JPH0463594A - Production of hormone-bonding region protein of growth hormone receptor - Google Patents

Abstract

NEW MATERIAL:A recombinant baculovirus containing a DNA fragment coding an amino acid sequence of the hormone-bonding region protein of human growth hormone receptor under controlled gene manifestation system. USE:Production of hormone-bonding region protein of human growth hormone receptor, etc. PREPARATION:The polyhedron gene part containing a polyhedron promoter is exclusively sectioned and inserted into pUC8. The objective gene is inserted to the downstream of the polyhedron promoter and the vector DNA is transduced together with a wild-type baculovirus DNA into an insect cell and cultured to induce the intracellular DNA recombination in the insect cell.

Description

Detailed Description of the Invention [Object of the Invention] The present invention provides an improved expression vector containing a gene encoding the amino acid sequence of the hormone binding region protein of the growth hormone receptor, and a method for producing the hormone binding region protein of the growth hormone receptor using the vector. It is related to the production of.

The present invention provides a means and method for efficiently producing a hormone binding region protein of a growth hormone receptor using recombinant DNA technology.

Using the present invention, by arbitrarily exchanging the amino acids and peptides that make up the growth hormone binding domain protein,
Improved human or other mammalian growth hormone receptors with enhanced binding to growth hormone can be produced. In addition to this, it is also possible to conduct research on the production of improved growth hormones. When the hormone-binding region protein of the growth hormone receptor produced by the method of the present invention is clinically administered, the protein may inhibit the function of growth hormone by antagonizing the growth hormone receptor in vivo. There is. This may be useful in treating diseases caused by some kind of growth hormone abnormality, such as gigantism or acromegaly. Furthermore, by administering the protein to animals, it is possible to create small animals.

[Prior art and problems to be solved by the invention] Complementary DNA + of growth hormone receptor gene;
It was cloned in humans and rabbits by Leung et al., and its hormone binding region protein has been expressed using human-derived or animal cells [
Nature, 330. 537 543 (198
7)]. More recently, Fuh et al. [J, Bio, Ch.
em, 265.3111-3] 15 (1990)]
It has been demonstrated that this disease is caused by intestinal obstruction in dogs.

However, those using animal cells have extremely low expression levels and are not suitable for production.

In addition, whereas expression in animal cells is extremely similar to the biosynthetic conditions of receptors in vivo, expression in E. coli does not involve the addition of sugar chains, so the expression products are not naturally occurring. considered to be different. Moreover, the expression product of Fuh et al.
It is difficult to say that it is the main product of bacterial cells. Therefore, even when using a human growth hormone affinity column, which is considered to be very effective, many impurities are still found, making purification extremely difficult. Furthermore, in the case of mutants of the same receptor, the above-mentioned affinity columns may not be usable, making purification even more difficult.

[Means for Solving the Problem] The present inventor used an insect-derived virus, noculovirus, as a vector to express a gene encoding the hormone binding region of a growth hormone receptor.
Insect-derived cells were used as hosts. As a result,
The present invention was completed by successfully secreting and producing the target product efficiently.

[Detailed Description] The present invention is for producing a hormone-binding region protein of a growth hormone receptor using a genetic engineering method, and a DNA fragment encoding the same region is used in a baculovirus-derived gene control system. Plasmite incorporated into
A method for producing a hormone binding region protein of a growth hormone receptor using insect cells as a host using a recombinant baculovirus vector in which the DNA is also integrated under this gene control system and a recombinant virus vector. This is what we provide.

In the present invention, the term growth hormone receptor refers to growth hormone receptors of all natural mammalian origin, including humans, and also includes variants of these natural growth hormone receptors produced by genetic engineering.

The DNA fragment encoding the hormone-binding portion of the growth hormone receptor is the amino acid sequence of the naturally occurring human growth hormone receptor (L eung
et al. (+987), Nature, 330.537-5
43) may be used, and the synthesized DNA thereof is also used in this embodiment. However, they are not necessarily limited to these. If the DNA fragment encoding this target portion also includes a signal peptide portion for secretion production to the outside of the cell, the hormone-binding portion of the produced growth hormone will be secreted and produced to the outside of the cell. In this state, the hormone-binding portion of growth hormone also accumulates within cells.

In order to express the l)NA fragment encoding the hormone-binding portion of the growth hormone receptor in insect cells as a host, this DNA fragment must be converted into the l)kl fragment in advance.
Is it necessary to connect it with the current line of Mitsuko?

