JPH0413632A - Nontoxic pseudomonas exotoxin a - Google Patents

Abstract

PURPOSE: To obtain nontoxic pseudomonas exotoxin A that is composed of polypeptide chains of pseudomonas exotoxin A (PE) having a carboxyl terminal from which a specific number of amino acid moieties are lost. CONSTITUTION: This nontoxic PE is obtained by partially digesting a plasmid (e.g. plasmid pJH4) which contains a complete code sequence of mature PE by using Hha I to isolate linear DNA, completely digesting the isolated DNA by Eco R1, connecting the obtained DNA fragments with a linker (e.g. synthesized oligonucleotide of two chains) to construct a plasmid that contains a PE gene from which a structural gene corresponding 36 or more amino acids at the carboxyl terminal are deleted, and expressing thus constructed plasmid in an expression host, thus obtaining a nontoxic PE which has completely lost ADP-ribosylation activity and cell injury and lacks in 36 or more amino acids at the carboxyl terminal.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to non-toxic Pseudomonas exotoxin A (PE), its production and its use for the production of PE vaccines.

Pseudomonas aeru 1nosa
is a Gram-negative bacillus. These are widely distributed in nature and account for about 10% of normal intestinal bacteria. Such bacteria rarely infect healthy humans, but they do infect immunocompromised humans, such as patients suffering from burns, cystic fibrosis, malignant tumors, or patients using immunosuppressive drugs. After infection, Pseudomonas aeruginosa is infected with Pseudomonas exotoxin A (PE
) is produced. PE toxin is active against liver cells and destroys liver function, with potentially fatal consequences. Pseudomonas aeruginosa is also a pathogen that often causes nosocomial infections.

Moreover, it is highly resistant to many antibiotics and post-infection treatment is generally difficult, and the best solution is vaccination.

PE is a single polypeptide chain containing 613 amino acids. The three-dimensional molecular structure of PE consists of the following three structural domains: domain I, consisting of domain Ia (J!!c groups 1-252) and domain Ib (residues 365-404), and domain ■ (residues 253-404). 364) and domain■
(residues 405-613).

The Ia n Ib m amino acid domain Im is a domain that allows the toxin to recognize receptors on the membrane of toxin-sensitive cells. Domain ■ is PE that moves the toxin from the endocyte to the cytoplasm.
This is the dislocation domain of The adjacent 20 amino acids (residues 385 to 404) of domain ■ and domain lb are P
This is the P-ribosylation enzyme domain of E. ((+
ray, G, L,, Sm1th, D, H,,
Baldridge, J. S., Harkins
, R.N., ,Vasil, M.L.

Chen, E., Y., & Heyneker, H.
, L. (1984) Proc.

Natl, ^cad, Sci, US 8 81.26
45-2649; Hwang, J.

FitzGerald, D.J., ^dhya, S.
,, & Pa5jan, 1 (1987) Ce1
l, 48. 129-138;Douglis,
C, H, & Collier, R, J. (1987
) J, 13acteriol, 169.
4967-4971; Chen, S. T., Jor
dan, E. M., Milson.

R, B, Draper, R, Y, &
C1oves, R.C. (1987) J.Cen.
Microbiol, 133.3081-3091
).

The mechanism of PE intoxication process is that PE binds to specific receptors on the surface of toxin-sensitive cells.
The PE-receptor complex is then taken up into cells by receptor-mediated endocytosis. Finally, PE moves into the cytoplasm, where PE is attached to HAD'^D
Catalyzes the binding of the P-ribose moiety to the elongation factor EF-2. As a result, EF-2 becomes inactivated and no longer synthesizes proteins, leading to the death of infected cells (Fitz
Gerald, D., Worries, R.E.

& Saelinger, C.B. (198G)
Ce1l 21. 867-8F3; Fitzl
;erild, D,, 1orries, R,E,
& Saelinger.

C.B. (1982) Infect, Im
munol, 35.715-720; Worries
, R.E., ,Nanhart, M.D., &S.
aelinger, C,B.

(1983) Infect', Immunol
, 40.806-811; EidelsL1, Pro
ia, R, L, & Hart, D, ^, (
1983) Microbiol.

Rev, 47. 596-620; Middleb
rook, J. L., & Dorland, R.
, B. (1984) Microbiol, R.
ev, 48. 199-221).

The equilibrium of P-ribosylation of EF-2 by PE is represented by the following equation.

PE EF-2+ HAD” (present in large amounts in cells)’; ＾D
P-ribose EF-2 + Nicotinamide + H Traditional vaccine production methods detoxify the pathogen with heat or chemicals, or purify the pathogen's surface antigen. There are several drawbacks to the gargar method, such as the presence of residual toxins. The antigenicity is not strong enough, and there is a risk of infection for people involved in vaccine production.

