JPH0459800A - Production of high-purity feline interferon - Google Patents

Abstract

PURPOSE:To obtain a feline interferon (FeIFN) expected to be useful s an antiviral agent and antitumor agent for cat in high purity by producing FeIFN in a mass utilizing a gene-recombinant silkworm nuclear polyhedrosis virus and contacting the produced FeIFN with a carrier containing bonded blue dye. CONSTITUTION:A recombinant silkworm nuclear polyhedrosis virus is produced by the gene recombination with a DNA coding the protein of FeIFN. A culture liquid containing the virus is injected into a silkworm larva, which is bred with mulberry leaf or a synthetic feed. After breeding, body fluid is collected from the larva and FeIFN is recovered from its supernatant. The solution containing FeIFN is brought into contact with a carrier bonded with a blue dye or C1 Reactive Blue 2. The adsorbed FeIFN is desorbed with an eluent to obtain FeIFN having high purity.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is to mass produce interferon, whose primary protein structure is derived from cat genetic information, by using a genetically modified nuclear polyhedrosis virus. The present invention relates to a method for producing highly purified feline interferon (hereinafter abbreviated as FeIFN) using a recombinant silkworm nuclear polyhedrovirus and silkworm larvae, which is intended to be used as a pharmaceutical (antiviral agent).

[Prior Art] Interferon is a physiologically active substance whose main component is a protein that exhibits antiviral activity, and is abbreviated as IFN.

Due to advances in genetic engineering technology, not only human IFN but also
It has become possible to mass-produce IFN from animals such as cows, horses, and dogs, and as a result, research is being conducted to develop the use of IFN as a therapeutic agent for viral diseases, tumors, etc.

α, β, and γ types of rFN have also been reported for cats (Reference 1).

A number of viral diseases are known in cats, including feline AIDS, feline leukemia, feline viral rhinotracheitis, feline kaliki virus disease, and feline infectious peritonitis.

Therefore, cases have been reported in which human α-IFN or bovine β-IFN was orally administered to prolong the life of cats infected with feline leukemia virus. Although more pronounced effects are expected if the drug is injected into the body rather than orally, there is a concern that neutralizing antibodies against foreign IFH may be produced. Allogeneic IFN
In other words, if feline IFN becomes easily available, it is expected that it will be used as an antiviral and antitumor agent for cats.

The present inventors have previously developed a method for producing FeIFN using genetic engineering technology, that is, using a DNA encoding FeIFN.
We cloned A and developed a production method using monkey C081 cells, Chinese hamster CHO cells, Escherichia coli, etc. (Reference 2).

[Problem to be solved by the invention] However, FeIFN of high purity for practical use is
A method for mass-producing it has not yet been established.

The purpose of the present invention is to utilize genetic engineering technology to
The objective is to provide a method for mass-producing the product with even higher purity.

[Means for Solving the Problems] In view of the above situation, the present inventors have developed a recombinant virus in which the DNA of the silkworm nuclear polyhedrosis virus has been recombined with the DNA encoding the FeIFN protein. F
They succeeded in producing eIFN and further established a method for purifying FeIFN to a high degree of purity, thus completing the present invention.

That is, the present invention provides D
F
After producing eIFN, a solution containing the produced FeIFN is brought into contact with a carrier to which a blue dye is bound, and then the adsorbed substance on the carrier is eluted with an eluent. A manufacturing method is provided.

The present invention will be explained in detail below.

The recombinant Bombyx mori nuclear polyhedrosis virus of the present invention can be obtained from a plasmid extracted from E, coil (pFeIFNl) (Kikoken Jokyo No. 1633) described in Reference 2, for example.
A recombinant plasmid and silkworm nuclear polyhedrosis virus D were prepared by cutting out the DNA encoding the e1FN protein and ligating it to a silkworm cloning vector (Reference 3).
It can be easily produced by co-transfecting established silkworm cells with NA. Therefore,
The recombinant virus of the present invention can be produced by an in v'iv''o method.