As a gene expression system for insect cells, nuclear polyhedrosis virus (N
uclear polyhedrosis virus
: NPV) expression system is known, and A u
tographa California N P
V (AcN P V) and Bombyx moriN
There are two types of PV (BmNPV). In the case of AcNpv, the host is cells derived from night robber moths (S podo
Ptera rrugiperda: S, f,
cells), and in the case of BmNPV, silkworm-derived cells (Bot
nbyx mori N: 8 mN cells) are used.

Both baculoviruses contain polyhedral genes, which are under the control of strong polyhedral promoters. Therefore, when expressing using these viral vectors,
A recombinant baculovirus with a desired gene inserted downstream of this polyhedral promoter is prepared, and a host cell is infected with the recombinant baculovirus and then cultured to express the desired protein. When preparing this expression virus vector, since this virus is large at about 130 kilobase pairs, it is only possible to directly insert the gene of interest into it. Therefore,
The actual procedure is to first cut out only the polyhedral gene region containing the gene expression control region, that is, the polyhedral promoter, and then use it to create a vector that uses E. coli as a host, such as p
Uc8, and then insert the gene of interest downstream of the polyhedral promoter. Next, this vector DNA
is cotransfected with wild-type baculovirus DNA into insect cells and cultured, and intracellular DNA recombination occurs in the insect cells to obtain a recombinant baculovirus. There is also a method of infecting larvae with the obtained recombinant/sj curovirus and extracting the target product from the larvae.

In the present invention, the AcNP■ vector system is used, S,
Although the present invention is not limited to a method of infecting r cells and extracting a target substance from the culture solution and cells after culturing, the present invention is not limited thereto. Genetic manipulation methods using E. coli are described in the author's work (Maniatis et al. (1982), Moleu
lar CIoning: A Laboratory
Manual, Co1d Spring)1ar
It is detailed in many recent experimental books such as bor laboratory). For experiments using baculovirus and its hosts, please refer to the author [Summers
and Sm1th (1987) + A manual of
methods for baculovirus
vectors and 1nsect cell c
culture procedures, Texas
Agricultural Experiment 5
Bulletin No. 1555.
Texas A&MU n1versity] or [Ibid. (1989), Experimental Medicine, Vol. 7, 146-151
page] provides a detailed description of the procedure.

Host cells infected with recombinant baculovirus can be cultured using known media, such as Brace insect cell culture medium (
KIBUCO, Grace's l n5ect Ce1l
It can be performed using culture medium to which fetal bovine serum has been added to a concentration of 10%. After removing cells from the resulting culture solution, it is subjected to affinity column chromatography using growth hormone as a ligand to obtain a pure hormone-binding domain peptide of the growth hormone receptor. The affinity column was developed by Spencer et al. (J.

Biol, Chem, 263.7862 7867 (
1988)). The resulting expression product has a larger molecular weight than predicted from the amino acid sequence encoded by the inserted DNA.

This indicates the addition of sugar chains to the product.

In fact, this molecular weight comes to match the predicted molecular weight after treatment with glycanase, an enzyme that removes sugar chains from proteins. During culturing, use EF Surcel 400 (J R5cie) as a serum-free medium.
ntific, EX-C, ELL400) can also be used to obtain the hormone binding region peptide of the growth hormone receptor in the same manner. Furthermore, the peptide can be further recovered by collecting the cultured cells and disrupting them with a homogenizer.

Effects of the Invention] As described above, the present invention establishes a new secretory production system for growth hormone receptor hormone binding region peptide using insect cells as hosts.

Using this system, it is possible to obtain much larger quantities of pure peptides than with conventional animal cells.

Compared to using Escherichia coli, this method not only has the effect of obtaining peptides in a state close to natural, but also makes it easier to obtain pure specimens.

Example 1 Construction of pA cY MhG HR plasmid pAcYMl containing the part encoding the polyhedral protein of AcNPV (Matsuura et al. J, gen, V 1ro
1. (1987), 68.1233-1250), DNA encoding the human growth hormone receptor hormone binding region (hereinafter abbreviated as hG HR) is inserted downstream of the polyhedral promoter.

