KR20010018852A - Plasmid(pGbc5.5hGH, KCTC 8949P) tranforming animals to produce human growth hormone from their milk - Google Patents

Abstract

PURPOSE: A plasmid pGbc5.5hGH(KCTC 8949P) used for transforming mammals is provided to stably produce milk containing human growth hormone from the mammary gland of the transformed mammals, in a high yield. CONSTITUTION: The plasmid pGbc5.5hGH(KCTC 8949P) is produced by inserting human growth hormone gene into plasmid pBluescript KSII to prepare plasmid pKShGH; inserting the black goat beta-casein gene into plasmid pKShGH to prepare plasmid pGbc0.8hGH; and inserting the 4.7 kb region located in front of -802 bp of black goat beta-casein genome clone, into pGbc0.8hGH to prepare plasmid pGbc5.5hGH. Mammals are transformed by inserting plasmid pGbc5.5hGH(KCTC 8949P) into fertilized eggs of mammals and produce human growth hormone from mammary gland of mammals, especially 70% or more of transformed mammals produce human growth hormone in the concentration of 3.1 to 16.1 mg/ml.

Description

Plasmid (pGbc5.5hGH, KCTC 8949P) tranforming animals to produce human growth hormone from their milk}

The present invention relates to a transforming plasmid pGbc5.5hGH (KCTC 8949P) for the production of human growth hormone, and more specifically, a novel plasmid for transformation having a beta-casein gene of 5.5 kb and a human growth hormone gene derived from black goat. pGbc5.5hGH (KCTC 8949P) and a method for transforming mammals using the same to stably produce milk containing tissue-specific high concentrations of human growth hormone in the mammary gland.

Human growth hormone is a single peptide consisting of 191 amino acids. When the human growth hormone is deficient due to genetic or physiological defects, adolescents appear dwarf, in order to treat it, the human growth hormone should be supplied from the outside. In addition, such human growth hormone is generally used for treatment of Turner syndrome, chronic kidney failure, digestive burns and fractures, and its application range is gradually expanding. However, in the related art, it is difficult to secure an absolute amount because the human growth hormone is extracted from the dead body and used.

In order to improve this problem, a gene recombination technology has been developed that can transform human growth hormone genes into Escherichia coli and culture the transformed Escherichia coli, thereby obtaining a large amount of human growth hormone [Genentech, US Patent 4,634,677]. ].

Another improvement is the use of mammalian mammary glands, which are useful biological organs that can continuously produce large amounts of protein, which can be transformed into mammalian milk by transforming mammals to specifically express foreign genes in the mammary glands. It is possible to mass produce useful proteins such as human growth hormone of interest (Gordon et al., Bio / Technol., 5, 1183 (1987)).

However, until now, the production frequency of the transgenic animals was very low, such as 10% pig, 1.3-10% sheep, and 5% cow [Hammer et al., Nature, 315, 680 (1985)]. The transformation of the subject evaluated the usefulness of the recombinant gene.

On the other hand, in order to produce the human growth hormone in milk, the first study was conducted using a mouse fusion of a promoter of the whey acidic protein gene (weakly referred to as a promoter) and the human growth hormone gene of rabbits There is a method of transformation (Devinoy et al., Transgenic Res., 3, 79 (1994)). Looking at the results of the above study, the concentration of human growth hormone in the milk of the produced mice was 0.01 ~ 22 mg / ㎖ depending on the strain, an average of 10.3 ± 9.1 ㎎ / ㎖ per individual. However, although the expression level was considerably high in this method, there were not only significant variations between openings, but also significant amounts of human body in other tissues (liver, kidney, thymus, brain, salivary glands, lungs, uterus, ovaries, etc.). The growth hormone gene has been expressed, and thus, most of the transformed mice have a problem in that a side effect of losing reproduction ability occurs.

