JPH03224491A - Production of pig growth hormone (pgh) - Google Patents

Abstract

NEW MATERIAL:Recombinant pBE274 DNA-containing pig growth hormone gene integrated in the polylinker region of transfer vector pBE274 containing the promoter region of a polyhedron protein structure gene of silkworm nuclear polyhedrosis virus DNA. USE:Production of pig growth hormone. PREPARATION:A DNA fragment of pig growth hormone gene is introduced into the EcoRI cutting site of transfer vector pBE274 to obtain recombinant transfer vector pBE274-pGH.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a transfer vector p derived from the silkworm nuclear polyhedrosis virus that is recombined with the porcine growth hormone gene.
The present invention relates to a method for producing porcine growth hormone, which comprises infecting cells and a host with BE274 DNA, a karyopolyhedrosis virus containing a porcine growth hormone gene recombined using the ρBE274 DNA, and a knee recombinant virus.

(Prior art) Recent advances in genetic engineering have made it possible to produce large amounts of heterogeneous tectal white matter.For example, a method for producing useful substances using recombinant plasmids in Escherichia coli and yeast. have been reported in large numbers.

On the other hand, in recent years, there has been increasing interest in the use of livestock growth hormones in the livestock industry, and there have already been many reports on the effects of administering livestock growth hormones to livestock. For example, Terry et al. administered porcine growth hormone to piglets and confirmed that it had a significant growth promoting effect (T
erry et al., J. Anim.

Sci, 64:433-443.1987).

Furthermore, this hormone is a peptide, so there is no problem of it remaining in meat, which is advantageous in terms of safety.

Therefore, in recent years, attempts have been made to mass produce livestock growth hormone using the above-mentioned genetic engineering techniques.

[Problems to be solved by the invention]

However, the conventional means of producing livestock growth hormone uses expression systems of prokaryotic cells such as Escherichia coli, and these prokaryotic cell systems are ineffective in addition of sugar chains, splicing, degradation by peptidase, etc., and secretion. Unlike the nuclear cell system, in many cases it is not possible to directly produce large amounts of useful substances derived from mammals, and even when producing livestock growth hormone, it is not possible to directly produce the growth hormone, and after expression, 2 Further processing was required, and the expression level was also low.

Therefore, the object of the present invention is to solve such problems,
The object of the present invention is to provide a means for efficiently producing a large amount of porcine growth hormone.

[Means to solve the problem]

That is, the present invention provides the following features: 1. A transfer vector p containing the promoter region of the polyhedron protein structural gene of the silkworm polyhedrosis virus DNA.
Recombinant pBE274 DNA that incorporates the porcine growth hormone gene into the polylinker region of BE274.

2. Recombinant pBE274 DNA described in 1 above,
Recombinant silkworm nuclear polyhedrosis virus containing the porcine growth hormone gene obtained by simultaneously infecting cultured silkworm cells with the silkworm nuclear polyhedrosis virus, 3. Silkworm nucleus containing the porcine growth hormone gene described in 2 above. A method for producing porcine growth hormone, which comprises obtaining porcine growth hormone by infecting and multiplying cultured silkworm cells or silkworm larvae with polyhedrosis virus;
Regarding.

The present invention will be explained in more detail below.

(1) The transfer vector pBE274 used in the present invention is derived from the silkworm nuclear polyhedrosis virus that has been adjusted by front pressure, etc., and has the structure shown in Figure 1 (Maeda Invertebrate CellS
system Applications, CRC Pre
ss C1eveland.

1988). Jhai transfer vector pBE274
In this method, a part of the polyhedral protein structural gene is replaced with a linker region having a cleavage site for EcoR1, and the promoter region of the polyhedral protein structural gene is located upstream of the 5' end of the linker. That is, from pBR322 (pBmE36) into which an EcoRI 10.5 kb fragment containing the polyhedral gene was cloned, the polyhedral gene was removed while leaving as much of the preserved sequence downstream of the promoter as possible, and a polylinker was inserted. Approximately 3 kb is left for the combination of upstream and downstream beams of the polyhedral gene.