The DNA encoding hGHR is a patent application filed by the present inventors (Japanese Patent Application No. 01-020182) (Synthetic Gene Encoding the Amino Acid Sequence of the Hormone Binding Region of Human Growth Hormone Receptor, January 30, 1999). hG integrated into the plasmid pUSR described in )
A DNA sequence was used in which a base sequence corresponding to 3 amino acids was deleted from the C-terminus of HR DNA, and the restriction enzyme recognition site was slightly modified to facilitate incorporation into pAcYMI. Its outline is shown in FIG. First, pUSR
hG by digestion with restriction enzymes Hindll and 5alI.
Cut out a fragment of HRDNA. Next, the same fragment was added to a plasmid pH3 that had also been digested with HindIII and 5alt.
V8 DNA, treated with T4 ligase, and transformed into E. coli JMI O5 [Yanisch-Perro
n et al., Gene, 33, 103 119 (1985)]
to create a plasmid pHS R8. pH
SV8 is a plasmid that has been slightly modified in the restriction enzyme site of the plasmid pUc118 (available from Takara), which is widely used in the technical field.
The Bbe1 site of Uc118 was removed by Klenow enzyme treatment, roughly deleting the region between the EcoR1 site and the Xba1 site). Then Bgl on plasmid pH5R8
In order to move the recognition site of the II enzyme downstream, the plasmid was digested with BglII and 5all to create a large NA.
I cut out the pieces. On the one hand, the Bg111 site and the 5
As a DNA fragment inserted between the al1 sites, the synthetic oligonucleotide U I4 shown in FIGS. 1 and 2 was used.
-BACSL 14-BAC.

U15, Li2, U16-3BGLSL16-3BGL
Each DNA fragment was ligated by T4 ligase treatment. Both DNAs were mixed, treated with T4'J gauze, and transferred to E. coli JM105 to create a plasmid pB.
R38-3 was created. When this DNA terminus shown in FIG. 2 (its N terminus forms a paired base with the BgIII enzyme cleavage site) is inserted, the BgIII enzyme recognition site disappears. On the other hand, the missing Bglll restriction enzyme recognition site is designed to move downstream of the termination codon.

In addition, in this case, the hG HR DNA of pUsR is
Compared to the fragment, the C-terminal phenylalanine of hGHR,
Proline, tryptophan 3 amino acid deletion form,
In other words, we use a product designed with a 3-amino acid deletion (Figure 2). Whether the desired DNA sequence was obtained was confirmed by DNA sequencing. For purposes of this example, all oligonucleotide syntheses in this invention were performed using Applied Biosystems (Model 381).
This was carried out using the DNA synthesizer shown in A).

The outline of the insertion of the hGHR (3 amino acid deletion type) DNA thus inserted into pBSR8-3 into pAcYMl is as follows.
It is shown in FIG. First, pAcYMl is BamH
Digested with I enzyme and cut out was used. pBsR8
-3, the hGHR DNA fragment inserted into restriction enzyme Mlul
Then, most of it except the signal sequence part is excised by BglII digestion.

Based on its base sequence, the Bg111 site can create base pairs that pair with a DNA fragment treated with BamHI enzyme. Synthetic nucleotide 8glll site U-signal and L-signal were used for the removed signal sequence portion (Figure 4). This synthetic nucleotide is
It encodes the signal sequence of hGHR, and the 5' end of its initiation codon ATG is designed to create complementary base pairs with the DNA fragment cut out with the restriction enzyme BamHI. Next, these three DNA fragments were mixed and ligated by treatment with T4 ligase, and E. coli KI2.
Transfer to JM105 and propagate to create plasmid, pA
cYMhGHR was obtained. E. coli K 12 JMl 05 (pAcYMhGHR) into which this plasmid was introduced
has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Higashi 1-1-3, Tsukuba City, Ibaraki Prefecture (Feikoken Bacteria No. 11564, date of deposit: June 28, 1990).

Example 2 Creation of recombinant virus AchGHR Example 1
A recombinant baculovirus is created from the plasmid pAcYMhGHR DNA obtained in . It is wild type A
This plasmid DNA along with cNPV DNA is introduced into insect cells by calcium chloride-phosphate precipitation method, and then recombined within the cells to create the plasmid DNA. Its outline is shown in FIG. First, μg of DNAI of wild-type baculovirus AcNPV (AcNPV
is available from M, D, Summers. D
part of Entomology, T
exas Agricultural Experiment
ent 5tation and Texas A&M
UniversityC.

