KR100375671B1 - Single chain bovine Luteinizing hormone and producing method thereof - Google Patents

Abstract

The present invention relates to a luteinizing hormone of a cow formed in a single chain by genetic recombination, a nucleic acid molecule encoding the hormone, and a method for producing the hormone, and a luteinizing hormone composed of two parts, α group and β group, By forming a large amount of hormones can be easily produced, luteinizing hormone which is a monolith of the present invention is stronger than the natural form of luteinizing hormone can be usefully used as an animal medicine.

Description

Single chain bovine luteinizing hormone and producing method

The present invention relates to luteinizing hormone and a method for producing the same, and more particularly, to a method for producing luteinizing hormone formed in a single chain by genetic recombination, and a hormone that is a single body.

Natural type of luteinizing hormone (hereinafter referred to as "LH") is a gonadotropin secreted by the pituitary gland and belongs to the glycoprotein hormone system.

This hormone, like follicle-stimulating hormone (FSH) derived from the pituitary gland, chorionic gonadotropin (hCG), and chromosome gonadotropin (eCG), is composed of noncovalent bonds of α and β groups.

Bovine luteinizing hormone (hereinafter referred to as "bLH") is also secreted from the pituitary gland of bovine gonadotropin involved in bovine over-ovulation and estrous organs and is composed of α and β groups.

The α group of bLH is composed of a signal peptide consisting of 24 amino acids and a peptide consisting of 96 amino acids, and has N-terminal sugar chain linkage sites at 56 and 82 [Biochemistry 22, 4856 (1983). ) Reference]. The nucleic acid base sequence of the cDNA encoding the α group of bLH is as described in SEQ ID NO: 1, and the amino acid sequence of the α group of bLH is as described in SEQ ID NO: 2.

The β group of bLH consists of a signal peptide consisting of 20 amino acids and a peptide consisting of 121 amino acids [JBC. 260, 7072 (1985). The nucleic acid base sequence of the cDNA encoding the β group of bLH is as described in SEQ ID NO: 3, and the amino acid sequence of the β group of bLH is as described in SEQ ID NO: 4.

The bLH of the cow as described above is necessary for the over-ovulation and estrous maintenance of the cow, but conventionally has a problem that must be imported and used because bLH was not produced domestically.

In order to reduce cost, instead of bLH, the LH may be purified from the pituitary gland of a pig slaughtered in a slaughterhouse, but the process of separating and purifying LH is difficult and the yield is low.

The present invention for solving the above problems is formed in a single chain by genetic recombination and is easily expressed in the expression cell system, so mass production is easy, and the luteinizing hormone of bovine luteinizing hormone is superior to the natural hormone of the hormone. It aims to provide.

It is also an object of the present invention to provide a method for producing a lutein forming hormone of bovine cattle.

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

2 is a plasmid map of pcDNA3-bLH.

In order to achieve the above object, the present invention provides a single chain of α group and β group of bovine luteinizing hormone by genetic recombination, and the amino acid sequence is identical to or substantially identical to positions 1-237 or 21-237 in SEQ ID NO: 6. It is characterized by the luteinizing hormone of bovine, which is a similar one.

Representative amino acid sequences of the recombinant monomeric bLH of the present invention are set forth in SEQ ID NO: 6.

As shown in SEQ ID NO: 6, bLH is composed of 20 signal peptides and peptides consisting of 217 amino acids.Amino acids 21 to 141 correspond to β groups and amino acids 142 to 237 are α groups. Corresponds to In addition, an N-terminal sugar chain is bonded to Asn at 33 and an N-terminal sugar chain is bonded to Asn at 197 and 223, respectively.

However, not only the amino acid sequence identical to the amino acid sequence set forth in positions 1-237 or 21-237 in SEQ ID NO: 6 is included in the scope of the present invention.

In another aspect, the present invention is characterized by an isolated nucleic acid molecule having a nucleic acid base sequence encoding the luteinizing hormone of the cow.

Representative nucleic acid sequences are shown in SEQ ID NO: 5. The DNA set forth in SEQ ID NO: 5 consists of 714 nucleic acids, positions 1-60 encode signal peptides, positions 61-711 encode peptide, and positions 712-714 represent terminators. In addition, 61-423 in positions 61-711 represent amino acid sequences for β groups, and 424-711 represent amino acid sequences for α groups.

However, not only the same nucleic acids as the positions 61-711 in the nucleic acid sequences set forth in SEQ ID NO: 5, but also substantially similar nucleic acid sequences are included in the scope of the present invention.