First, the old ndI of pEmE36 [[3,9 kb and 3.1
kb, pUc9 (p9H1B) and pUc, respectively.
8 (p8H255). In order to remove the polyhedral gene from the p9818 plasmid containing this polyhedral gene, after cutting with EcoRI, exonuclease treatment was performed with Bal 31, and then the old ndlI [After cutting, the old nd m -
Clone into the Sma ■ site. In this case, Bal 3
By changing the reaction time in step 1, DNA fragments of various lengths can be cloned, and the base sequences of these plasmids can be determined from the Sma I side by the dideoxy method. By using the dideoxy method, which directly seafences plasmids, the nucleotide sequence of the polyhedral gene is not determined, so a plasmid that has been digested to the desired length can be selected and used as is for the construction of the next vector. p9B31
The 0 plasmid is obtained by removing up to 18 bases upstream of the methionine ATG at the start of translation of the polyhedral gene.

On the other hand, the downstream of the polyhedron gene was cloned into pLlcs (p8H225). This is to take advantage of the fact that the puc 8 and puc 9 plasmids have the same backbone and the polylinkers are in opposite directions. A plasmid (p89B3
10), each p
It is derived from uc 9 and pUc 8, and contains 3 kb upstream of the polyhedral gene including the promoter, the polylinker derived from puc 8, and 3 kb downstream in the forward direction.

Finally, pBE274 is completed by removing cutting sites other than EcoRl from a portion of the polylinker.

(2) In the present invention, the DNA fragment of the porcine growth hormone gene to be inserted into the transfer vector pBE274 is prepared, for example, as follows. That is, mRNA is extracted from pig pituitary gland by a conventional method, and a cDNA library is created using it as a template. Next, from the library, E and c with growth hormone genes are selected.
oli was selected, and EcoRI was added to both sides of the plasmid.
A porcine growth hormone gene with a truncated surface is obtained. The cDNA base sequence of the porcine growth hormone gene is shown in FIG.

(3) Next, the DN of the porcine growth hormone gene of (2)
Transfer the A fragment into (1) transfer vector pBB274
into the EcoRI cutting site to create a recombinant transfer vector pBE274-pGH.

In this recombination, transfer vector pB
Cut E274 with EcoRI, insert the porcine growth hormone gene that already has the EcoR1 cleavage plane, and create T4.
The recombinant transfer vector pBE274-pG)l can be obtained by ligating with ligase, transforming E. coli, and selecting E. coli having a plasmid containing the porcine growth hormone gene in the correct orientation.

Figure 3 shows the recombinant transfer vector pBB2 when pBE274 was cut with EcoRI and a porcine growth hormone gene DNA fragment was connected to the cut site.
74-pG)t structure.

(4) Transfer the recombinant transfer vector pBE274-pGH obtained in (3) to the silkworm nuclear polyhedrosis virus DNA.
Together with this, cultured silkworm cells are infected using, for example, the calcium phosphate method. This allows the porcine growth hormone gene D on the recombinant transfer vector pBE274 to
NA and the polyhedrosis gene DNA of the silkworm nuclear polyhedrosis virus are replaced, and a recombinant silkworm nuclear polyhedrosis virus containing the porcine growth hormone gene region can be obtained. Cloning of the virus can be performed by plaque assay or limiting dilution.

(5) Thereafter, the recombinant silkworm nuclear polyhedrosis virus is infected and propagated in a host such as cultured cells of silkworms or silkworm larvae to produce porcine growth hormone.

When using cultured silkworm cells, the recombinant virus solution is added to the medium in which Bm-N cells are cultured so that there are two infectious virus particles per cell, and the number of infectious virus particles is 2 to 6 days after the 4th to 6th day. 50 μg/rnl of porcine growth hormone was found in the medium.