Therefore, the present inventors have registered a patent based on the results in mice transformed by designing and transforming the plasmid pBCN1GH using the beta-casein promoter of rats in order to express and secrete tissue growth-specific human growth hormone only in the mammary gland. [Lee Kyung-Kwang et al., Republic of Korea Patent Registration No. 184754]. In this patent, the plasmid pBCN1GH showed very strict tissue specificity as expected, and was found to be mainly expressed only in the mammary gland, but the average expression level was 1.9 ± 3.2 mg / ml in the analyzed result of three lines of the transformed mouse. Only one line is overexpressed and the other two lines have very low levels of expression. Eventually, tissue specificity was improved, but the expression pattern of each strain still shows severe variation. This phenomenon is known to be due to a position effect in which the expression level of the foreign gene is changed depending on the position on the chromosome into which the recombinant gene is inserted [Jaenisch et al., Cell, 32, 209 (1983); Al-Shawi et al., Mol. Cell. Biol., 10, 1192 (1990)].

As such, in the related art, since the expression level of genes is severely changed from opening to opening, it is very inefficient for mass production of useful proteins in animal milk, and thus more efficient improvement measures are required.

Therefore, the present inventors further studied to solve the above problems, using a novel plasmid pGbc5.5hGH (KCTC 8949P) having a human growth hormone gene and a black goat-derived beta-casein gene transformed the mouse, and transformed By stably producing a large amount of human growth hormone in the milk of mother rats, the present invention has been completed.

Accordingly, the present invention uses a novel plasmid pGbc5.5hGH (KCTC 8949P) for transformation to stably produce milk containing a high concentration of human growth hormone in tissues in a mammalian mammary gland, and produces human growth hormone using the same. The purpose is to provide a way to.

1 shows the structure of plasmid pGbc5.5hGH (KCTC 8949P) according to the present invention.

Figure 2 shows the results confirmed by the electrophoresis of 12% SDS-PAGE the presence and content of human growth hormone in the milk of the transformed mice according to the present invention.

Figure 3 shows the results confirmed by the northern blot (northern blot) to determine whether the tissue of the human growth hormone gene is specifically expressed in the tissues of the transformed mice.

The present invention is characterized by a novel plasmid pGbc5.5hGH (KCTC 8949P) for transformation with a beta-casein gene derived from a black goat of 5.5 kb size and a human growth hormone gene.

In addition, the present invention is characterized by a method of transforming mammals using the novel plasmid pGbc5.5hGH (KCTC 8949P), and producing human growth hormone in the mammary gland.

Referring to the present invention in more detail as follows.

The present invention is a novel plasmid pGbc5.5hGH (KCTC 8949P) for transformation, which can stably mass-produce human growth hormone, which is used as a useful medicine, and also maintains strict tissue specificity, and transforms a mammal by using the same. Gene expression is stable according to mammalian strains, and when it is inorganic, it relates to a method for continuously producing human growth hormone only in the mammary gland.

Therefore, the present inventors examined various genes related to the mammary gland, which is a living organ that can continuously produce a large amount of protein, and thus, beta-derived from black goats with respect to a promoter capable of expressing genes specifically in the mammary gland of mammals A novel plasmid pGbc5.5hGH (KCTC 8949P) having a casein gene was prepared. A process for preparing the plasmid will be described in detail as follows.

That is, the plasmid pKShGH is inserted into the Hind II position of the plasmid pBluscript KSII by separating the 2.1 kb human growth hormone gene including the transcription termination signal and the 3'-flanking sequence of the human growth hormone gene from the plasmid phGH. Manufacture. Then, a plasmid pKShGH was inserted into the plasmid pKShGH using a 5.5-kb black goat-derived beta-casein gene as a promoter to prepare a new plasmid pGbc5.5hGH (KCTC 8949P) and to the Biotechnology Research Institute Gene Bank 11 June 1999. Deposit dated accession number KCTC 8949P.

On the other hand, the present invention is another feature of the method for transforming a mammal using the novel plasmid pGbc5.5hGH (KCTC 8949P), which will be described in detail as follows.