S, Nirburg, R,L, Tolman
and T. Callahan in 1983 proposed the use of PE) xoids prepared by photoaffinity inactivation as vaccines. The principle is
The purpose of υV irradiation is to form a covalent bond between NAD+ and PE, resulting in the loss of PE's DP-ribosylation enzyme activity (S, Marburg, et al.
R, L, -Tola+an & L, T, Ca
! 1ahaall (1983) Proc, Natl.
, ^cJLd, Sci, ■S^Vo1.80゜287
However, a drawback of this method is that residual toxins are present since such a reaction cannot guarantee photoaffinity deactivation of all PE molecules. Therefore,
PE) xoids prepared in such a manner are not suitable.

The currently best method is to link the structural gene of the surface antigen of the pathogen into a vector by genetic engineering, express it by transforming E. coli, yeast or mammals through the vector, and then purify it. Now, with this technique, non-toxic PE fragments will be prepared and used in the production of new vaccines against PE.

Light 1 and U The present invention relates to non-toxic PE. In such modified PE, 36 or more amino acids at the carboxyl terminus are deleted, and as a result, P-ribosylation activity and cytotoxicity are completely lost.

Such non-toxic PE is isolated by expressing in an expression host a plasmid containing a PE gene in which a series of structural genes corresponding to the carboxyl terminus of PE have been deleted.

PE that has lost its cytotoxicity is a native type (intact)
Since it can block the cytotoxicity of PE, it can be used as a vaccine for diseases caused by PE.

Therefore, an object of the present invention is to provide a non-toxic PE lacking 36 or more amino acids at the carboxyl terminal.

Another object of the present invention is to provide a plasmid containing a PE gene in which a series of structural genes corresponding to the carboxyl terminus of PE have been deleted.

Another object of the present invention is to provide a method for manufacturing PE.

Yet another object of the present invention is to provide non-toxic PE for producing new vaccines against PH.

The above and other objects, features, and advantages of the present invention will become apparent from the following description of preferred embodiments of the present invention based on the accompanying drawings.It is understood that the scope of the present invention is not limited to the following description. It is to be understood that the scope of the invention is limited by the claims.

The invention will be explained in more detail below on the basis of the accompanying drawings.

FIG. 1A is a schematic diagram of the PE structural gene. FIG. 1B is a schematic diagram showing the construction of plasmids used to express PEs with various deletions at the carboxyl terminus. p
JII4 was partially cut with HhaI. 5.9k
The linear DNA of b was completely cut with EcoRl. 4.4
DNA fragments ranging from ~5.9 kb were isolated and ligated with synthetic oligonucleotide linkers. The linker
contained stop codons in three open reading frames.

Figure 2 shows a clone of deleted PE expressed by a modified plasmid.

Figure 3 shows the DP-ribosylation enzyme activity of PE obtained by expression of a modified plasmid in the host BL21(DE3). It was used as a positive control and had a DP ribosylation activity of 100%. BL21(
DE3)/- was used as a negative control. BL21(
DE3)/pJJ1-BL21(DE3)/pJJ14
The relative hep-ribosylation activity of all other PE fragments obtained from . BL21(DE3)/pJ
The specific activity of hep-ribosylation of full length PE obtained from H4 was compared.

Details J141''1 The non-toxic PE of the present invention is produced by translating 36 or more amino M residues at the carboxyl terminus of the PE polypeptide into recombinant DNA.
This was caused by technology. Although non-toxic PE retains toxin binding activity, it has lost its P-ribosylation enzyme activity and therefore its cytotoxicity.

The non-toxic PE of the present invention can be obtained by transforming a host with a plasmid containing a PE gene in which a structural gene corresponding to 36 or more amino acids at the carboxyl terminal has been deleted, and expressing the transformed plasmid in the host. can be manufactured. Construction of the plasmid consists of partially digesting the plasmid containing the complete coding sequence of mature PE with Hha I to isolate the linear DNA; completely digesting the linear DNA with EcoRI; and separating the DNA fragments. It consists of combining with a linker.

The domains of PH he○P-ribosylation enzyme activity were identified as domain ■ and a portion of domain Ib. In order to determine in more detail the carboxyl terminal portion of the toxin required for the P-ribosylation activity, a plasmid constructed according to the method of the present invention was transferred to E. coli and expressed. The size of the deletion in the plasmid was determined by restriction analysis and size mapping.