When using silkworm larvae, if 3 x 10' infectious virus particles are percutaneously injected into the 5th instar larva, 4~
On day 6, 500 μg/ml of porcine growth hormone is found in the body fluids. These porcine growth hormones are purified from culture fluids or body fluids by general methods such as gel filtration chromatography, ion exchange chromatography, and affinity chromatography.

〔Effect of the invention〕

According to the present invention, there is no need for secondary processing of the obtained porcine growth hormone as in conventional prokaryotic cell systems, and growth hormone that has biological activity that is effective for pigs as it is can be produced directly and in large quantities. I was able to do it. As a result, the present invention greatly contributes to the development of livestock farming.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 (1) Preparation of transfer vector pBE274 Transfer vector pBE274 was prepared as shown in FIG. 1. That is, 5O3 (sodium lauryl sulfate, 1 .W/W%) and protease K (manufactured by Merck & Co., Ltd., 1+ng/mf) were added and incubated at 37°C for about 2 hours.To this solution was added an equal volume of phenol solution (10mM l~Lis-HCl). Buffer (pH 7,6)
-1mM EDTA saturation) was added, gently shaken for about 5 minutes, and then centrifuged at 12,000 rpm for 5 minutes. The aqueous layer was taken and the same phenol treatment was repeated twice. This D
An equal volume of chloroform was added to the aqueous layer containing NA, gently shaken for about 5 minutes, and then centrifuged at 12.00 rpm for 2 minutes. After taking the aqueous layer and repeating the same chloroform treatment twice, the aqueous layer was dissolved in Tris-HCl buffer (pH 7,
6) Dialyzed against 1 mM EDTA for about 2 days.

The obtained viral DNA was digested with various restriction enzymes and
．． DNA fragments separated by 7% agarose gel electrophoresis were analyzed using the satin method, and EcoRI containing the polyhedral gene was detected.
A 10.5 kb fragment was cloned into pBR322 (manufactured by Takara Shuzo Co., Ltd.).

The polyhedral gene is removed from this pBR322 (pBmE36), leaving as much of the preserved sequence downstream of the promoter as possible, and a polylinker is inserted.

Approximately 3 kb is left for the combination of upstream and downstream beams of the polyhedral gene.

First, pBmE36 Hind m 3.9kb and 3.
1 kb, respectively pUC9 (p9) 11B), pU
Clone into C8 (p8H255). In order to remove the polyhedral gene from the p9H18 plasmid containing this polyhedral gene, after cutting with EcoRI, exonuclease treatment was performed with Bal 31, and after cutting with old ndI [l, the old nd m - S
Clone into the ma I site. In this case, Bal 31
By changing the reaction time, D'NA fragments of various lengths can be cloned, and the base sequences of these plasmids can be determined from the Sma I side by the dideoxy method. By using the dideoxy method, which directly seafences plasmids, it is possible to select plasmids that have been digested to the desired length while determining the base sequence of the polyhedral gene.
It can be used as is for the next vector construction experiment. p9B
The 310 plasmid is obtained by removing up to 18 bases upstream of the methionine ATG at the start of translation of the polyhedral gene. On the other hand, the downstream part of the polyhedron gene was cloned into pUcs (p8H225). This is pUc 8 and pUc 9
This is to take advantage of the fact that the plasmids have the same backbone but the polylinker is in the opposite direction. A plasmid for foreign gene insertion (p89B
310) are derived from puc 9 and pUc s, respectively, with AatII of puc as the border, and include 3 kb upstream of the polyhedral gene including the promoter, the polylinker derived from puc 8, and 3 kb downstream in the forward direction. There is.

This plasmid p89B310 was further processed into p89B310.
It is BE274. It differs from p89B310 in that about 18 bases upstream of the polyhedral gene are left almost unchanged and the cloning site is only EcoRIl.

(2) Preparation of porcine growth hormone gene DNA Porcine growth hormone cDNA was prepared from porcine pituitary gland by a known method. That is, c
A DNA library was created (niato, Mo1.C
e11. Endocrinol).

