another object of the present invention is to increase the economics through export to the world market by producing a large amount of expensive hematopoietic accelerator (EPO).

1 is a schematic diagram showing a process of producing a transgenic pig,

Figure 2 is a polymerase chain reaction (PCR) photograph,

Figure 3 is a diagram showing the cDNA sequence of the transformed pig.

In order to achieve the above object, in the present invention, a transgenic pig having a human hematopoietic promoter (EPO) gene by injecting and transforming a hematopoietic promoter (EPO) gene present in human kidney into a fertilized egg of pigs (aka: Salom) produces and provides a method for producing a large amount of expensive hematopoietic promoters (EPO) through the pig's milk.

Hereinafter, the production of the transgenic pigs of the present invention will be described in more detail with reference to the drawings, and in the following description of the present invention, a detailed description of related known functions or configurations will unnecessarily obscure the gist of the present invention. If it can be determined that the detailed description will be omitted. Terms to be described later are terms defined in consideration of functions in the present invention, and may be changed according to intentions or customs of users or operators. Therefore, the definition should be made based on the contents throughout the specification.

Figure 1 is a schematic diagram of the process until the present invention was established, according to the description, in order to produce a transgenic pig for the production of human hematopoietic promoter (EPO) of the present invention, the hematopoietic promoter which is a genetic recombinant of the present invention first To prepare (EPO), a human hematopoietic promoter (EPO) genome DNA was provided by Professor Kim Jin Hoe of the Faculty of Animal Science, Gyeongsang National University, Korea, which produced a very small amount of 2.6kb of WAP promoter from mouse mammary gland. After cloning using a polymerare chain reaction (PCR) that amplifies and copies a large number of genes, human erythropoietic factor (hEPO) and SV40 poly A genes are prepared and plasmid A hematopoietic promoter (EPO) transformed expression vector was constructed. As a result, it was as shown in <Table 1>.

Hematopoietic promoter (EPO) transformation expression vector 2.6 kb 2.5kb 2.6 kb rat WAP promoter hEPO genome SV40 Poly A

Porcine fertilized eggs were intramuscularly injected with ovum serum gonadotropin (eCG) and placental maternal gonadotropin (hCG), which are hyperovulation inducer hormones, to induce overovulation and to establish natural estrus after confirmation of estrus. The above-described gene (DNA) was injected into the male pronucleus using a manipulator, and then immediately implanted into surrogate sows, and the tail, blood, and semen (sperm) of one of the pigs delivered through the surrogate sows. It was confirmed through the polymerare chain reaction (PCR) photographic result of FIG. 2 that the gene (DNA) encoding human hematopoietic promoter gene (EPO DNA) was transferred from.

Table 2 shows primer sequences according to the polymerare chain reaction (PCR).

primer Sequences Expected Size hEPO-304 F 5'- CGA GAA TAT CAC GGT AGA ACC -3'R 5'- CTC ATT CAA GCT GCA GTG TTC -3 ' 304 bp hEPO-567 F 5'- AAG TGG TGC ATG GTG GTA GTC -3'R 5'- TTA CAG AAA GGG CAA GCA GAA -3 ' 567 bp

Table 3 shows the results of analysis of blood collected from hematopoietic promoters (EPO) transgenic pigs.

division Erythrocyte count (x10 ⁶ / ul) Erythrocyte volume (%) Control transformation 4.63 (100) 5.25 (113) 66.5 (100) 78.3 (118)

The transfer of DNA encoding human hematopoietic promoter gene (EPO DNA) in pigs delivered through surrogate sows was confirmed through the polymerare chain reaction (PCR) photograph of FIG. 2. The nucleotide sequence is as follows.

(Sequence list)

SEQ ID NO: 1

Sequence length: 582

Type of sequence: nucleic acid

Number of chains: 2 chains

Topology: linear

Type of molecule: cDNA

origin

Biological name: EPO cDNA isolated from human liver gene (DNA)

Characteristic of the sequence

Characteristic symbols: sig peptide

Location: 1-81

Characteristic symbols: mat peptide

Location: 82-579

Characteristic symbols: terminator

Location: 580-582

(SEQ ID NO: 1)

