JPH03190900A - Cytokine-inducible neutrotaxis-insective protein - Google Patents

Abstract

NEW MATERIAL:A cytokine-inducible neutrotaxis-insective protein having following properties; Capable of induction of neutrotaxis at 10<-9> M concentration, showing single peak at 15500 molecular weight by the gel filtration method using the high-performance liquid chromatography technique, showing single band at 6300-6500 molecular weight by SDS polyacrylimide electrophoresis and having >=9000000 unit/mg protein specific activity. USE:A medicine for prevention and remedy of infectious diseases. PREPARATION:For example, a rat kidney-derived cell line NRK-52E (ATCC CRL-1571) is cultured in a culture medium containing fetal bovine serum and a supernatant of the cultured material is subsequently collected. To the collected supernatant, ammonium sulfate is added to 80% saturation and the generated precipitate is separated by centrifugation. The separated precipitate is dissolved in distilled water and the resultant solution is fractionated by the gel filtration method and the ion-exchange column chromatography method and then concentrated. The concentrated solution is subjected to SDS polyacrylamide electrophoresis, thus obtaining the objective cytokine-inducible neutrotaxis-insective protein.

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to cytokine-induced neutrophil attracting protein (hereinafter referred to as rCINCJ).

[Conventional technology]

In local infections, neutrophils accumulated at the site of inflammation exhibit a bactericidal effect against foreign bacteria. The in-vivo substance that induces this accumulation of neutrophils, that is, fugetaxis and chemotaxis of neutrophils, is complement fragment C5a (J, Biol, Chem, 253.6955
-6964. (1978)) and interleukin B4
(Prostaglandins, 20, 411-4
18. (1980)), human monocyte-derived neutrophil chemoattractant (
ProcNatl, Acad, Sci, USA 84
9233-9237. (1987)) are known.

[Problems to be solved by the invention]

The present inventors discovered CINC, a novel protein that induces chemotaxis of neutrophils, from the extracellular fluid of the rat kidney cell line NHK-52H induced by various cytokinetics, and completed the present invention. Ta.

[Means to solve the problem]