11Cge 5tation, Texas, 77
843 2475゜The DNA extraction method is experimental medicine Vo.
17, No. 13.146-151.1989, supra) and pAcYMhGHR DNA precipitate g (purified twice by cesium chloride density gradient method) in a hepes phosphate buffer (pH 7.05) containing calcium chloride. , precipitate. Insect cell S podop on the one hand
tera frugiperda9 (Americu
n Type Culture Co11ection
, 12301 Parklawn Drive,
Rockvlle, MD20852, available from 1776, hereinafter SJ, 9. 1 to 1.5 x 10 cells/2jl were cultured in Brace medium (Gibco) containing 10% tallow serum (abbreviated as ``Tallow Baby Serum'') in a 35 mm cell culture dish (Falcon 3001) for 1 hour. f
tLif and culture the cells. Next, the cell supernatant is removed, the above DNA precipitate is added dropwise, and the cell supernatant is allowed to stand for 1 hour. Furthermore, remove the cell supernatant and add 10% fresh
Add 21C of Brace medium containing beef tallow serum to 26.5
Culture at 9C for 4 days. The resulting culture solution contains wild type A
In addition to cNPV, a recombinant baculovirus AchG II R is also included.

A plaque assay is performed to extract AchGHR from this culture solution. First, S, f grown in the same manner as above
, remove the culture medium of 9 cells (10" cells/35 mm petri dish) and dilute the culture solution approximately 4 times to 10,000 times to 0.11.
Add Iρ. After leaving it for about 1 hour, remove the culture solution and add 1
% agarose solution [a 1:2 mixture of 3% agarose (low melting point G-Plaque agarose, F.M.O. Co., Ltd.) and Brace medium containing 10% tallow serum and kept at 37°C] was layered in 1 ml layer. . After the agarose solidifies]
Brace medium containing 10% yQ tallow serum is further layered and kept at 26.5°C for 4 days to generate virus plaques. Among the plaques that have formed, use a pipette tip to scoop up the one with a clear center, including the agarose, and place it in a 1 m
The virus is isolated by suspending it in Brace's medium containing 10% tallow serum of ff (the wild type virus has a white turbidity in the center of the plaque, and the recombinant virus is clear). If it is difficult to find clear plaques, dilute the original culture medium to an appropriate concentration to make it easier to identify the plaques, and repeat the plaque assay. The plaque suspensions of the collected transparent plaques were each diluted 100 to 1000 times, and 0.2tRQ of this was diluted in the same manner as above.
, f, repeat the plaque assay by overlaying on 9 cells to ensure that the plaques are free from other wild-type viruses. The product thus isolated is the recombinant baculovirus AchGHR.

Example 3 Secretory expression of human growth hormone receptor hormone binding region using AchGHR The thus obtained recombinant virus AchGHR was expressed as S.

f, Infect 9 cells. First, approximately 106 S, f, 9 cells grown in the same manner as above were transferred to a 25 cm cell culture flask, and after culturing for several days (the cells were grown at approximately 5X
Remove the culture medium and inoculate the recombinant virus (primary Q solution containing approximately 5x107 plaque-forming ability).After standing still for 1 hour, remove the cell supernatant and add 5mQ of the culture medium. Brace medium containing 10% tallow serum was added, cultured at 26.5°C for 3 days, and the culture solution was collected.A similar operation was performed using the wild type virus AcNPV.

The hGHR activity in the culture solution was measured by the method of Leung et al. [Nature, 330.537 543 (1987
)] with some modifications. That is, 90mM Tris-HCl buffer, 5QmM chloride 01 tn/
human growth hormone (Tyna Hot Co., Ltd., approximately 30ttC) containing 25% iodine in bovine serum albumin solution
1/1 tfl, approximately 4000 cpm (hereinafter, human growth hormone is abbreviated as hGH) and the hGHR fraction are mixed, and the pH is finally adjusted to 8.2 and the solution volume to 200 μa. This is kept warm at room temperature overnight. Subsequently, 0.02 mC of anti-hGHR antibody diluted 500 times with 50 mM Tris buffer (pH 7.5) and 10 mM calcium chloride is added, and the mixture is allowed to stand at room temperature for 2 hours. The anti-hGHR antibody is Le
3, which has a slightly modified amino acid sequence based on the amino acid sequence of hGHR revealed by ung et al.
Antibodies against the two peptides (Figure 6) were created using rabbits. This antibody is also an antigen. The peptides are purified using a column bound to each peptide, and then mixed together. Next, add 0.22x (OI% bovine γ-globulin solution, 0.44 m12 of 30% polyethylene glycol solution, mix well, and then press in a horizontal rotor at 600 m
Centrifuge at 0 rotations, then centrifuge at 15,000 rpm using an angle rotor.
Centrifuge at 0 rpm, remove the supernatant, and count the precipitate using a γ-counter (Cobra, Pa-Nocard). The results are shown in Table 1.

