JPH0459799A - Production of high-purity feline interferon - Google Patents

Abstract

PURPOSE:To obtain a feline interferon (FeIFN) expected to be useful as 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 copper bonded to the carrier through a chelate bond. 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 containing copper bonded to the carrier through chelate bond, preferably an insoluble polysaccharide carrier containing chelate-bonded copper. The adsorbed FeIFN is desorbed with an acidic buffer solution such as phosphoric acid and acetic acid to obtain FeIFN having high purity.

Description

[Detailed Description of the Invention] [Industrial Application Fields] The present invention is directed to the mass production of interferon, whose primary protein structure is derived from cat genetic information, 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 IFN 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 IFN 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 copper is chelated, and then the adsorbed substance on the carrier is eluted with an eluent. It provides a manufacturing method.

The present invention will be explained in detail below.

The recombinant silkworm nuclear polyhedrosis virus of the present invention is obtained by cutting out the DNA encoding the FeIFN protein from plasmid extracted from E, coil (pFeIFNl) (FeIKEN Jyoki No. 1633) described in Reference 2, for example. hand,
It can be easily produced by co-transfecting silkworm nuclear polyhedrosis virus DNA with a recombinant plasmid produced by ligation to a silkworm cloning vector (Reference 3) into established silkworm cells. Therefore, the recombinant virus of the present invention can be produced by an in vivo method.

That is, from an Escherichia coli transformant containing the plasmid pFe I FNI containing DNA encoding the FeIFN protein (FeIKEN Jyoyori No. 1633), for example, reference 4
Plasmid pFe extracted by a common method such as
A recombinant plasmid can be produced according to general genetic manipulation in which a DNA portion encoding the proteins IFN1 to Fe1FN 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 combined with
After co-transfecting established silkworm cells, such as the BM-N strain (Reference 3), by the method described above, culture is continued, and non-recombinant (wild type) and recombinant viruses appear in the culture solution. Recombinant viruses can be cloned from among them by general methods such as limiting dilution method 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.

The copper-chelated carrier used in the present invention (hereinafter referred to as
The copper chelate carrier (abbreviated as copper chelate carrier) is a carrier in which an exchange group having a chelating ability such as biscarboxymethylimino group [N(CH2C00H)2] is bound to agarose, cellulose, polyacrylamide gel, etc., and a carrier such as copper sulfate is used. Examples include carriers treated with solutions of copper salts. Preferably, "chelating sepharose" (Pharm
A carrier prepared by chelating copper to an insoluble polysaccharide carrier such as Acia Co., Ltd.) is used.

The purification operation of FeIFN using a copper chelate carrier is carried out as follows.

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

Elution is 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, elution at even higher pH becomes possible.

Ionic strength can be increased by increasing the concentration of buffers such as phosphoric acid, acetic acid, citric acid, etc., or by adding neutral salts such as sodium chloride, potassium chloride (0.2-1.0M).

The composition and volume of the eluent are not particularly limited, and the optimal elution conditions are to remove contaminant proteins and FeIFN.
It is determined as appropriate depending on the amount of water, column dimensions, etc.

By adsorbing and eluting Fe1FN onto the copper chelate 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 a blue dye is bound (hereinafter abbreviated as blue carrier) is preferable, and it is particularly preferable to use the blue carrier before the copper chelate carrier. Moreover, you may use these continuously.

As the blue carrier, the following are used.

The blue pigment for general customers is called CI Reactive Blue 2, for example, "Cibacron Blue F3GA" or "Cibacron Blue 3GA" from CI BA-GE IGY.
Examples include blue dyes commercially available under the trade name . The blue carrier used in actual chromatography is “Blue Sephas CL-6B” (P
Pharmacia), “Blue-Sepharose 6 Fast Flow” (Pharmacia), “Madrex Gel Blue A” (Amieon), and “
Blue agarose gel is commercially available under trade names such as Affigel Blue (Biorad), or blue agarose gel is commercially available under trade names such as "Blue-Trisacryl-M" (LKB) and "Blue Cellulose Gel" (Chisso). Blue cellulose gel and the like are suitable and easily available.

Elution from the blue carrier 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. High ionic strength can be achieved by increasing the concentration of buffers such as phosphoric acid, acetic acid, citric acid, boric acid, Tris/HCl, or by adding neutral salts such as sodium chloride, potassium chloride, calcium chloride (08
2 to 1° OM). Also,
When the eluent contains a solvent that weakens hydrophobic interactions, such as ethylene glycol or propylene glycol, the pH is 5.
Elution at ~8 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 IFN was used.

20 μg of plasmid pFe, IFN, 1 extracted from transformed Escherichia coli E, colt (pFeIFNl) (Kaikoken Jokyo No. 1633) using the method described in Reference 4 was treated with restriction enzyme 5f.
Completely decomposed with aN1 and HincII, the obtained multiple D
The NA fragments were separated by agarose gel electrophoresis and were approximately 750 b
A DNA fragment of P was removed by electroelution, and about 2 μg was recovered. In this way, a 5faN1-HincII 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. HindI of plasmid extracted by alkaline miniscreen method from colonies growing on LB plate containing 1100tt/ml ampicillin.
Cloning vector pBM was isolated by restriction analysis with [I
A plasmid in which a DNA fragment of about 750 bp was integrated into 030 was obtained. Approximately 100 baie of D containing the initiation codon of the FeIFN-encoding DNA of the plasmid.
Perform NA sequencing and add FeIFN to pBMO30.
A plasmid containing DNA encoding the was obtained. This recombinant plasmid was named pBmFeIFNl. The method for producing pBmFeIFNl is shown in FIG.