C,INC of the present invention has the following properties: (1)1
At a concentration of 0-'M, chemotaxis of neutrophils is induced (2) A single peak with a molecular weight of 15.500 is shown by gel filtration method using high performance liquid chromatography. (3) By SDS polyacrylamide gel electrophoresis,
(4) Showing a single band with a molecular weight of 6,300-6.500. Showing a specific activity of more than 9,000,000 units/mg protein. Moreover, CINC of the present invention has the following amino acid sequence: (N)-APVANEL RCQCLQ
TVAGIHF KNIQSLKVMP PGPHCTQTEV I AT L K N G RE ACLDPEAP
MVQ K I VQKMLKGV PK-(C) ((N) indicates the N-terminus, (C) indicates the C-terminus). Note that the description of amino acids in this specification is as follows. A: Alanine R: Arginine N: Asparagxne D: Aspartic acid B: Asparagine or Aspartic
acidC: Cysteine Q: Glutamne E: Glutamic acidZ: G
]utamj, ne or Glutamic acid
G: Glycine H: Histidine I: l5oleuc1ne L: 1euctne K: Lysine M: Methionine F: Phenylalantne P:
Proline S: 5erine T: Threonine W: Tryptophan Y: Tyrosine V: Valine The method for producing CING of the present invention is described below. CING is produced from the rat kidney-derived cell line NHK-52E (ATCC CRL-i571). The cytological properties of this cell line were determined by DeLar
reported by co et al. (J, Ce1l Ph
ysjol, 94°335-342. (1978))
. NHK-52H can be cultured in a medium for culturing normal animal cells. That is, the medium is Dulbecco's modified Eagle's medium (DM
EM), RPMI-1640 medium, etc. can be used,
DMEM is preferably used. These media containing 2 to 5% fetal calf serum (hereinafter referred to as "serum-containing media") are used for normal culture. Cultivation is performed using a CO2 incubator at a CO2 concentration of approximately 5%. The culture concentration is approximately 37
℃ is preferable. The production of CING from NHK-52E is due to interleukin-1β, tumor necrosis factor (Tumor N
This can be carried out by culturing NHK-52E in a serum-containing medium containing cytokines such as Ecrosis Factor)-α, riboborizacharide, and bovine serum albumin. The concentration of cytopotins may be 1-100 βg/ml, but is preferably Oug/ml. Cultivation time may be 1 to 5 days. The neutrophil-inducing activity of CINC was determined using the method of Watanabe et al.
pan, J., Pharmacol. And, 102. (1985)) by measuring the chemotaxis of neutrophils. CING can be obtained by using the extra-culture fluid containing CING thus obtained as a material and using a method commonly used for protein separation and purification. For example, CING can be obtained by appropriately combining a salting-out method, a centrifugation method, and various chromatography methods. As various chromatographies, hydrophobic chromatography, ion exchange chromatography, gel filtration method, affinity chromatography, etc. are used. The amino acid sequence of purified CINC can be determined by automated Edman degradation using a gas phase protein sequencer. The CINC of the present invention can be used for various purposes. For example, it can be used as a control substance for CINC present in samples obtained from humans, such as serum or plasma. That is, it can be used as a labeled antigen when quantifying CINC of the present invention by competitive immunoassay. Furthermore, by administering the CINC of the present invention to patients with infectious diseases, preventive and therapeutic effects can be expected.
In this case, the present invention
of CINC and a suitable diluent and a pharmacologically acceptable carrier. The effective amount is
The dose is 1 to 1.000 βg/Kg body weight/day, and is administered once to several times a day. The present invention will be explained in more detail below with reference to Examples. Chemotaxis of neutrophils in CINC was measured by Watanabe et al.'s method using a multi-well type Boyden chamber. The upper and lower chambers were separated by a polycarbonate filter (thickness) with 2 μm diameter holes (OALm). 0.3m in the upper chamber
] rat neutrophil suspension (107 cells/m 1 )
, 0.4 ml of test sample solution was added to the lower chamber, respectively. After incubating the Boyden chamber at 37°C for 60 minutes in a CO2 incubator, the number of neutrophils that migrated to the lower chamber was counted using a Coulter counter. The neutrophil-inducing activity is expressed by the fugue rate calculated by the following formula. (Number of cells that migrated to the lower chamber)/(Number of cells initially delivered to the upper chamber) x 1.00 (%) Activity to induce 1% (3x1.O') cells to the lower chamber after 60 minutes of incubation was taken as 1 unit. 15 above 2. NHK-52E cells were prepared from CING by NHK-52E cells, and D-52E cells were incubated with D-52E cells containing 5% fetal calf serum.