Table 1 (a value of 616 cpm when 1 μg of culture fluid without infectious virus was added to the reaction solution as a symmetric value was subtracted).

) This result shows that when infected with AchGHR virus,
The culture fluid shows hGHR activity or secretion.

Furthermore, the binding constant between hGH and hGHR was determined for the culture supernatant in the presence of labeled hGH and unlabeled natural hGH (Tainabonoto). The results are shown in FIG. 7, and a high result constant of about 3×10 9 M1 was obtained from the Scachiard plot.

Secretory production of hGHR into the culture solution can also be detected by Western blotting. The Western plot method can be carried out using the method described in the book (Zoku Biochemistry Experiment Course Volume 1, Nippon Biochemistry Complete Edition, Tokyo Kagaku Doujin). This is Nogubio 9. A kit from F1 ([mmun-Blot,
Goat Anti-Rabbit IgG (
H+L) Alkaline Phosphatase
Conjugate) and follow the instructions attached to it. According to this, an hGHR band is detected at a position corresponding to approximately 38 kilodaltons. Based on the amino acid sequence encoded by the imported DNA, it is expected that hGHR with a size of about 32 kilodaltons will be detected. A value larger than this suggests that a sugar chain is added to the hGHR produced here, and hGHR was actually detected at about 32 kilodaltons by treatment with glycanase, an enzyme that removes sugar chains. will be done.

Example 4 Purification of hGHR The hGHR in the culture solution was derived from hGH with hGH as a ligand
-Can be purified by affinity column chromatography. The preparation method for hGH-affinity column chromatography is described by Spencer et al. [J, Biol, Che.
M., 263.7862-7867 (1988)]. In this case, glycerin control bore glass (Sigma 1
hGH bound to 20-200 mesh) was described by Tokunaga et al. .

153.445-449 (1985)], a large amount of synthetic hGH gene is expressed in an insolubilized state in Escherichia coli, and the gene is rolled up using guanidine hydrochloride. Magnesium chloride was added to 3 mQ of the cell culture solution infected with ΔchG II R prepared in Example 3 to give a concentration of 10 mM, and the mixture was added to a 06 mQ hGH-affinity column.

The liquid that passes through the column is added to the column again.

Repeat this operation once more. This was followed by 30z (!50mM containing 1M sodium chloride) Lis-HCl buffer (
Wash the column with pH 7.5). hGI (R activity is
50mM containing 1.2Mg of 4.5M magnesium chloride
Elute with Tris-HCl buffer. The results of analysis using 10% polyacrylamide gel electrophoresis containing SDS showed that the eluted protein was a single sample, and its molecular weight was approximately 3.
The molecular weight of 8 bovine lotalton completely matched the molecular weight previously detected by Western blotting. According to the N-terminal amino acid sequence method, the N-terminal amino acid sequence of this preparation was determined by Leung et al. [Nature, 330.
537-543 (1987)], which corresponds to the N-terminal amino acid sequence of natural hGHR from which /gunarbebutite was cleaved. The above results indicate that the purified protein is the desired hGHR. hG I-I as a single preparation
Elution of R is also done with 50mM Tris-HCl buffer containing 8M urea.

Example 5 Secretion expression of hGHR using a serum-free medium Production of hGHR using a serum-free medium is carried out according to Example 3. In other words, approximately 106 S, f, 9 cells cultured in Brace medium containing 10% tallow serum were transferred to a 25 cm cell culture flask and cultured for several days, resulting in a cell count of approximately 58 cells.
The cell supernatant (increasing to 10') was removed and recombinant virus AchG I-I R (1 positive solution containing about 5XlO' plaque-forming ability) was inoculated. After standing still for another hour, the cell supernatant was removed and added to 5ff12 serum-free medium EExcel 400 (J RS scientific
EX-CELL 400) (Company C, EX-CELL 400) was added thereto and cultured stationary at 26.5°C for 3 days, and the culture solution was collected.

