JPH04505402A - Schistosoma mansoni glutathione-S-transferase SM26 - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 6. The species according to claim 5, which is a species of Escherichia coli (E coli). Cells on board.

7. Culturing the cells according to claim 5 or 6, and producing the protein according to claim 1 or 2 from the culture. The method for producing a protein according to claim 1 or 2, which includes an act of collecting white matter.

8. Claim 1 or 2 as a drug having glutathione transferase activity Application of the proteins described in .

9. As a therapeutic agent, the amino acid sequence starts with an alanine residue at position 1 and starts at position 217. The sequence shown in the identifier sequence ending with a garicine residue and less A predictor of schistosomiasis containing at least one protein with 90% homology with both Preventive or therapeutic pharmacological composition.

10. As a therapeutic agent, the amino acid sequence starts with an alanine residue at position 1 and 217 The sequence shown in the identifier sequence ends with a lysine residue. at least one protein or a fragment of said protein containing at least 22 amino acids A pharmacological composition for the prevention or treatment of schistosomiasis, comprising a schistosomiasis fragment.

Specification Schistosoma mansoni glutathione-8-transferase 5M26 The present invention is directed to the development of Schistosoma mansoni. , p26 or Sm26, glutathione s-trans with a molecular weight of 26Kd. Ferrase and methods of producing the protein in substantially purified form.

Schistosomiasis (Schistosomiasis) miasis) (or bilharziosis) It is a parasitic insect that is the cause of a parasitic disease in tropical and subtropical regions. this disease affects between 200 million and 400 million people. Effective drugs against adult parasites, viz. Praziquantel exists, but it is not commonly used. However, the cost is too high to be considered in developing countries. this To date, the only serious hope for preventing disease is the use of effective treatments against the parasite. In the development of vaccines.

At least two species of glutathione S -) It is already known that it contains transferase. One side is a molecule ff126Kd, and the other is the main one, called p28 or Sm28. The molecular weight is 28Kd. The presence of these two proteins indicates that Sch. mansoni Affinity chromatography of fluke extract on a glutathione-agarose column After electrophoresing a sample of the resulting eluate, the 5DS-polyacrylate (Tiu et al.).

Parasije Immunol, (1988) 10: 693) o However, in this paper, two types of glutathione S-transfection were detected from the eluate. There is no indication of how to obtain a sample containing each enzyme in separate form.

Furthermore, 5m28 obtained by recombinant DNA technology has been shown to be a vaccine against schistosomiasis. It is also known that it is a candidate of choice as a drug of choice.

Finally, a parallel study of Schistosoma japonicum (S. iaponicum) Therefore, similar antigens in the living body of this Schistosoma japonicum, namely 5p26 and 5p28, are similar. (Smith et al., Mol, Bioc. hem, Paras Hol, (1988) 27+249).

A DNA fragment encoding Sm26 has now been discovered. This makes it possible to produce this protein using recombinant DNA technology. Ru. In particular, this allows Sm26 to be purified in the absence of Sm28. You can get enough.

In conclusion, the present invention provides: (i) the amino acid sequence is Sequence sequence starting with alanine residue and ending with lysine residue at position 217 An array as shown in the identifier, Proteins having at least 90% homology, advantageously at least 95% homology. the protein in a substantially purified form, and (11) The amino acid sequence starts with an alanine residue at position 1 and a lysine residue at position 217. Identifier array ending with (identifier 5equenc e) or the protein containing at least 22 amino acids a fragment of white matter, the protein or the fragment being in a substantially purified form; gement.

This includes in particular Sm26 and its analogs. Sm2 As in 6, these analogs inhibit glutathione S-transferase activity. or for infection by Schistosoma in mammals. Characterized by activity in inducing a protective immune response.

For example, starting from the lysine residue at position 45 and ending at the histidine residue at position 80. Analogs (45-80) may be mentioned.

The protein of the invention, especially its amino acid sequence, is shown in the identifier sequence. Such proteins are monomers or multimers (muHimers). It exists in the form of To illustrate the case of multimers, intermolecular disulfide bonds (i +Nerchain disulfide bridges) An example of this is the dimeric form that is maintained.

Glutathione S-transferase activity was determined by hahig and jacopy (hahi). g & Jakoby,) d, J:r, Methos in Enzymology ( Methods in Enz) moloB), 77:398. This can be clarified by the following method. Preferably a near-neutral buffer (butt About 10 μm test solution prepared in er medium) was mixed with 4.7 mM Reduced glutathione and 360 μM 1-chloro-2,4-dinitrobenze 50mM potassium phosphate buffer solution (pH 6.5) further containing 820 Add to μm. negative control are performed in parallel. The appearance of the reaction product is observed at 340 nm until complete conversion of the substrate. spectrophotometry (specjrophojomefly) monitored by Glutathione S-transferase activity is μM/m It is given in/mg.