YANG et al.'s method (Proc, Natl, Acad, S
ci, USA, 80°1194-1198),
Several growth hormone expressing clones were identified from 5×10' plaques expressed on nitrocellulose membrane using anti-sheep growth hormone rabbit antibody. From these clones, one that is thought to completely encode the structural gene for porcine growth hormone is selected, and after extracting the target gene, EcoR
A DNA fragment having I 5ite at both ends was prepared.

(3) Recombinant transfer vector pBE274-
Preparation of pGH This DNA was ligated with EcoRI-treated ptlc9, and the resulting plasmid was used to infect E. coli with 12JM106.
was transformed and propagated to produce a large amount of plasmid. This plasmid was treated with EcoRI, the fragment containing the porcine growth hormone cDNA was separated by agarose electrophoresis, and the resulting DNA containing the porcine growth hormone gene was ligated to EcoR using a Takara ligation kit (manufactured by Takara Shuzo).
12) IBIOI was transformed into Escherichia coli using the resulting plasmid integrated into pBE274 treated with IBIOI. Plasmids were extracted from these E. coli clones, treated with several types of restriction enzymes, and then subjected to agarose electrophoresis to select clones containing the porcine growth hormone gene in the correct orientation.

This clone was propagated to produce a large amount of plasmid.

This was named pBE274-pGH. This follows the upstream part of the polyhedral gene 1. Next to 5 bp of EcoRI residues and 32 bp of the 5' untranslated region of the pGH gene obtained above, the pGH gene starting with ATG is bound in the correct direction, and the downstream part of the polyhedral gene (approximately 300 bp downstream from the stop codon) is bonded in the correct direction. bρ to approximately 3.1 kbp).

This plasmid pBE274-pGH has been deposited as FERM P41128 at the Institute of Microbial Technology, Agency of Industrial Science and Technology.

(4) Preparation of recombinant nuclear polyhedrosis virus Transformation of Bm cells was performed using a commercially available transfection kit (manufactured by Pharmacia). 3 μg of viral DNA obtained by the method shown in (1) above and porcine growth hormone c
Transfer vector pBE2 containing DNA
A DNA solution containing 15 μg of 74 DNA was mixed with the buffer provided with the same kit to obtain a coprecipitate of both DNAs and calcium phosphate. 480 µm of this coprecipitate was added to 4 times less 8m cell culture medium, and after culturing for 20 hours, the medium was replaced with fresh medium and culture was continued for an additional 5 days. Next, 10-4 to 10 of this culture solution
-7 dilution solution was mixed with an equal volume of 1 x 105 cells/ml solution, and 2
After dispensing 00 μβ into microplates and culturing for another 5 days, each plate was distinguished between non-infected and infected states, and clones with virus infection and no polyhedrosis formation, i.e., recombinant silkworm nuclear polyhedrosis virus. were selected.

(5) Production of porcine growth hormone If the recombinant virus solution is added to the medium in which Bm-N cells are cultured so that the number of infectious virus particles per cell is 2, the medium will be in the medium on the 4th to 6th day. 50 μg/ml of porcine growth hormone was found in the sample.

On the other hand, 17 5th instar and 1 day old silkworms were injected percutaneously with the medium 0105ml after 5 days of culture as described above, and after rearing with the artificial sample at 25°C for 5 days, the pig intestines were cut and the body fluid was drained. Collect into an ice-cold polystyrene centrifuge tube, add an equal volume of TC-10 medium, centrifuge at 10,000 rpm for 5 minutes), and remove the supernatant.
Qualitative and quantitative measurements of porcine growth hormone were performed.

(6) Confirmation of porcine growth hormone gene expression Confirmation of porcine growth hormone gene expression was performed by Western bronting method. First, the Bm cell expression culture medium and the total expression body fluid shown in (5) above were collected and diluted 10 to 100 times to prepare an analysis sample.