That is, the present invention is transformed by micro-injecting the new plasmid pGbc5.5hGH (KCTC 8949P) having the beta-casein gene and the human growth hormone gene derived from the black goat into the fertilized mouse and transplanting the fine-injected fertilized egg into the surrogate mother, Produce mice.

Then, the organic milk is collected from the transgenic mouse females obtained by breeding the transgenic mice and the expression capacity and content of the human growth hormone gene are examined.

As a result, more than 70% of the transformed mice of the present invention express human growth hormone in milk at high concentrations of 3.1-16.1 mg / ml, resulting in a high expression level of 6.2 ± 5.6 mg / ml on average. In addition, in the present invention, by expressing the human growth hormone gene while maintaining a very strict mammary tissue specificity that is rarely expressed in other tissues, blood concentration can be maintained normal. Therefore, the present invention can achieve more advanced results that can prevent infertility caused by side effects by the expression of human growth hormone in tissues other than the mammary gland.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Genomic DNA Library Construction of Black Goats

Genomic DNA isolated from the liver of the black goat was partially digested with restriction enzyme Sau3AI, and only about 40 kb of DNA fragment was isolated and linked to Lambda FIX II (stratagene). Then, genomic DNA libraries of black goats were prepared by in vitro packaging of the DNA linked to Lambda FIX II.

Example 2 Cloning of the Black Goat Beta-casein Gene

As a result of Southern blot using a probe region (-1760 to -802 bp) of about 0.9 kb previously reported in the genomic DNA library of the black goat prepared in Example 1, it was about 9.3 kb. A clone containing the promoter of the black goat beta-casein gene of was obtained.

Example 3: Preparation of Plasmid pGbc5.5hGH

① Preparation of plasmid pKShGH

PhGH containing human growth hormone gene (Namung Wook et al., Korean Journal of Biochemistry, 21, 226 (1988)) was digested with restriction enzymes BamHI and EcoRI to isolate human growth hormone genes. Plasmid pKShGH was prepared by filling the ends of the isolated genes with the Klenow enzyme and inserting them into the HindII position of the plasmid pBluescript KSII.

② Preparation of plasmid pGbc0.8hGH

In order to introduce the promoter region of the black goat beta-casein gene into the plasmid pKShGH prepared above, PCR (polymerase) using the genomic DNA of the black goat as a template and primer 1 described in SEQ ID NO: 1 and primer 2 described in SEQ ID NO: 2 A chain reaction) synthesized a promoter region of about 800 bp from -802 bp to +27 bp in beta-casein gene and isolated. Plasmid pGbc0.8hGH was prepared by inserting the isolated black goat beta-casein gene between XbaI and ClaI of plasmid pKShGH.

③ Preparation of the plasmid pGbc5.5hGH

In the black goat beta-casein genomic clone obtained in Example 2, a region corresponding to about 4.7 kb in front of -802 bp was separated by restriction enzyme XbaI and inserted into the XbaI position of pGbc0.8hGH in accordance with the transcription direction, thereby resulting in a novel novel plasmid. pGbc5.5hGH was prepared and deposited with the Korean Institute of Biotechnology Gene Bank under accession number KCTC 8949P as of June 11, 1999. Figure 1 shows the structure of the novel plasmid pGbc5.5hGH.

Example 4 Transformation of Mice

Plasmid pGbc5.5hGH was digested with restriction enzymes NotI and XhoI, electrophoresed with 0.7% agarose gel and purified by passing through an Ellutip-D column (Schleicher & Schuell, Germany). The purified human growth hormone gene was adjusted to a concentration of 4 μg / ml with 10 mM Tris solution to which 0.1 mM EDTA was added. Purified 4 μg / mL human growth genes were finely injected into 1-cell embryos obtained from C57BL / 6 × CBA hybrid mice, and the micro-implanted embryos were implanted into pseudo-pregnant ICR surrogate mothers. The baby rats were produced. A part of the ear tissue of the produced little mouse was cut and digested, and the digested tissue solution was subjected to PCR analysis using the primer of Example 3. Selected transformed mice were named # 3, # 6, # 7, # 8, # 11, # 13 and # 15.