That is, plasmid pFerF extracted from an E. coli transformant containing plasmid pFeIFN1 (FeIFN1) containing DNA encoding the FeIFN protein by a general method as described in Reference 4, for example.
A recombinant plasmid can be produced according to general genetic manipulation in which the DNA portion encoding the N1 to Fe1FN proteins is ligated downstream of the expression control portion of a silkworm cloning vector such as pBMO30 (Reference 3). . This recombinant plasmid and silkworm nuclear polyhedrosis virus DNA (Reference 3) were added to established silkworm cells, such as BM-N strain (Reference 3), by the method described in Reference 3.
), continue culturing,
Recombinant viruses can be cloned from the non-recombinant (wild type) and recombinant viruses that appear in the culture medium by common methods such as limiting dilution or plaque method. Recombinant viruses can be easily distinguished from wild-type viruses because they do not have the ability to form polyhedra.

FeIFN is produced by propagating the recombinant silkworm nuclear polyhedrosis virus in living silkworms. Silkworm larvae are injected with a culture solution containing the recombinant virus and raised with mulberry leaves or synthetic feed. After rearing, body fluid is collected and FeIFN is collected from the supernatant.

The recombinant silkworm nuclear polyhedrosis virus present in body fluids is p.
H1-4 can be inactivated by storing at 4°C for 1 day.

As the carrier bound to the blue dye (hereinafter abbreviated as blue carrier) used in the present invention, the following are used.

The common name of blue dye is CI Reactive Blue 2.
For example, a blue pigment commercially available from CI BA-GE IGY under the trade name "Cibacron Blue F3GA" or "Cibacron Blue 3GA" may be mentioned.

The blue carrier used in actual chromatography is 'Blue Sepharose CL-6B' (Pbarma
cia), ``Blue-Sepharose 6 First Flow'' (Phatmacia), ``Madrex Gel Blue A'' (Amicon), and ``Affigel Tatami Blue'' (Biorad). Agarose gel or blue cellulose gel commercially available under trade names such as “Blue Trisacryl-M” (LKB) and “Blue Cellulose Gel” (Chisso) are suitable and easily available. I can do it.

The purification operation of FeIFN using the blue carrier is carried out as follows.

That is, first, a solution containing FeIFN is contacted and adsorbed onto a blue carrier. Adsorption can be performed by either a batch method or a column method, but the column method has higher adsorption efficiency.

Elution is determined by the pH value, ionic strength, and hydrophobicity of the eluent. For example, FeIFN is eluted at pH 6-8 under high ionic strength. This ionic strength is
Increase the concentration of buffer solutions such as acetic acid, citric acid, boric acid, Tris/HCl, or add neutral salts such as sodium chloride, potassium chloride, and calcium chloride (0.2M to 1.0M
) can be increased. Furthermore, when the eluent contains a solvent that weakens hydrophobic interactions, such as ethylene glycol or propylene glycol, elution at pH 5 to 8 becomes possible.

The composition, concentration, and liquid volume of the eluent are not particularly limited, and each is effective for removing contaminant proteins contained in the crude FeIFN material, and has a concentration necessary to maintain the pH and adsorption. The amount necessary to substantially recover the depleted FeIFN is used.

By adsorbing and eluting FeIFN to the blue carrier in this manner, contaminant proteins mixed in the crude FeIFN can be removed and FeIFN can be selectively purified and separated.

The method of the present invention can also be used in combination with other affinity chromatography methods. As another affinity chromatography, a method using a carrier to which copper is chelated (hereinafter abbreviated as copper chelate carrier) is preferably used. Moreover, these may be used consecutively.

As a copper chelate carrier, a carrier in which an exchange group having a chelating ability such as a biscarboxymethylimino group [-(CH2C00)l) 2 ] is bonded to agarose, cellulose, polyacrylamide gel, etc. is used as a copper salt such as copper sulfate. Examples include carriers treated with a solution of Preferably, "chelating sepharose" (Pharmae
A carrier in which copper is chelated to an insoluble polysaccharide carrier such as (manufactured by IA) is used.

Further, elution from the copper chelate carrier is usually performed with an acidic buffer such as phosphoric acid, acetic acid, or citric acid, preferably at a pH of 5 or less. However, under high ionic strength, even higher p
A solution in H is possible.