These samples were subjected to 15% polyacrylamide electrophoresis using a commercially available porcine growth hormone reagent (Funakoshi Pharmaceutical Co., Ltd.) as a control, and then the porcine growth hormone in this gel was transferred to a transfer membrane using a transfer device. After applying a commercially available anti-pig growth hormone rabbit antibody (Funakoshi Pharmaceutical) to the porcine growth hormone on the transfer film, and then applying an enzyme-labeled anti-rabbit antibody,
The presence of porcine growth hormone was confirmed by the color reaction caused by the action of this enzyme. Other coloring solutions containing enzyme-labeled anti-rabbit antibodies are commercially available enzyme antibody method Kinol (manufactured by Bio-Rad).
I used

The results are shown in Figure 3, and the ability to express lid growth hormone was confirmed.

(9) Confirmation of expression level of porcine growth hormone Most of the porcine growth hormone synthesized by the recombinant virus in cultured cells or silkworm body cells is secreted into the culture solution or body fluid.

Therefore, a portion of the culture fluid or body fluid was collected over time and used as a sample for confirming the expression level. In addition, the amount of porcine growth hormone in the sample was measured by a commonly used enzyme antibody method.

That is, commercially available pure porcine growth hormone (Funakoshi Pharmaceutical Co., Ltd.) is used as a standard product, and it is appropriately diluted together with the obtained sample solution and injected into a microplate to serve as an antigen. Next, anti-porcine growth hormone rabbit antibody, enzyme-labeled anti-rabbit antibody,
Color formers were injected in order, and the color density was measured using a commercially available dedicated measuring device. Figure 4 shows the measurement results.

There have been no known examples of such a large amount of expression.

(11) Biological test of expressed porcine growth hormone Using silkworm body fluid containing porcine growth hormone as a starting material, a dry powder of porcine growth hormone with a purity of about 98% was obtained by a protein purification method used in boat fishing. 4mg/ml of this dry powder
It was dissolved in physiological saline and used as a sample for the biological tests described below.

Twenty-four castrated Landrace x Large Yorkshire x Duroc male pigs (average weight 30 kg) were divided into two groups, a test group and a control group, and raised for about 6 weeks.

During that time, pigs in the experimental group received 100 μg per kg of body weight daily.
porcine growth hormone dissolved in saline was administered to the control group, while the control group received saline alone. Administration was performed by intramuscular injection near the neck. Dosage for individual pigs in the study area was adjusted by weighing each weekend.

The results are shown in Table 1.

(Margins below this page) A A Q Δ-NO-E SOYI SHAN CH The administration of the porcine growth hormone according to the present invention resulted in significant improvements in both body weight gain and feed efficiency.

[Brief explanation of the drawings] Figure 1 is a construction diagram of pBE274 and pBE274-pG)I, Figure 2 is a nucleotide sequence diagram of the porcine growth hormone gene, Figure 3 is an expression diagram of the porcine growth hormone gene, and Figure 4 is a diagram of the porcine growth hormone gene. FIG. 2 is a diagram showing the production of porcine growth hormone in silkworms and cultured cells infected with a recombinant virus.

Claims (1)

[Scope of Claims] 1. Transfer vector p containing the promoter region of the polyhedron protein structural gene of the nuclear polyhedrosis virus DNA
Recombinant pBE274 DNA in which the porcine growth hormone gene is integrated into the polylinker region of BE274. 2. A recombinant silkworm nuclear polyhedrosis virus containing the porcine growth hormone gene obtained by simultaneously infecting cultured silkworm cells with the recombinant pBE274 DNA according to claim 1 and the silkworm nuclear polyhedrosis virus. 3. A method for producing porcine growth hormone, which comprises obtaining porcine growth hormone by infecting cultured silkworm cells or silkworm larvae with the silkworm nuclear polyhedrosis virus containing the porcine growth hormone gene according to claim 2.