The transformant obtained by expressing the deletion plasmid in a host was appropriately cultured, and the resulting PE clone was assayed for hep-ribosylation activity (Coll.
ier, R.J., & Kandel, J. (1
971) JBiol, Chell, 246.1
496-1503). ^rg-609 to Lys613
The carboxyl-terminal amino acid residues up to ^rg-
Clones substituted with ^sn are ADP-
It retained ribosylation activity. ^Ia-596 to L
The terminal amino'tIi residues up to ys-613 are deleted and V
In clones substituted with aille-^sn, ^DP-
Ribosylation activity was reduced by 75%. In addition, clones in which 36 or more amino acid residues were deleted from the carboxyl terminus completely lost the hep-ribosylation activity.

The cytotoxicity of these modified PEs and their ability to inhibit PE cytotoxicity were also tested. According to these results, modified PE that completely lost DP-ribosylation activity
There was also a corresponding loss of cytotoxicity. Furthermore, extracts containing PE fragments without He○P-ribosylation activity had the ability to inhibit the cytotoxic activity of native PE. It is the best choice to use PE in which 36 or more amino acids are deleted from the carboxyl terminus as a new vaccine for diseases caused by PE. Furthermore, PE with 36 amino acids deleted from the carboxyl terminus
The plasmid containing the gene is expressed in E. coli to produce large amounts of modified PE. Regarding safety, the vaccine produced by this genetic engineering method is absolutely safe;
Absolutely effective in terms of antigenicity. Furthermore, the manufacturing cost is low and the manufacturing process is simple.

Examples are described below to help understand the invention.

The scope of the invention is not limited to the following description.

Plasmid pJH4, which contains the complete coding sequence of mature PE with an additional methionine group at the amino terminus, and a plasmid containing the PE structural gene (pJJl-pJJl4) resulting in a series of deletions at the carboxyl terminus, were prepared using the procedure shown in Figure 1. Built with.

Using the sequence data reported by Cray et al.
ray, G, L,, Sm1th, D, H,,
Baldridge, J.S.

Harkins, R.N., Vasil, M.
L., Chen, E., Y., & Heyneke
r, H, L, (1984) Proc, Natl.
,^ead, Sci.

USA 81.2645-2649), pJH4 was first partially digested with Hhal. 5.9kb linear DNA
was isolated and then completely digested with EcoRI. 4.4～
A DNA fragment ranging from 5.9 kb was isolated. H
The linear fragments were ligated using double-stranded synthetic oligonucleotides with cohesive ends for haJ and EcoRl. The double-stranded synthetic oligonucleotide used to join the two non-complementary ends consisted of a universal sequence for protein synthesis termination. It contained stop codons in all three open reading frames.

Plasmids pJJ1 to pJJ14 were each transferred side by side into a host (E. coli), and the size of the deletion in each plasmid was determined by restriction analysis and recombinant DMA methods of size mapping. The results are shown in FIG.

Italy 2: None PE Davis et al. (L, G, Davis, M, D,
Dibner & J, F.

Battey (1986) Ba5ic Method
s in Molecular Bi.

1ogy, pp, 90-92. Elsevier
5Ccientific Publishing Co.
,, New York) by the method described in ]acUV
Plasmids pJJ1~ to the host BL21 (DE3) that had been lysogenized in advance with a phage (DE3) carrying the T7 RN^ polymerase gene under the control of the 5 promoter.
pJJ14 was transfected on each side. The obtained transformant was immediately transferred to LB supplemented with 50 μg/xl ampicillin.
Cultured in dibroth at 37°C. When the absorbance of 650nn+ reaches 0.3, isopropyl-1-thio B-D
- galactopyranoside (IPT(:) final concentration 0.5
+M to induce synthesis of T7 polymerase localized to BL21 (DE3) lysogenic phages. Cells were harvested after 90 minutes and PE clones expressed in BL21 (DE3) and containing a series of deletions at the carboxyl terminus were obtained.

^To assay DP-ribosylation activity, Co!
1ier &Kandel's General Law (J, Biol
, Cheta, 246°1496~1503.19
71) was used. Wheat germ extract rich in EF-2 is
It was used as an F-2 source. The assay (in a total volume of 500 μl) consisted of approximately 10 pmol of EF-2 and 37 pmol of [
Lysate of BL21 (DE3) containing U-IC] MAD” (Q, 06 μCi) and various PE clones and buffer (40 mM dithiothreitol, 1 mH EDT^,
50mM Tris, pH 8.1>.

Cells were incubated with 2 mg 7x1 lysozyme at room temperature for 50 μm.
M Tris, pi (8,0 and 50mM EDTA
The supernatant and pellet were separated by centrifugation. The pellets were then dissolved in 8M urea. 3 in each reaction to measure the activity in the supernatant and pellet.
Picomole of DP-ribose bound to EF-2 was measured in 0 minutes. After incubation for 30 minutes at 37°C, 0.5z1 of 12% trichloroacetic acid was added to each assay mixture.The assay mixtures were then kept in a water bath for 15 minutes and centrifuged at 3000x, 4°C, for 10 minutes. The pellets were washed with 6% trichloroacetic acid in IWl and centrifuged as above. The content of 14(' in Benit was measured using a liquid scintillation counter and used as an index of he○P-ribosylation activity.

