KR20010077538A - Single-chain human chorionicgonadotropin(hCG) and method for producing the same - Google Patents

Abstract

PURPOSE: A human chorionic gonadotropin (hcg) consisting of single chain by genetic recombination is provided, which has same physiological activity as natural type human chorionic gonadotropin (hcg), and is thus used for animal medicines and clinical medicines. Also, nucleic acid molecule coding the hormone and its preparation method are provided. CONSTITUTION: A single chain-human chorionic gonadotropin (hcg) is composed of α-unit and β-unit and is identical with or similar to 1-257 site or 21-257 site of sequence list 6. A single chain-human chorionic gonadotropin (hcg) is prepared by the following steps of: (a) amplifying nucleic acids coding α and β units of human chorionic gonadotropin (hgc) by PCR; (b) cloning amplified nucleic acids coding α and β units; (c) coupling the resultant nucleic acids; (d) constructing an expression vector including the coupled nucleic acid; (e) transforming an eukaryotic cell using the expression vector; (f) expressing human chorionic gonadotropin in the eukaryotic cell; and (g) separating the prepared human chorionic gonadotropin.

Description

Single-chain human chorionicgonadotropin (hCG) and method for producing the same}

The present invention relates to chorionic stimulating hormone and a method for producing the same, and more particularly, to a method for producing a chorionic stimulating hormone formed in a single chain by genetic recombination and hormones of such a single body.

Natural chorionic gonadotropin (hCG) is a gonadotropin secreted from the placenta of pregnant women and belongs to the glycoprotein hormone system.

This hormone, like follicle-stimulating hormone (FSH), luteinizing hormone (LH) and gonadotropin-like hormone (eCG), is composed of α- and β-groups covalently coupled.

The α group of hCG is composed of a signal peptide consisting of 24 amino acids and a peptide consisting of 92 amino acids, and has an N-glycoside-linked sugar chain addition site at 52 and 78 [Nature. 281, 351 (1979). The nucleic acid base sequence of the cDNA encoding the α group of hCG is as described in SEQ ID NO: 1, and the amino acid sequence of the α group of hCG is as described in SEQ ID NO: 2.

The β group of hCG consists of a signal peptide consisting of 20 amino acids and a peptide consisting of 145 amino acids [Nature. 286, 684 (1980). The nucleic acid base sequence of the cDNA encoding the β group of hCG is as described in SEQ ID NO: 3, and the amino acid sequence of the β group of hCG is as described in SEQ ID NO: 4.

As described above, hCG was used to purify the hCG secreted from the urine of pregnant women for the promotion of ovulation and infertile women as well as the estrous period of large and small cattle, but there were various problems in use because the physiological activity is not constant depending on the individual.

The present invention for solving the above problems is formed in a single chain by genetic recombination is easily expressed in the expression cell system, so mass production is easy, provides a chorionic gonadotropin which is a single body having the same physiological activity as a natural hormone It aims to do it.

In addition, an object of the present invention is to provide a method for producing a single chorionic gonadotropin.

1 shows a process for combining cDNA of α group and cDNA of β group,

2 is a plasmid map of pcDNA3-hCG.

In order to achieve the above object, the present invention provides a single chain of α group and β group of chorionic gonadotropin by genetic recombination, wherein the amino acid sequence is the same as position 1-257 or 21-257 in SEQ ID NO: It is characterized by chorionic gonadotropin, which is a substantially similar entity.

A representative amino acid sequence of hCG, the recombinant monolith of the present invention, is shown in SEQ ID NO: 6.

As shown in SEQ ID NO: 6, hCG consists of a peptide consisting of 20 signal peptides and 237 amino acids.Amino acids 21 to 165 correspond to β groups and amino acids 166 to 257 belong to α groups. Corresponds to N-glycosidic sugar chains are added to Asn at 33 and 50 and Asn at 217 and 243, respectively.

However, not only the amino acid sequence identical to the amino acid sequence set forth in positions 1-257 or 21-257 in SEQ ID NO: 6 is included in the scope of the present invention.

In addition, the present invention is characterized by an isolated nucleic acid molecule having a nucleic acid base sequence encoding the homozygous gonadotropin.