It was passaged in MEM. These cells were cultured for 2 days in DMEM containing 0.1% bovine serum albumin and 0.1βM interleukin-1β (manufactured by Tenba Pharmaceutical Co., Ltd.). After culturing, the culture supernatant was collected and used as a material for purification of CING.The neutrophil-inducing activity of CINC was measured by the method described in Example 1. CING was purified from the culture supernatant described in Example 2. All of the following operations were performed at 4°C.Ammonium sulfate was added to the culture supernatant of 231,
It was saturated. Centrifuge to collect the precipitate, and centrifuge this at 600 m.
1 in distilled water cooled to 4°C. Next, add this to 0.05M containing 0.05M sodium chloride.
Dialysis was performed with M Tris-HCl buffer (pH 7.2, hereinafter referred to as "preparation buffer"). This fraction was added to DEAE-3ep.
hadexA-50 column (2,5x], 44cm:
(manufactured by Pharmacia) and the passed-through fraction was equilibrated with a preparation buffer.
It was directly applied to an xA-50 column (2.5 x 14 cm, manufactured by Pharmacia). The adsorbed fraction is 0.05-1.0M
It was eluted with a linear concentration gradient of sodium chloride. Both fractions showing neutrophil-inducing activity were collected and concentrated to 2 ml using an Amicon diaflow membrane (YM5 manufactured by Amicon). This concentrated solution was applied to a 5ephadex G-50 (fine) column (1.5 x 85 cm, manufactured by Pharmacia) equilibrated with PBS (O, 01M phosphate buffer containing 15M sodium chloride (pH 7,4)). Gel filtered. Both fractions showing neutrophil-inducing activity were collected, dialyzed against distilled water, and then lyophilized. The dried samples were dissolved in 50111 distilled water. This sample C-18 reverse layer UnisilNQ column (
0.46 x 15 cm: manufactured by Gas Kuro Kogyo Co., Ltd.). 0.01% trifluoroacetic acid (TFA) as solution A.
) with 60% acetonitrile containing 0.01% TFA as solution B, followed by a gradient of 0 to 40% acetonitrile in the first 10 minutes (1 to 40 to 60% in 50 minutes). The adsorbed protein was eluted using a gradient. The eluted fraction was monitored at 228 nm. The flow rate was 1 ml/min, and 1. ml each was collected. Both fractions showing neutrophil-inducing activity were collected and lyophilized. The final purified CING had a specific activity of 9.130,000 units/mg protein, which was calculated to be 30,300 times more purified.The overall recovery rate was 10. 6%.Also, this C
ING induced chemotaxis of neutrophils at a concentration of 10-9M. Furthermore, this purified CINC was dissolved in a preparation buffer and subjected to gel filtration using a TSKgel G200O8W column (manufactured by Tosoh Corporation). Measurements of neutrophil attracting activity showed a single peak at min%i 15,500. 1 & Isamu A, SDS polyacrylamide electrophoresis 0.1%
Electrophoresis was performed on the sample obtained in Example 3 using a separation gel (17.5% acrylamide) containing SDS. 2 Tris-HCl (pH 8,8) buffer containing 0.1% SDS was used as the buffer solution in the reservoir, and 0.0% as the buffer solution in the reservoir.
．． A 0.025M Tris-0,2M glycine (p+-16,8) buffer containing 1% SDS was used. 1.5 m
After 1 hour of electrophoresis in A, the proteins were silver stained (Bio-Rad). A single band was confirmed at the position of 63 kilodal]. In combination with the results of Example 3, it was concluded that CINC is a dimer of the same subunit. Amino acid composition of CING Amino acid composition of CINC can be determined by pretreatment with phenyl isodiocyanate (AnalBiochcm,
136.65-74° (1,984)). The number of amino acids per molecule is shown below. R: 2.4 H: 4.3 'T', :'1.9 S: l l Z・8 ・1 P: 6.9 G: 4. ] A: 5.5 V: 6.2 M: 2 2 I: 3.5 L: 5. ]O F: ], 2 K: 6 9 H co1.8 Note that cysdine and tryptophan were not quantified. Determination of the amino acid sequence of CIN
C was fragmented by bromocyanic decomposition, and each peptide fragment was fractionated by reverse phase high performance liquid chromatography. Add each Vepgood PI to 47OA gas-phase protein supplement (
Arise Biosystems Co., Ltd.) for automated engineering 1~
I did a man analysis. 1 of the unobtained PTH amino acids
It was identified and sequenced using 20 AP Ti (Analyzer (Arise Biosystems)). The determined amino acid sequence is shown below. (N)-APVANELRCQCL
Q T V A G I N FK N I Q S
L K V M PPGPHCTQTEV I A T L K N G RE ACL D P
E A P M V QK I V Q K M L
K G VPK-(C) ((N) indicates the N-terminus, (C) indicates the C-terminus).

Claims (1)

[Claims] 1. Cytokine-induced neutrophil-attracting protein having the following properties: (1) Induces neutrophil chemotaxis at a concentration of 10^-^9M (2) High-speed liquid Chromatographic gel filtration shows a single peak with a molecular weight of 15,500. (3) By SDS polyacrylamide gel electrophoresis,
(4) Showing a single band with a molecular weight of 6,300-6,500. Showing a specific activity of 9,000,000 units/mg protein or more. 2. The cytokine-induced neutrophil-inducing protein according to claim 1, which has the following amino acid sequence: (N)-APVAN
ELRCQ CLQTVAGIHF KNIQSLKVMP PGPHCTQTEV IATLKNGREA CLDPEAPMVQ KIVQKMLKGV PK-(C) ((N) indicates the N-terminus, (C) indicates the C-terminus).