JPH0123447B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to subunit proteins of pertussis toxin. More specifically, the present invention relates to a protein having antigenic activity for protecting against pertussis infection, which is obtained by separating LPE-HA, a toxin isolated and purified from a culture solution of Bordetella pertussis, into its constituent components (subunits). The present invention also relates to a protein that can exhibit insulin secretagogue activity in combination with the same. Whooping cough is caused by Bordetella pertussis.
Pertussis is a contagious disease caused by infection, and pertussis vaccine is used to prevent it (usually used as a triple vaccine to prevent diphtheria and tetanus). It is used as a killed whole bacteria vaccine, and although its effectiveness is well recognized, side effects such as redness, swelling, tingling or fever at the vaccination site, diarrhea, vomiting, and in rare cases, symptoms of shock may also occur. There are many problems. Therefore, in order to solve this problem, attempts have been made to develop a purified vaccine, that is, a pertussis protective antigen fractionated vaccine (component vaccine) (see, for example, Japanese Patent Publication No. 57-5203 and Japanese Patent Application Laid-open No. 57-50925). . LPF-HA, a toxin produced by Bordetella pertussis, is a protein with a molecular weight of approximately 107,000. It has sensitizing activity (HSA), insulin secretagogue activity (IAA) that promotes insulin secretion in the body, or hemagglutinating activity (HA activity). Then, endotoxins and heat-labile toxins that are involved in side effects are removed from the B. pertussis culture solution.
The above-mentioned component vaccine is prepared by obtaining a fraction with HA activity and treating it with formalin to inactivate (toxoidize) LPA and HSA, which are also thought to be the cause of side effects. There is. The endotoxin and LPA of the component vaccine obtained in this way are reduced to 1/10 to 1/25 of that of the whole-cell vaccine, and clinical evidence shows that the side effects during vaccination are also greatly improved. It's confirmed. However, even in this component vaccine, the reduction in LPA is not necessarily sufficient, and in particular, the reduction in HSA is insufficient, and improvements have been desired. As a result of intensive research into the relationship between the molecular structure of pertussis toxin and the expression of various activities, the present inventors found that only a specific subunit has antigenic activity that protects against pertussis infection.Using this subunit protein can reduce side effects. It was discovered that a vaccine containing no pertussis can be obtained, and also that when this subunit protein is combined with another subunit protein, insulin secretagogue activity is expressed, leading to the present invention. That is, the present invention has a molecular weight of approximately 32,000 determined by the sucrose density gradient method, and an amino acid composition and composition ratio (wt%).
are Asp7.0, Thr5.6Ser11.1, Glu13.6, Gly8.4,
Ala6.0, Cys2.8, Val5.7, Met3.5, Ile4.0,
Leu7.0, Tyr5.0, Phe5.1, Lys4.0, His3.4,
Arg4.6, Pro3.2, gives a single band in polyacrylamide gel electrophoresis, does not have leukocytosis activity, histamine sensitizing activity, insulin secretagogue activity, and has pertussis infection protective antigen activity. However, it is a subunit protein of pertussis toxin, T234. In addition, for subunit proteins, polyacrylamide gel electrophoresis and SDS
Together give a single band in polyacrylamide gel electrophoresis; alone, leukocytosis activity,
It has neither histamine sensitizing activity nor insulin secretagogue activity, but subunit protein T234
There are two subunit proteins of pertussis toxin, T100 and T500, which can exhibit insulin secretagogue activity when combined with pertussis toxin. T100 is
The molecular weight as determined by SDS polyacrylamide gel electrophoresis is approximately 25,000, and the amino acid composition and composition ratio (weight%) are Asp8.5, Thr4.9, Ser14.0, Glu15.6,
Gly10.7, Ala5.7, Cys2.5, Val4.8, Met3.0,
Ile4.0, Leu5.1, Tyr4.2, Phe4.5, Lys3.3,
It is a subunit protein of His3.7, Arg5.4, and T5
00 has a molecular weight of about 10,000 as determined by SDS polyacrylamide gel electrophoresis, and the amino acid composition and composition ratio (wt%) are Asp9.0, Thr4.7, Ser13.0,
Glu16.5, Gly10.9, Ala5.2, Cys2.6, Val4.4,
Met3.0, Ile3.6, Leu6.8, Tyr3.6, Phe4.8,
It is a subunit protein of Lys4.0, His3.8, and Arg4.0. The pertussis toxin subunit protein of the present invention is a toxin isolated and purified from a culture solution of Bordetella pertussis,
Obtained from LPF-HA. From the viewpoint of ease of culturing, it is preferable to use Pertussis I phase bacteria among Bordetella pertussis. Any known culture method may be used, but the so-called stainer
Scholte medium (referred to as SS medium) (Journal of General Microbiology (J.
gen.Microbiol) volume 63, pages 211-220, 1971)
However, in recent years, it has been widely used for mass culture of Bordetella pertussis. A medium containing cyclodextrin or its derivative proposed by the present inventors, for example,