Representative nucleic acid sequences are shown in SEQ ID NO: 5. The DNA set forth in SEQ ID NO: 5 consists of 774 nucleic acids, positions 1-60 encode signal peptides, positions 61-771 encode peptides, and positions 772-774 represent terminators. In addition, 61-495 of the positions 61-771 represent the amino acid sequence for β group, and 496-771 represents the amino acid sequence for α group.

However, the nucleic acid base sequences set forth in SEQ ID NO: 5 above are identical to positions 61-771 as well as substantially similar nucleic acid base sequences within the scope of the present invention.

In addition, the present invention comprises the steps of amplifying nucleic acid molecules encoding α groups and β groups of chorionic gonadotropin by polymerase chain reaction (PCR); Cloning the nucleic acid molecule encoding the amplified α-group and the nucleic acid molecule encoding the β-group; Binding a nucleic acid molecule encoding a cloned α group and a nucleic acid molecule encoding a β group by a polymerization chain reaction; Forming an expression vector comprising the bound nucleic acid molecule; Transforming eukaryotic cells by introducing the expression vector; Expressing chorionic gonadotropin in the transformed eukaryotic cell; There is a characteristic of the method for producing a monolithic chorionic stimulating hormone, which comprises the step of separating the expressed chorionic stimulating hormone.

In the above method, it is preferable to use a plasmid as the expression vector.

In addition, eukaryotic cells, which are host cells for introducing hormones by introducing expression vectors, are preferably cultureable cells derived from mammalian animal cells, and particularly preferably CHO-K1 cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples, however, illustrate the invention and do not limit the scope of the invention.

<Example 1>

Preparation of monochain hCG

(1) Formation of Transition Vector of Monolithic hCG

First, PCR was performed as shown in FIG. 1 to connect DNA encoding α group of hCG and DNA encoding β group.

In this case, four oligonucleotide groups having the following nucleotide sequences were used as the primer.

Primer-1, M13 RV, 5'-CAGGAAACAGCTATGAC-3 '

Primer-2, 5'-CACATCAGGAGCTTGTGGGAGGATCGG-3 '

Primer-3, 5'-ATCCTCCCACAAGCTCCTGATGTGCAG-3 '

Primer-4, M13 M4, 5'-GTTTTCCCAGTCACGAC-3 '

cDNA of α group of hCG and cDNA of β group {Nature. 281, 351 (1979) and Nature. 286, 684 (1980)} was amplified by PCR. PCR for β-group cDNA was performed using Primer 1 and Primer 2, and PCR for α-group cDNA was performed using Primer 3 and Primer 4.

The cDNA of α group and the cDNA of β group amplified by PCR were cloned into pUC119 (Takara, Japan). PCR was performed on the cloned DNA using Primer 1 and Primer 4 to form hCG, which is a combination of cDNA of α group and cDNA of β group.

The monolith formed, hCG, was cleaved with EcoRI / SalI and linked to the same site of the pUC119 vector. After linking, the entire base sequence was checked to identify the incorrect nucleotide sequence.

After confirming the nucleotide sequence, the fragment was cut with EcoRI / SalI and the fragment was connected to the EcoRI / XhoI site of the expression vector pcDNA3 (Invitro) to form the expression vector pcDNA3-hCG as shown in FIG. Was confirmed by restriction enzyme.

(2) cell culture

The expression vector pcDNA3-hCG was introduced into CHO-K1 cells using lipofectamine, a liposome preparation.

800 μg / ml G418 was added to Ham F-12 medium, a stable clonal growth medium containing 50 units / ml penicillin, 50 ug / ml streptomycin, 2 mM glutamine and 10% FCS, and the expression vector pcDNA3-hCG Introduced CHO-K1 cells were introduced and cultured for 2 weeks at 37 ℃ under a mixed atmosphere of 5% CO ₂ and 95% air. After incubation, only stable cells were selected.

(3) Expression of hCG, a recombinant monomer

Selected stable cells (1 × 10 ⁶ ) were placed in 20 ml CHO-S-SFM-II medium containing 50 units / ml penicillin and 50 ug / ml streptomycin and incubated at 37 ° C. for 48 hours.

After incubation, the culture supernatant was collected and centrifuged at 100,000xg for 60 minutes to remove cell debris. Monomer hCG is expressed in the upper fraction of the centrifuged fraction.

<Example 2>

Activity measurement of single chain hCG

(1) Quantification of single chain hCG using RIA

Native hCG and the monoclonal hCG of the present invention were quantified by RIA using polyclonal antibodies (A555 / R1H).