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

Example 1 (1) Antiviral activity measurement method The virus is Vesicular Stomatitis.
Antiviral activity was measured according to the CPE method using cat FC9 (Reference 1) as virus-susceptible cells. As a standard reference, the NIH human natural α
Hu I FNα converted to rFN was used.

20 μg of plasmid pFeIFN1 extracted from transformed Escherichia coli E. coli (pFeIFNl) (Feikoken Jokyo No. 1633) using the method described in Reference 4 was treated with restriction enzyme S f
Completely decomposed with aNl and HincII, the obtained multiple D
The NA fragments were separated by agarose gel electrophoresis and were approximately 750 b
The p DNA fragment was removed by electroelution and approximately 2 μg was recovered. In this way, a 5faN1-HinclI fragment containing DNA encoding FeIFN was obtained.

5 μg of cloning vector pBMO30 (Reference 3) was completely digested with restriction enzymes Bgln and SmaI, and the above 5f
The aN1-HincII fragment was ligated with T4 DNA ligase. E, which made this reaction solution competent,
The mixture was mixed with E. coli HBIOI (manufactured by Zenshuzo Co., Ltd.) and transformed. Hind of plasmids extracted by alkaline miniscreen method from colonies growing on LB plates containing 100 μg/ml ampicillin.
Restriction analysis of the cloning vector pB
A plasmid in which a DNA fragment of about 750 bp was integrated into M030 was obtained. Approximately 100 base DNA sequences including the start codon of the FeIFN-encoding DNA of the plasmid were performed, and FeIF was inserted into pBMO30.
A plasmid containing DNA encoding N was obtained. This recombinant plasmid was named pBmFeIFNl. The method for producing pBmFeIFNl is shown in FIG.

(3) A recombinant virus was produced by recombining with DNA encoding FeIFN using the method described in Reference 3.

i.e. 50mM HEPES buffer y-pH7,
1,0,28M NaCl1,0,7mM Na2HPO
2.5 ml consisting of 4.0.7mM NaH2PO4
Add 2.5 ml of DNA mixture (0.25 M
CaCl2, Bombyx mori nuclear polyhedrosis virus BmNPV
(containing DNA mixture g of Ta strain (Reference 3) and DNA mixture g of recombinant plasmid pBmFelFN1) was added dropwise, and 0.5 ml of the resulting suspension was added to 5 ml of 10% FBS.
The DNA was introduced into the silkworm cells in addition to the culture medium of about 3×10 5 BM-N cells (Reference 3) that were cultured on a flat surface in a 25aIr flask in TC-10 medium (Reference 5) supplemented with . After 20 days, the medium was replaced with fresh medium, and after culturing for an additional 5 days, the culture solution was collected. The culture solution was centrifuged and the clarified supernatant was diluted and added to the culture medium of BM-N cells cultured on a flat surface. After culturing for 7 days, virus infection was observed by microscopic observation, and polyhedra were formed. (limiting dilution method). A recombinant virus was cloned by repeating the limiting dilution method twice, and the recombinant virus produced here containing DNA encoding FeIFN was designated as BmFe I FNI.

BmFeIFNl is European Co11ec
tion of Animal Ce1l Cuture
Deposited under the Budapest Treaty at
session No. V89062701).

(4) Preparation of recombinant virus solution In the bottom of a 75 cof flask, add 15 ml of 10% FBS.
About 3 x 106 BM-N cells cultured in a flat surface in TC-10 medium containing
-N cells and cultured at 27°C for 5 days, then the culture solution was added to 3. The mixture was centrifuged at OOOrpm for 5 minutes to obtain a centrifuged supernatant as a recombinant virus solution. 1 of each virus solution
When 0-7 dilution was made, 1 ml of the diluted solution was added to a culture medium of BM-N cells, and the culture was continued at 27°C for 7 days, virus infection was observed in the BM-N cells in the culture medium by microscopic observation.