SS medium containing methyl β-cyclodextrin (Meβ.CD) is particularly preferred (see, for example, Japanese Patent Application No. 163478/1983). The culture method and conditions are not particularly limited, and conventionally known methods and conditions can be adopted, but shaking culture is preferable to static culture, the culture temperature is around 35 ° C, and the culture time is 10 to 100 °C. The time is appropriate. produced from the culture (culture medium and bacterial cells)
The method and means for collecting LPF-HA are not particularly limited, and known methods and means can be used. For example, pertussis I phase bacteria (Bordetella patassis Higashihama strain) is cultured in SS medium containing 300 μg/ml Meβ-CD at 35°C for 18 hours, and the centrifuged supernatant (PH8.6) of the resulting culture is Pass through a hydroxyapatite column equilibrated with 0.01M phosphate buffer at pH 8.0. After adjusting the resulting permeate to pH 6.0, this time it was adsorbed onto a hydroxyapatite column equilibrated with 0.01M phosphate buffer at pH 6.0, and this was adsorbed onto a hydroxyapatite column containing 0.5M sodium chloride.
Elute with 0.1M phosphate buffer (PH7.0) to obtain protein fraction. LPF-HA can be obtained by adsorbing this protein fraction on affinity chromatography using haptoglobin-Sepharose as a support and desorbing it with a 0.1M phosphate buffer containing 0.5M NaCl and 3M potassium thiocyanide. The method for separating and collecting subunit proteins T234, T100, and T500 from LPF-HA separated and purified as described above is not particularly limited, and may be carried out by appropriately combining known methods and means. Can be done. For example, purified LPF
- HA was separated into its subunits in a 0.015M phosphate buffer at pH 7.5 containing 8M urea, and the resulting solution was mixed with CM sepharose equilibrated in the same buffer.
Apply to CL-6B column. Then, the unadsorbed fractions are collected, concentrated using a dialysis membrane, and then subjected to gel filtration using Sephacryl S-200. By such gel filtration, subunit protein T
100 and T500 are separated. In the above CM Sepharose CL-6B column, the adsorbed proteins were washed in a pH 7.5 solution containing 4M urea and 0.5M NaCl.
Effluent is performed using 0.1M phosphate buffer to collect a fraction containing protein, which is concentrated and gel-filtered in the same manner as above. Through such processing,
A small amount of undissociated LPF-HA and subunit protein T2
34 are separated. The subunit protein T234 of the present invention has a molecular weight of about 32,000 (32,000±
3000). Further, the amino acid composition and composition ratio (wt%) are as shown in Table 1. And polyacrylamide gel (polyacrylamide concentration
Gives a single band in 7.5%, 1N KOH-glacial acetic acid buffer (PH4.3) electrophoresis (disk electrophoresis). Details of the methods for measuring various biological activities will be described later, but the outline thereof is as follows. Specifically, subunit proteins were intravenously injected into mice, LPA was administered by measuring white blood cell counts 3 days later, IAA was administered by glucose loading and blood insulin levels were measured 4 days later, and histamine was administered 6 days later. Inject into the peritoneal cavity and check HSA by measuring body temperature 30 minutes later.
The subunit protein T234 of the present invention substantially does not have any of the above three types of activities. Pertussis infection protective antigen activity is measured as follows. That is, first, a rabbit is immunized with an antigen (toxoidized LPF-HA or subunit protein) in incomplete Freund's adjuvant, and an antibody (IgG fraction) is obtained from the immune serum by ammonium sulfate fractionation or the like. Next, with the antibody
Incubate LPF-HA at 37℃ for 1 hour,
In addition to examining the neutralizing ability of various antibodies by intravenously injecting them into mice, we will examine the antigenic activity that protects against pertussis infection through infection experiments by intraperitoneally administering the above antibodies to mice and intracerebral inoculation with pertussis I phase bacteria. . The subunit protein T234 of the present invention has the same antigenic activity for protecting against pertussis infection as LPH-HA. As described above, the subunit protein T23 of the present invention
4 is the cause of side effects of pertussis vaccine
Since it does not contain LPA or HSA and has sufficient antigenic activity to protect against infection, it can be used to produce a whooping cough vaccine with almost no side effects. Subunit proteins T100 and T500 of the present invention
The molecular weight measured by SDS polyacrylamide gel electrophoresis is approximately 25000 (25000±2000).
and about 10000 (10000±1000). The amino acid composition and composition ratio (wt%) are as shown in Table 1 (hydrolysis was performed with 6N hydrochloric acid at 110°C for 24 hours and analyzed using a Hitachi-835 high-speed amino acid analyzer). And both polyacrylamide gel electric