200 ul of 0.02 M phosphate buffer [0.017 M Na ₂ HPO ₄ .12H ₂ O, 0.003 M KH ₂ PO ₄ , 0.9% (w / v) NaCl, 0.5% (w / v) BSA, 0.02% (w / v) NaN ₃ ], 100 ul of natural hCG standard (6.25, 12.5, 25, 50, 100, 200, 400 ng), anti-hCG polyclonal antibody (7 ug / ml) and 100 ul Chloramine-T method of 100 ul rabbit A ¹²⁵ I hCG probe (20,000 cpm / 100 ul) labeled according to the above was added to each tube and incubated overnight at 4 ° C.

After incubation, 500ul anti-rabbit precipitated antibody was put in each tube and incubated for another 30 minutes. After incubating for 30 minutes, 2 ml of phosphate buffer was added, and the upper layer was removed by centrifugation at 3,000 rpm for 15 minutes, and then measured by a counter.

(2) Determination of Recombinant hCG Activity by Intracellular cAMP Quantitation

Cells expressing the receptor of the antibody-forming hormone (HEK293-LHR: Min et al., JBC, 273: 34911-34919, 1998) were used to confirm the biological activity of the recombinant hormone.

3 × 10 ⁵ cells were put in six wells, incubated in an incubator for about 72 hours, and washed twice with WA / BSA culture. After washing with WA / BSA culture, 1 ml of 0.5 mM MIX was added and incubated for 30 minutes by adding 0.075-250 ng of native and recombinant hCG.

After the incubation was completed, put on ice and trichloroacetic acid (Trichloroacetic acid) was added to the final concentration of 5% and stored at -20 ℃ until analysis.

cAMP was quantified using the cAMP enzyme immunoassay kit (Cayman Chemical, MI, USA).

That is, incubate the culture tank for 18 hours by adding 50ul of buffer, 50ul of sample, 50ul of cAMP acetylcholinesterase tracer, 50ul of cAMP antiserum, and 50ul of cAMP acetylcholinesterase tracer. Washed 5 times. After washing, 200ul of Elman reagent (Ellman's Reagent) was added and left for 60 minutes, and the plate was read at 405 nm to measure the activity of cAMP.

The results of measuring the activity are shown in Table 1 below.

Biological Activity of Recombinant hCG Sample amount (ng / ml) 0.25 0.75 2.5 7.5 25 75 250 Release cAMP amount (pMol / 10 ⁶ cells) Natural hCG 0.80 16.4 48.1 158.8 315.4 395.6 462.7 Recombinant hCG 0.72 14.5 49.7 149.7 319.2 406.5 459.3

The present invention as described above has the effect of easily producing a large amount of hormones by forming a chorionic gonadotropin consisting of two parts of α group and β group in a single chain.

In addition, chorionic gonadotropin, which is a monolith of the present invention, has the same cAMP activity as a natural hormone, and thus may be usefully used as an animal medicine and a clinical medicine.

Claims (7)

A chorionic gonadotropin, wherein the amino acid sequence is made of a homolog of the α and β groups of the chorionic gonadotropin as a homolog or substantially similar to positions 1-257 or 21-257 in SEQ ID NO: 6. An isolated nucleic acid molecule having a nucleic acid base sequence encoding the chorionic gonadotropin which is a monolith according to claim 1. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule comprises a nucleic acid base sequence identical or substantially similar to position 1-771 or 61-771 in SEQ ID NO: 5. 4. Amplifying nucleic acid molecules encoding α groups and β groups of chorionic gonadotropin by polymerase chain reaction (PCR); Cloning the nucleic acid molecule encoding the amplified α-group and the nucleic acid molecule encoding the β-group; Binding a nucleic acid molecule encoding a cloned α group and a nucleic acid molecule encoding a β group by a polymerization chain reaction; Forming an expression vector comprising the bound nucleic acid molecule; Transforming eukaryotic cells by introducing the expression vector; Expressing chorionic gonadotropin in the transformed eukaryotic cell; A method for preparing chorionic stimulating hormone which is a monolith comprising the step of separating the expressed chorionic stimulating hormone. 5. The method of claim 4, wherein the expression vector is a plasmid. The method of claim 4, wherein the eukaryotic cell is a cultured cell derived from a mammalian animal cell. The method of claim 6, wherein the eukaryotic cell is CHO-K1.