A recombinant virus was produced by the method described in Reference 3.

i.e. 50mM HEPES buffer pH 7,
1,0,28M NaCl1,0,7mM Na2HPO
+, 2.5 ml consisting of 0.7mM NaH2PO4
Add 2.5 ml of DNA mixture (0.25 M
CaCl2, Bombyx mori nuclear polyhedrosis virus BmNPV
DNAlopg of strain 73 (Reference 3) containing 65 μg of recombinant plasmid pBmFeIFN1 DNA was added dropwise, and 0.5 ml of the resulting suspension was added to 5 ml of 10% FBS.
The DNA was introduced into silkworm cells in addition to the culture medium of about 3×10 5 BM-N cells (Reference 3), which were cultured on a flat surface in a 25 cnf 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 containing the FeIFN-encoding DNA produced here was designated as BmFeIFNl.

BmFeIFNl is European Co11ec
tion ofAnimslCulture
Deposited under the Budapest Treaty at
tion No. VB9062701).

η) Preparation of recombinant virus solution Approximately 3
50μ of the BM-N cell culture solution containing the recombinant virus cloned in (3) above to the MN cells! BM-
After adding it to N cells and culturing at 27°C for 5 days, the culture solution was
．． The mixture was centrifuged at OOOrpm for 5 minutes to obtain a centrifuged supernatant as a recombinant virus solution. 10 of each virus solution
-7 dilution, 1 ml of the diluted solution was added to the 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 5th instar and 1 day old silkworm larvae, and incubated at 25°C for 4 days. After rearing on 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, and antiviral activity was examined.

7.7 x 107 units/ml (body fluid) of Fe1FN was produced.

(6) Inactivation of recombinant silkworm nuclear polyhedrosis virus 0.
Silkworm hemolymph containing 4×108 TC×D5o per ml of recombinant silkworm nuclear polyhedrosis virus BmFelFN1 was mixed with 0. The pH was adjusted to 1.5 with IN hydrochloric acid and stored at 4°C for 1 day. Neutralize with 2N NaOH solution and add 1 ml of BM
Although it was added to the culture medium of -N cells, BM-N cells were not infected with the virus.

Recombinant virus BmF e I FNl by hydrochloric acid
was deactivated and neutralized with a NaOH solution to obtain a silkworm body fluid and a crude FeIFN solution.

(7) FeIFN using a carrier bound with blue dye
Purification of 3. of item (6) above. 8 x 106, F in U/ml
Contains eIFN activity, FeIFN specific activity is 1.2X10
6U/mg protein of crude FeIFN solution 14.5
A' was applied to a column containing 270 ml of "Blue Sepharose (Fast Flow Type)" and the column was subsequently washed with 1250 ml of 50 mM) Lis-HCl buffer (pH 8) containing 0.5 M potassium chloride, followed by 1 M potassium chloride. 50mM) Lis-HCl buffer (pH 8) containing
) 1.081 and 1M potassium chloride, 50mM) Lis-HCl buffer containing 40% ethylene glycol (pH-
8) FeIFN was eluted at 1.081. Eluted F
eIFN contains 4.2X107U/ml of Fe1FN activity, with a specific activity of 1. It was 1×108 U/■ protein. The FeIFN activity recovery rate at this time was 82.4%, and the specific activity increased 92 times.

(8) FeI using a carrier chelated with copper
Purification of FN FeIFN eluate i from the blue carrier in item (7) above,
o8i was applied directly to a column containing 150 ml of ``chelating Sepharose'' containing 0.0.
20mM acetate buffer (pH 4) containing 3M sodium chloride
, 2), then 20 m containing 0.3 M sodium chloride.
FeIFN was eluted with 750 ml of M acetate buffer (pH 3,9). The eluted FeIFN was 2.7X107U/
Contains ml of FeIFN activity, specific activity 1.4x108U
/■It was 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, Yxmamoto et al.: Vet,
Immunol, and Immun.

patholl, 11. 1-19. (198
6).

(2) A, Yanai et al.: European Published Patent No. 322870 (3) T, Horiuchi et al.: ^gric,
Riot, Chew, 51. 157.3
-1580. (1987).

(4) Edited by T., Maniatis et al.: Mo1ecul
er Cloning, A Laboratory
Manual, (1982) pH 6-96.
Co1d Spring Harbot La
botator7. New Work.

(5) G, R, Gardinet and H6
5tockdale: J, Invert
brxte PatbologT, 25. 36
3-370. (1975).

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of an example of the construction of a recombinant plasmid pBmFelFN1 containing DNA encoding feline interferon. Patent applicant Toshi Co., Ltd. Figure 1

Claims (1)

[Claims] (1) DN encoding the protein of feline interferon
After the recombinant silkworm nuclear polyhedrosis virus genetically modified by A is propagated in living silkworms to produce feline interferon, a solution containing the produced feline interferon is transferred to a carrier to which copper is chelated. 1. 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.