【table】

[Table] Electrophoresis and SDS polyacrylamide gel electrophoresis (polyacrylamide gel concentration 10%, SDS 0.1%,
A single band can be obtained in both cases (0.1M phosphate buffer (PH7.2)). In addition, none of them, when used alone, has substantially no protective antigen activity against LPA, HSA, IAA, or pertussis infection. However, both have the characteristic that they express approximately the same level of IAA when combined (coexisting) with the subunit protein T234. The subunit protein T100 or T500 of the present invention does not substantially have LPA or HSA, which causes side effects, and when combined with T234, it comes to have IAA.
It may be possible to use it in combination with 4 as a therapeutic drug for diabetes. The various measurement methods adopted in the present invention are as follows. (1) Sucrose density gradient method A sample solution is placed on a sucrose solution with a density gradient from 10% to 20%, and ultracentrifuged at 65,000 revolutions per minute at 4°C for 20 hours. bovine serum albumin, chymotrypsinogen)
Molecular weight was measured based on. (2) Polyacrylamide gel electrophoresis Using polyacrylamide concentration 7.5% and 1N KOH-glacial acetic acid buffer (PH4.3), it was performed according to the method of Davis (BJ Davis, Amr. NYAcad. Sci.,
(See No. 121, P404, 1964). (3) SDS polyacrylamide gel electrophoresis Polyacrylamide concentration 10%, SDS 0.1%,
Fairbanks using 0.1M phosphate buffer (PH7.2)
(GTFairbanks)
etal, Biochem., Vol. 10, p2606, 1971). (4) Biological activity (i) Leukocytosis activity The sample was injected intravenously into mice (ddY, SPF, 4W, ♀) through the tail vein, and 3 days later, 5 μl of blood was collected from the tail vein and diluted 2000 times with a commercially available diluent. The number of white blood cells was measured using a cell counter (manufactured by Toa Denpa KK). Leukocytosis activity is calculated by calculating the difference (A) between the number of white blood cells in the sample group and the number of white blood cells in the control group.
- It was expressed as a relative titer to the difference (B) between the number of leukocytes when using HA and the number of leukocytes in the control group. (ii) Insulin secretion promoting activity After measuring the leukocytosis activity, that is, fasting on the 3rd day, intraperitoneal administration of 50% glucose on the 4th day, and 15 minutes later, 20 μl of blood was collected from the tail vein and diluted 10 times. IRI (Lmmuno) was obtained using a radioimmunoassay kit manufactured by Dynatec.
Reactive Insulin) was measured. The relative potency was then determined using the same method as in (i). (iii) Histamine sensitizing activity After measuring the insulin secretagogue activity, that is, on the 6th day, 0.8 mg/mouse of histamine hydrochloride was intraperitoneally administered to the mouse, and the decrease in rectal body temperature was measured 30 minutes later. The relative potency was then determined using the same method as in (i). (5) Binding ability of anti-subunit antibody to LPF-HA Add anti-subunit antibody to a microplate coated with LPF-HA, T100, T234, and T500, react for 30 minutes at room temperature, and evaluate the binding ability of the antibody using MICRO ELISA. It was measured by the method.
The binding ability of each antibody was determined by the binding ability of anti-LPF-HA to LPF-HA.
It is expressed as a relative value when the binding ability of the HA antibody is set as 100. As for the antibody, rabbits were immunized with the antigen (LPF-HA was toxoidized and the subunit protein was intact) in incomplete Freund's adjuvant.
An IgG fraction obtained from the serum by ammonium sulfate fractionation was used. (6) Neutralizing ability of anti-subunit antibodies against LPF-HA LPF-HA and each antibody at a constant concentration were mixed, incubated at 37°C for 1 hour, and then intravenously injected into mice. I saw Kazuno. The value of neutralization ability is the relative value of the antibody dilution required to neutralize each activity by 50% using the arbitrarily determined standard LPF-HA. I asked for it as. Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 (1) Separation and purification of LPF-HA Bordetella pertussis