Example 5 Confirmation of Human Growth Hormone in Milk of Transgenic Mice

When the female rats of the transgenic mouse strains # 3, # 6, # 7, # 8, # 11, # 13 and # 15 obtained in Example 4 were bred and fed on the 10th day of lactation, the milk was collected and milked. The presence of human growth hormone was confirmed by the following method.

First, after 3 hours of isolation from the rats to collect milk, oxytocin 10 IU was intraperitoneally injected, and milk was collected while massaging the mammary gland. The collected milk was centrifuged at 14,000 g for 30 minutes. When 1 μl of whey obtained by centrifugation was electrophoresed in 12% SDS-PAGE and stained with Coomassie Blue, a large amount of human growth hormone protein of 22 kDa was detected (FIG. 2).

Example 6 Confirmation of the Content of Human Growth Hormone in the Milk of Transgenic Mice

In order to quantitatively analyze the content of human growth hormone in milk, the whey obtained in Example 5 was used to quantify by RIA assay (radioimmunoassay; manufactured by DPC), and the results are shown numerically at the bottom of FIG. 2 in the accompanying drawings. . Female mice of the transgenic mouse strains # 3, # 8, # 11, # 13 and # 15 were found to produce human growth hormone at high concentrations of 3.1 mg / ml to 16.1 mg / ml. The other two lines # 6 and # 7 had concentrations of up to 1 mg / ml at concentrations of 0.6 mg / ml and 0.3 mg / ml, respectively, but also expressed significant amounts of human growth hormone. As a result, the human growth hormone was expressed at a considerably high level in all lines, and 5 out of 7 (more than 70%) were expressed at high concentration.

Example 8 Tissue Specific Expression of Human Growth Hormone Gene

To determine whether tissue growth hormone genes are expressed in a tissue-specific manner, female rats of the transgenic mouse strains # 3, # 8, # 11, # 13, and # 15 were treated with mammary gland and liver on day 10 , Spleen, pancreas, kidney, lung, heart, salivary gland and brain tissues were extracted, total RNA of the tissues was extracted according to Samburk's method, and each extracted 15 μg RNA was extracted on 1% formaldehyde-agarose gel. After electrophoresis, transfer to nylon membrane [Sambrook et al., Mol. cloning: a laboratory manual, pp. 7.3 to 7.23, CSH Press (1989)]. Human growth hormone genes were labeled with [α- ³² P] dCTP (Amersham), cleaved with restriction enzyme PvuII, and subjected to Northern blot using this as a probe.

Figure 3 shows the results of the northern blot, showing very stringent tissue specificity in which human growth hormone is overexpressed mainly in the mammary gland. Human growth hormone was expressed in the heart of the # 15 strain, but was extremely low. It was not detected in other tissues or in the control tissues. No abnormality was found in any of the strains.

As described in detail above, the present invention is a novel plasmid pGbc5.5hGH (KCTC 8949P) was prepared by fusing human growth hormone gene to a beta-casein promoter derived from a black goat of about 5.5 kb in size, and transformed into the plasmid prepared above. All mouse strains produce tissue-specific high levels of human growth hormone in the mammary glands, and in particular, more than 70% of the transformed mice produce high concentrations of human growth hormone of 3.1-16.1 mg / ml in the mammary gland.

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Claims (4)

Transgenic plasmid pGbc5.5hGH (KCTC 8949P), characterized in that it contains a beta-casein gene and a human growth hormone gene derived from a black chlorine of 5.5 kb. Transformed mammal, characterized in that it is transformed with plasmid pGbc5.5hGH (KCTC 8949P) to produce human growth hormone from the mammary gland. The transformed mammal according to claim 2, wherein at least 70% of the transformed mammal produces human growth hormone at a concentration of 3.1-16.1 mg / ml from the mammary gland. A method of producing human growth hormone, characterized by microinjecting plasmid pGbc5.5hGH (KCTC 8949P) into a fertilized egg of a mammal to produce a transformed animal, and then breeding the animal to separate human growth hormone from milk.