Full-length PE obtained from BL21 (DE3)/pJH4 was used as a positive control, giving a DP-ribosylation activity of 100%. BL21 as a negative control
(DE3)/- was used. BL21(DE3)/p
The relative activity of P-ribosylation of all other PE fragments obtained from JJ1-BL21(DE3)/pJJ14 was determined. The specific activity of DP-ribosylation of the deleted PE was compared to that of the full length PH obtained from BL21 (DE3)/pJH4. The results are shown in Figure 3.

A PE fragment obtained from plasmid pJJ1 with a deletion of the carboxyl-terminal amino acid residues from ^rg-609 to Lys-613 and a ^rg-^sn substitution retains the ^DP-ribosylation activity of wild-type PE. Was. However, from 81a-596 to Lys-613
Deletion of carboxyl-terminal amino acid residues up to Val-
The 11e-^sot conversion showed a dramatic decrease in P-ribosylation activity of 75%. Deletion of 36 or more amino acids from the carboxyl terminus reduced all enzyme activities to the level of BL21 (DE3) host cells under similar induction conditions.

3: Assay PE fragment protection test for PE cytotoxicity.
Performed in w1ss 3T3 cells. 24 of the protection assays
5wlss 3T3 cells were inoculated an hour before. Each well contained 2x 10'll cells of different concentrations of P
After 72 hours of incubation in the presence of E alone or with 8121 (DE3) cell extracts containing PE fragments, the monolayers were stained with methylene blue to detect viable cells.

〇P ~ pJJ3 that retains no ribosylation activity
It is thought that the pJJ4-deficient fragment also lost its cytotoxicity. However, these deleted fragments retained the ability to competitively inhibit the cytotoxicity of native PE on 5wlss 3T3 cells. The modified fragments produced from plasmids pJJ3 and pJJ4 effectively prevented PE poisoning when present at higher concentrations.

Although the present invention has been described above based on preferred embodiments and examples, the scope of the present invention is limited not by the above description but only by the claims.

[Brief explanation of the drawing]

Figure 18 is a schematic diagram of the PE structural gene, Figure 1B is a schematic diagram showing the construction of plasmids used to express PE with various deletions at the carboxyl terminus, and Figure 2 is a schematic diagram of PE structural genes expressed by modified plasmids. Deficient PE clone, 3rd
The figure shows the DP-ribosylation enzyme activity of PE obtained by expression of a modified plasmid in host BL21 (DE3). U wa wa rko Nori 1'') I+) U tsu false QI QI QiQ 1, Amendment of title procedure for the indicated invention in the case June 1990 1S Date 1990 Patent Application No. 116670 Non-toxic Pseudomonas spp. A 8. Contents of amendment (1) The scope of claims in the specification of the present application is amended as shown in the attached sheet. The use of such non-toxic PE as a vaccine is completely safe, and non-toxic PE also has strong antigenicity. ”. 3. Relationship with the person making the amendment

Claims (9)

[Claims] (1) A non-toxic Pseudomonas exotoxin comprising a polypeptide chain of Pseudomonas exotoxin A lacking 36 or more amino acid residues from the carboxyl terminus and capable of inhibiting the cytotoxicity of natural Pseudomonas exotoxin A. Toxin A. (2) Use of Pseudomonas exotoxin A according to claim 1 for the production of Pseudomonas exotoxin A vaccine. (3) A plasmid containing a Pseudomonas exotoxin A gene in which a structural gene corresponding to 36 or more amino acids at the carboxyl terminus has been deleted. (4) (A) A plasmid containing the complete coding sequence of mature Pseudomonas exotoxin A was partially digested with Hha I to isolate the linear DNA, and (B) the linear DNA was completely digested with EcoR I. , (C) The method for constructing a plasmid according to claim 5, comprising the step of ligating the DNA fragment with a linker. (5) The method according to claim 4, characterized in that the plasmid containing the complete coding sequence of the mature Pseudomonas exotoxin is pJH4. (6) The method according to claim 4, wherein the linker is a double-stranded synthetic oligonucleotide. (7) Linker is 3'TAATTAATTAGCCAT
7. The method according to claim 6, characterized in that TAATTAATCTTAA5'. (8) A method for producing non-toxic Pseudomonas exotoxin A, which comprises transforming an expression host with the plasmid according to claim 3 to obtain a defective Pseudomonas exotoxin. (9) The host is Escherichia coli￥E. 9. The method according to claim 8, characterized in that it is E. coli.