(Bacillus pertussis) 1% freeze-dried bacterial cells of Higashihama strain I phase bacteria
20% defibrinated horse blood suspended in casamino acid solution
The cells were cultured in Bordet Gengou medium (hereinafter referred to as BG medium) at 35°C for 3 days. Scrape off one platinum loop of this bacteria and add it to BG medium.
Suspend the bacteria refreshed for 24 hours in SS medium,
An inoculum suspension was obtained. This inoculum suspension was
Meβ-CD was suspended in SS medium containing 500 μg/ml to a concentration of 10 ⁷ colonies/ml, and was incubated at 35°C under shaking conditions.
Culture was performed for 18 hours. After culturing, LPF- produced by the following method
HA was collected. Centrifuged culture supernatant (PH8.6),
Pass through a hydroxyapatite column equilibrated with 0.01M phosphate buffer at pH 8.0 (other proteins F-HA with red blood cell agglutination ability are removed by adsorption to the column), and the flow-through containing LPF-HA is filtered to pH 6. After adjusting to
LPF-HA was adsorbed onto the column and eluted with 0.1M phosphate buffer (PH7.0) containing 0.5M sodium chloride to obtain a protein fraction containing LPF-HA. This protein fraction was passed through an affinity chromatography column with haptoglobin-Sepharose 4B as a support, LPF-HA was selectively adsorbed onto haptoglobin, and this was transferred to a 0.1M phosphate buffer containing 0.5M NaCl and 3M potassium thiocyanide. A fraction containing LPF-HA was obtained by desorption using a liquid (PH7.0). The obtained fraction was dialyzed against 0.1M phosphate buffer to remove potassium thiocyanide to obtain pure LPF-HA. (2) Separation and purification of subunit proteins of LPF-HA A solution of the purified LPF-HA obtained in (1) above in 0.1M phosphate buffer (PH7) containing 0.5M salt was added to a solution of 0.1M phosphate buffer (PH7) containing 4M urea. The resulting solution (14.8 ml, protein concentration 0.2 mg/ml) was dialyzed against 0.015 M phosphate buffer.
5.7 g of urea was added to make an 8M urea solution, and the solution was left at room temperature for 30 minutes. Add an equivalent amount of 0.015M phosphate buffer with pH 7.5 to make a 4M urea solution, then add 4M urea to the pH containing 4M urea.
It was applied to a column of CM Sepharose CL-6B equilibrated with 0.015M phosphate buffer at 7.5. The outflow pattern was as shown in FIG. Fractions 4 to 30 containing protein were collected, concentrated using a dialysis membrane, and subjected to gel filtration using Sephacryl S-200. The outflow pattern was as shown in FIG. By such gel filtration, subunit protein T100 (fraction 65-7
2) and T500 (fractions 75-78) were separated. In the above CM Sepharose CL-6B column, for fractions 40 onwards, a 0.1M phosphate buffer with a pH of 7.5 containing 4M urea and 0.5M salt was used, and fractions 51 to 55 containing protein were collected, and the same procedure as above was carried out. Then, concentration and gel filtration were performed (Figure 3). Through such treatment, a small amount of undissociated LPF-
HA and subunit protein T234 (fractions 62-72) were separated. (3) Subunit molecular weight and subunit protein T10 obtained by electrophoresis (2)
0, T500, T234 and LPF-HA 1%
The mixture was treated with sodium dodecyl sulfate and 1% 2-mercaptoethanol at 100°C for 2 minutes, and electrophoresed on a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate, yielding the results shown in FIG. Using standard substances such as bovine serum albumin, ovalbumin, and chymotrypsinogen, the molecular weights of subunit proteins T100 and T500 were calculated from FIG. 4 to be 25,000±2,000 and 10,000±1,000, respectively. on the other hand,
T234 was separated into three bands in SDS polyacrylamide electrophoresis, but showed a single band in 7.5% polyacrylamide electrophoresis at PH4.3 (Figure 5). The molecular weight of this stuff is 10% to 20
It was calculated to be 32000±3000 by the sucrose density gradient method up to %. (4) Properties of biological life, etc. Subunit proteins T100, T234 and T
500 LPA, HSA and IAA are as shown in Table 2.