(5) Production of FeIFN in living silkworms 50 μl/head of the virus solution of the recombinant virus obtained in (4) was injected into each silkworm larva on the 1st day of the 5th instar, and incubated at 25°C for 4 days.
After rearing on a commercially available synthetic feed (Vitamilk Sales Co., Ltd.), the tail legs were cut, and the body fluid was collected in an ice-cold Eppendorf tube, centrifuged, and the supernatant was obtained to examine antiviral activity.

7.7 x 107 units/ml (body fluid) of FeIFN was produced.

(6) Inactivation of recombinant silkworm nuclear polyhedrosis virus 0.
Silkworm hemolymph containing 4X108 TCID5o of recombinant silkworm nuclear polyhedrosis virus BmFe I FNI per ml was added to 0. The pH was adjusted to 1.5 with IN hydrochloric acid and stored at 4°C for 1 day. It was neutralized with a 2N NaOH solution and 1 ml of it was added to the culture medium of BM-N cells, but the BM-N cells were not infected with the virus.

Recombinant virus BmFelFN1 was inactivated with hydrochloric acid, and silkworm body fluid and crude F were neutralized with NaOH solution.
This was used as an eIFN solution.

(7) FeIFN using a carrier bound with blue dye
Contains FeIFN activity of 3.8X106U/ml as described in item (6) above, and has FeIFN specific activity of 1.2X106U/mg.
14.5 l of crude FeIFN solution, which is a protein, was added to 270 ml of “Blue Sepharose (Fast Flow Type)”.
The column was then soaked in 50mM) Lis-HCl buffer (pH 8) containing 0.5M potassium chloride.
After washing with 1250 ml,
5 containing 0mM Tris-HCl buffer (pH 8) 1.081 and 1M potassium chloride, 40% ethylene glycol.
0mM) Lis-HCl buffer (pH 8) 1,081
IFN was eluted. The eluted FeIFN is 4.2X1
Contains Fe1FN activity of 07 U/ml and has a specific activity of 1. lX
It was 108 U/■ protein. The Fe1FN activity recovery rate at this time was 82.4%, and the specific activity increased 92 times.

(8) FeIFN using a carrier with copper chelate bonded
Purification of FeIFN eluate from the blue carrier in item (7) above 1.
081 was applied directly to a column containing 150 ml of "Chelating Sepharose" containing chelated copper.
．． 20mM acetate buffer containing 3M sodium chloride (pH
After washing with 4,2), 20 containing 0.3M sodium chloride
FeIFN in 750 ml of mM acetate buffer (pH 3,9)
was eluted. The eluted FeIFN is 2.7X107U
Contains FeIFN activity/ml, specific activity 1.4X108
It was U/■ protein. The FeIFN activity recovery rate at this time was 45%, and the specific activity increased 1.3 times.

[Effects of the Invention] According to the present invention, FeIFN, which is expected to be used as an antiviral agent and an antitumor agent for cats, can be mass-produced with high purity.

[References] (1) J, K, Yamamoto et al.: Yet
, 1mmuno1. and Immun.

pathol, If, 1-19. (1
986).

(2) A. Yanai et al.: European published patent
No. 322870 (3) T, Hotiuchi et al.: Agric, B
iol, Chem, 51. 1573-158
0. (1987) (4) Edited by T., Maniatis et al.: Mo1ecul
er Cloning, A Laboratory
Manual, (1982) p86-96.
Co1d Spring Hatbor Labo
ratory, New York.

(5) G, RlGardiner and Hls
to, ckdale: 1. Inverteb
rate PathologF, 25. 363-
370. (1975).

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of an example of the construction of a recombinant plasmid pBmFeIFN1 containing DNA encoding feline interferon. Patent applicant Toshi Co., Ltd. Figure 1

Claims (1)

[Claims] (1) DN encoding the protein of feline interferon
The recombinant silkworm nuclear polyhedrosis virus genetically modified by A is propagated in living silkworms to produce feline interferon, and then the solution containing the produced feline interferon is transferred to a carrier bound to a blue dye. A method for producing high-purity feline interferon, which comprises contacting the carrier, and then eluting the adsorbed substance onto the carrier with an eluent.