The IICI (R activity) in the culture solution was determined in the same manner as in Example 3. The results are shown in Table 2. This shows that hG l-IR is secreted and produced (the amount produced is also the same as when serum is included).

Table 2 (a value of 616 cpm when 1 μe of culture fluid without infectious virus was added to the reaction solution as a symmetric value was subtracted).

) Western blot analysis also supports this finding, as hGI-IR was detected in approximately 38 kilocrotons, similar to the serum-containing culture solution. The serum-free culture solution was directly added to SDS polyacrylamide gel, electrophoresed, and stained with Kumasi. As a result, protein hands were detected at positions that do not exist in the corresponding wild-type AcNPV infection. Since this band corresponds to the position detected by Western blotting and the position of hGHR purified in Example 4, it is considered to be the hGHR of interest.

When compared with Kumasi staining of the target protein, the production amount in the serum-free medium is estimated to be several m9/(l).

hGHR produced in this way is also hGH as in Example 3.
- Can be purified into a single standard by affinity column chromatography.

Example 6. Intracellular expression of hGHR Intracellular expression of hGf(H) can be carried out according to Example 3. That is, as in Example 3, recombinant virus A
Infect S, f, 9 cells (approximately 5 x 106 cells) with chG HR (5 mf2 culture medium/25 cm2 flask), 26
．． The cells were cultured at 5°C for 3 days and collected. cell is 0.1
ml 50mM Tris-HCl buffer (pH 7°5),
250mM sodium chloride, 10mM calcium chloride,
Homogenize in 10 mM benzamide, 50,0 OOKIU/Q abroteinin solution. The obtained extract was diluted to a total concentration of 5 mO and measured by the method of Example 3. The results are shown in Table 3, and as expected, Example 4 was able to achieve a precision of 92.

Table 3 (value 613c when no cell fraction is added as a symmetrical value)
pm is subtracted)

[Brief explanation of the drawing]

Figure 1 is a schematic diagram of the construction of plasmid pBSR8-3, Figure 2 is a schematic diagram of the sequence of synthetic oligonucleotides used in the construction of plasmid pBSR8-3, Figure 3 is a schematic diagram of the construction of plasmid pAcYMhGHR, and Figure 4 is a diagram of the construction of plasmid pBSR8-3. Synthetic oriconucleotide N'Bglll site U-signal,
Schematic diagram of the sequence of the L-signal, Figure 5 is a model for constructing recombinant baculovirus, Figure 6 is a diagram of the sequence of the peptide used to create anti-hGHR antibody, and Figure 7 (a) is hGH and hGH.
Graph showing R and bond amount, Figure 7(b) is Figure 7(a)
This is a graph showing the results of Scachiard Pronotation. Patent Applicant Patent Attorney Aoyama Aoyama (1 other person) Representative of Protein Engineering Research Institute Co., Ltd. Maki 5 AcNPV DNA ◆ Supernatant ↓ Plaque AchG) - IR 86-Kucho Danrus Diagram Cys-3er -Gly - 5er-Glu-Al
a -Thr-Ala -Ala-11e -Lcu-
Ser -Arg -Ala -Pro -Trp -
5etSer -Leu-Gin -Ser -Val
-Asn-Pro-Gly-11e-Lys-
Thr −Asn −Ser −Ser −Lys −
Glu-Pr. Lys-Phe-Thr-Lys-CysCys-1
1is-Trp-Thr-Asp-Glu-V
at -11is -11is -Gly -Thr
-Lys -Asn -Leu -Gly -Pro-
11eGin-Leu

Claims (1)

[Scope of Claims] 1. A recombinant plasmid containing a DNA fragment encoding the amino acid sequence of the hormone binding region protein of human growth hormone receptor under the control of a baculovirus gene expression system. 2. A recombinant baculovirus containing a DNA fragment encoding the amino acid sequence of the hormone binding region protein of the human growth hormone receptor under the control of a gene expression system. 3. A method for producing a hormone binding domain protein of human growth hormone receptor, which comprises infecting and culturing insect cells with the recombinant baculovirus according to claim 2. 4. The production method according to claim 3, which comprises culturing in a serum-free medium. 5. The production method according to claim 4, which comprises recovering the hormone binding region protein of human growth hormone receptor from the cell extract after culturing.