The present invention also comprises amplifying a nucleic acid molecule encoding α group and β group of bovine luteinizing hormone by a 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 bovine luteinizing hormone in said transformed eukaryotic cell; There is a characteristic of the method for producing a luteinizing hormone of bovine which is a monolith comprising the step of separating the expressed bovine luteinizing 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 bLH

(1) Formation of transition vector of monolithic bLH

First, a polymerase chain reaction (hereinafter, referred to as "PCR") as shown in FIG. 1 was performed to connect the DNA encoding the α group of bLH and the DNA encoding the 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'-TCCATCAGGAAAGAGGAAGAGGATGTC-3 '

Primer-3, 5'-ATCCTCTTCCTCTTTCCTGATGGAGAG-3 '

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

cDNA of α group of bLH and cDNA of β group {Biochemistry. 22, 4856 (1983) and PNAS. USA. 83, 6618 (1986)} 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 bLH, a monoclonal combination of cDNA of α group and cDNA of β group.

The monolith formed, bLH, was cleaved with Kpnl / Xbal 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 was cut with Kpnl / Xbal and connected to the same site of the expression vector pcDNA3 (Invitro) to form the expression vector pcDNA3-bLH as shown in FIG. The direction of the expression vector was confirmed by restriction enzymes.

(2) cell culture

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

To a medium in which 800 μg / ml G418 was added to Ham F-12 medium, a stable clone growth medium containing 50 μg / ml penicillin, 50 μg / ml streptomycin, 2 mM glutamine (2 mM) and 10% FCS. CHO-K1 cells into which the expression vector pcDNA3-bLH was introduced were added and incubated for 2 weeks at 37 ° C. under a mixed atmosphere of 5% CO ₂ and 95% air. After incubation, only stable cells were selected.

(3) Expression of bLH, a recombinant monomer

Selected stable cells (1 × 10 ⁶ ) were placed in 20 ml of CHO-S-SFM-11 medium containing 50 units / ml penicillin and 50 µg / 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. The monolayer bLH is expressed in the upper fraction of the centrifuged fraction.

<Example 2>

Activity measurement of single chain bLH

(1) Quantification of monolithic bLH using RIA

Natural bLH and the monoclonal bLH of the present invention were quantified by RIA (Radio Immuno Assay) using polyclonal antibodies (A558 / P1H).

200 μl 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 μl of native bLH standard (0.09, 0.19, 0.390, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50 ng), 100 μl of rabbit antibLH polyclonal antibody (7 μg / ml ) And ¹²⁵ l bLH probe (20,000 cpm / 100 μl) labeled according to 100 μl chloramine-T method were added to each tube and incubated overnight at 4 ° C.

After incubation, 500 μl of anti-rabbit precipitated antibody was placed in each tube and incubated for another 30 minutes. After culturing 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 quantified by measuring with a counter.

(2) Measurement of Recombinant bLH Activity by Intracellular cAMP Quantitation

The physiological activity of the recombinant hormone was confirmed using cells expressing the receptor of follicle stimulating hormone (HEK293-FSHR: HEK293-FSHR).

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 with addition of 0.075-250 ng of native and recombinant bFSH.

After the incubation was completed, put on ice and 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).

In other words, 50 μl of buffer, 50 μl of sample, 50 μl of Cyclic AMP Acetylcholinesterase Tracer and 50 μl of Cyclic AMP Antiserum were added to each culture tank, followed by incubation for 18 hours. Was washed 5 times with washing buffer. After washing, 200 μl of Elman reagent (Ellman's Reagent) was added thereto, and left for 60 minutes. Then, 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 Genetically Modified bLH Sample amount (ng / ml) 0.075 0.75 2.5 7.5 25 75 250 Release cAMP amount (pMol / 10 ⁶ cells) Natural bLH 0.31 10.20 38 110 250 345 390 Recombinant bLH 0.41 15.43 46 154 301 389 451

As described above, the present invention has an effect of easily producing a large amount of hormones by forming a luteinizing hormone composed of two parts of α group and β group in a single chain.

In addition, luteinizing hormone, which is a monolith of the present invention, has a stronger activity than that of a natural luteinizing hormone, and thus may be usefully used as an animal medicine.

Claims (7)

A single chain bovine luteinizing hormone having an amino acid sequence having the amino acid sequence of positions 1-227 or 21-227 in SEQ ID NO: 6 in a single chain of the α and β groups of bovine luteinizing hormone. An isolated nucleic acid molecule encoding the luteinizing hormone of the single chain bovine of claim 1 and having the nucleic acid nucleotide sequence of SEQ ID NO: 5. delete (1) amplifying a nucleic acid molecule encoding α group of bovine luteinizing hormone and a nucleic acid molecule encoding β group by polymerase chain reaction (PCR); (2) cloning the nucleic acid molecule encoding the amplified α group and the nucleic acid molecule encoding the β group; (3) binding the nucleic acid molecule encoding the cloned α group and the nucleic acid molecule encoding the β group by polymerase chain reaction; (4) forming an expression vector comprising the bound nucleic acid molecule; (5) transforming eukaryotic cells by introducing the expression vector; And (6) separating the expressed bovine follicle stimulating hormone from the bovine luteinizing hormone of claim 1. delete The method according to claim 4, wherein the eukaryotic cell is a cultured cell derived from a mammalian animal cell, and preferably a CHO-K1 cell. delete