ATG GGG GTG CAC GAA TGT CCT GCC TGG CTG TGG CTT CTC CTG TCC 45

Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser

-27 -20

CTG CTG TCG CTC CCT CTG GGC CTC CCA GTC CTG GGC GCC CCA CCA 90

Leu Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Prp Pro

-10 +1

CGC CTC ATC TGT GAC AGC CGA GTC CTG GAG AGG TAC CTC TTG GAG 135

Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu

10

GCC AAG GAG GCC GAG AAT ATC ACG ACG GGC TGT GCT GAA CAC TGC 180

Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys

20 30

AGC TTG AAT GAG AAT ATC ACT GTC CCA GAC ACC AAA GTT AAT TTC 225

Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe

40

TAT GCC TGG AAG AGG ATG GAG GTC GGG CAG CAG GCC GTA GAA GTC 270

Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val

50 60

TGG CAG GGC CTG GCC CTG CTG TCG GAA GCT GTC CTG CGG GGC CAG 315

Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln

70

GCC CTG TTG GTC AAC TCT TCC CAG CCG TGG GAG CCC CTG CAG CTG 360

Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu

80 90

CAT GTG GAT AAA GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT CTG 405

His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu

100

CTT CGG GCT CTG GGA GCC CAG AAG GAA GCC ATC TCC CCT CCA GAT 450

Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp

110 120

GCG GCC TCA GCT GCT CCA CTC CGA ACA ATC ACT GCT GAC ACT TTC 495

Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe

130

CGC AAA CTC TTC CGA GTC TAC TCC AAT TTC CTC CGG GGA AAG CTG 540

Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu

140 150

AAG CTG TAC ACA GGG GAG GCC TGC AGG ACA GGG GAC AGA TGA 582

Lys Leu Tyr Thr Gly Gly Ala Cys Arg Thr Gly Asp Arg

160

The present invention can be variously modified and can take various forms and only the specific embodiments thereof are described in the detailed description of the invention. It is to be understood, however, that the present invention is not limited to the specific forms referred to in the description, but rather includes all modifications, equivalents, and substitutions within the spirit and scope of the invention as defined by the appended claims. Should be.

As such, the present invention can not only produce a transgenic animal usefully producing hematopoietic promoter (EPO), which is a human hematopoietic factor, but also a hematopoietic promoter (EPO) which is a human hematopoietic factor through the milk of the transformed pig. It is obvious that it is a very advantageous invention that can be produced in large quantities and can increase economics through export by producing hematopoietic promoters (EPO), which are expensive drugs, in large quantities.

Another effect of the present invention is to promote human health and welfare through the mass production of hematopoietic accelerators, and to turn the livestock industry into a high value-added industry.

Claims (9)

In the production of transgenic animals, Human hematopoietic promoter (EPO) genome DNA collected from humans was cloned from the mouse mammary gland using a polymerase chain reaction (PCR) of 2.6 kb of WAP promoter, followed by human erythrocytes. Hemopoietic gonadotropin (eCG) and placental maternal gonadotropin (hCG), which are hyperovulation-inducing hormone hormones (eCG) and placental maternal gonadotropin ) Intramuscular injection to induce superovulation to confirm estrus spontaneous cultivation, 1 cell stage fertilized egg was recovered and the gene (DNA) shown above was injected into male pronucleus using a manipulator and immediately transplanted into surrogate sows. And then to produce a transgenic pig that produces a human hematopoietic promoter with a gene represented by the nucleotide sequence of SEQ ID NO: 1, which is delivered through surrogate sows. Way. Transgenic pigs produced by the method. The method according to claim 1, A method of producing a transgenic pig that produces a hematopoietic promoter in human whose expression vector is the DNA plasmid shown in Table 1. The method according to claim 1, Method of producing a transgenic pig that produces a human hematopoietic promoter having a nucleotide sequence (DNA) sequence shown in Figure 3. The method according to claim 2, Transgenic pigs that produce human hematopoietic promoters with semen to which the DNA of WAP-EPO has been transferred. The method according to claim 2, A transgenic pig that produces a human hematopoietic promoter whose human hematopoietic promoter is WAP-EPO. The method according to claim 2, A transgenic pig that produces a human hematopoietic promoter whose name is Sarome. The method according to claim 2, Transgenic pigs whose offspring through transgenic pigs produce human hematopoietic promoters with the WAP-EPO gene (DNA). The method according to any one of claims 1 to 8, Transgenic pigs that produce human hematopoietic promoters for making medicines comprising hematopoietic promoters produced by the pigs.