[Table] From Table 2, T100, T234, T500
It can be seen that none of them have LPA, HSA, and IAA. On the other hand, it can be seen that when T100 and T234 are combined, or when T500 and T234 are combined, IAA is expressed. The binding ability of anti-subunit antibodies and the like to LPF-HA is shown in Table 3.

[Table] From Table 3, anti-T234 antibody is anti-LPF-
It can be seen that it has a binding ability to LPF-HA that is equivalent to about 60% of that of the HA antibody. This result indicates that T234 is LPF-HA in vitro.
This indicates that it has the same activity as an antigen. The neutralizing ability of anti-subunit antibodies and the like against LPF-HA is shown in Table 4.

[Table] It can be seen that anti-T234 has a considerable ability to neutralize LPF-HA in terms of LPA, HSA, and IAA activities. Infection protection experiment: Using 10 to 15 mice per group (4W, SPF, female),
Anti-LPF-HA antibody or anti-T234 antibody intraperitoneally
Passive immunization was carried out in 0.5 ml doses, and 30 minutes later, 5 x ¹⁰⁴ pertussis phase I bacteria were inoculated into the brain, and the survival rate after 14 days was determined. Survival rate when using anti-LPF-HA antibody is 50
%, and the survival rate when using anti-T234 antibody is 60%.
%, and it was confirmed that the anti-T234 antibody has almost the same ability to protect against infection as the anti-LPF-HA antibody. The survival rate of the control group (group without antibody) was 0%.

[Brief explanation of drawings]

Figures 1, 2, and 3 are graphs related to (2) of Example 1, and Figure 1 is a graph of CM Cef Arrows CL.
-6B column chromatography, Figure 2,
Figure 3 is Sephacryl S-200 gel perchromatography. Protein concentration was determined by the Lowry method. Figure 4 is an SDS polyacrylamide electropherogram of LPF-HA and each subunit protein, and Figure 5 is an SDS polyacrylamide electropherogram of LPF-HA and T-234.
It is a polyacrylamide electropherogram of PH4.3.

Claims (1)

[Claims] 1 The molecular weight as determined by the sucrose density gradient method is approximately 32,000,
Amino acid composition and composition ratio (weight%) are Asp7.0,
Thr5.6, Ser11.1, Glu13.6, Gly8.4, Ala6.0,
Cys2.8, Val5.7, Met3.5, Ile4.0, Leu7.0,
Tyr5.0, Phe5.1, Lys4.0, His3.4, Arg4.6,
Pro3.2, giving a single band in polyacrylamide gel electrophoresis, leukocytosis activity,
T234, a subunit protein of pertussis toxin, which has neither histamine sensitizing activity nor insulin secretagogue activity, but has pertussis infection protective antigen activity.