In another aspect, the invention also relates to: i) co-coating the protein of the invention; Isolated DNA fragment coded. Special DNA fragments of the present invention An example is shown in the identifier array. isolated DNA flag Ment means that it is produced in the organism in which it was obtained, that is, in the present case, within Schistosoma mansoni. refers to a DNA fragment that is no longer associated with the region that controls the expression of be understood.

■) Expression of the DNA fragment of the present invention and its expression in host cells ( expression cassette containing the elements necessary for transcription and translation) asset). These elements essentially consist of a suitable transcriptional promoter ( jranscriptionpromote') and start and end for translation. It is a stop codon. In some cases, a transcription terminator (trans"1pt It is advantageous to add the ion Ierminajot).

111) Cells that have been transformed with the expression cassette of the invention. This expression case The cut may, for example, be integrated into the genome of the cell, or may be integrated into the genome of the cell, or may be Replicative expression vector (atuonomouly replicaB exp reaction vector), for example, a plasmid. It may be kept as such.

iv) The act of culturing the cells of the present invention and recovering the protein of the present invention from the culture A method for producing the protein.

Techniques for cloning and expressing foreign genes in prokaryotic or eukaryotic host cells The technique is well known to experts. These are illustrated in the examples below, but other It should be understood that vectors and other host cells may also be used.

Finally, the invention also relates to: i) amino acid combinations as therapeutic agents; The column starts with the alanine residue at position 1 and ends with the lucine residue at position 217. Indicated in the identifier array (identifier 5 sequence). at least one protein having at least 90% homology with the sequence A pharmaceutical composition for the prevention or treatment of schistosomiasis.

il) As a therapeutic agent, the amino acid sequence starts with the alanine residue in position 1 and starts with the 217 Identifier sequence ending with a lucidic acid residue 5 sequence), or is a schistosome containing a fragment of the protein containing at least 22 amino acids. A pharmacological composition for preventing or treating insect diseases.

1ii) administering a therapeutically effective amount of the protein of the invention to a patient in need of treatment for schistosomiasis; A method for preventing or treating schistosomiasis, including the act of administering schistosomiasis.

iv) Protein of the present invention as a therapeutic agent intended for prevention or treatment of schistosomiasis Use of.

The proteins of the present invention or fragments thereof have therapeutic activity. activeNy), e.g. to retain the activity of the vaccine and, as a result, to maintain the pharmacological product. It has the value of This activity is similar to that described in Example 3. shown in the test.

The pharmaceutical composition of the present invention can be manufactured by a known method.

i In particular, the protein of the invention or a fragment of the protein may be prepared in a diluent or pharmaceutically Combined with an acceptable base.

Furthermore, for use in the compositions of the invention, the protein may be in monomeric or dimeric form. In particular, the latter form is shown to be of particular value. Book The composition of the invention may further include other compounds such as 5p26.5p28, Sm2 Preferably dimeric glutathione S-)transferases of other schistosomes such as 8 or one of their analogues. Finally, the present invention The composition may contain a vaccination adjuvant (ad1) such as arun. uvanf). Alternatively, this adjuvant can be used directly may be added to the composition of the invention beforehand.

The compositions of the invention can be prepared by any conventional means used in the field of vaccines. In particular, it may be administered subcutaneously, for example in the form of an injectable solution or suspension. stomach. Administration can be done in a single dose or repeated one or more times after regular intervals. . The appropriate dosage depends on various parameters, such as the individual being treated and the method of administration. and change.

In a final embodiment of the invention, the protein of the invention or a fragment thereof is particularly It has value as a drug with rutathione transferase activity. As a result For example, it is a bioconversion process (bioconversion process). processes).

The following examples will illustrate other features and advantages of the invention with reference to the following figures: It is intended.

Figure 1 represents vector pTG959.

Figure 2 shows the 5DS-polymer colored by staining with Coomassie blue after electrophoresis. Represents an acrylamide gel. Lanes a, b and C, respectively, after 5 hours of induction at 42°C. Unpurified soluble fraction and insoluble fraction of TGE901/pTG4170 extract and This corresponds to a preparation purified on a glutathione-agarose column.

Figure 3 allows comparison of the amino acid sequences of Sm26 and 5j26 proteins. It is. Spaces are used to guide them to obtain the optimal arrangement graphically. This is what was entered.

Example 1: Clone IA of complementary DNA encoding Sm26, anti-8m26 anti Preparation of serum , Biomphalaria glabrata (Biomphalaria glabrata) a) (aquatic 5nail) and its Afterwards, they are maintained in golden hamsters.

Adults of the parasite were collected from blood obtained from the portal vein of hamsters infected for 40 days. Collect from liquid. After washing in minimal Eagle's medium (MEM), the ends are formulated as follows: 10mM potassium phosphate p117. o, 1.15% KCI, 0.5mM PMS F (phenylmethylsulfonyl fluoride) and 1mM EDTA (ethylene diaminetetraacetic acid, disodium salt) in the buffer. This preparation After sonication and homogenization, a HB-4 sawpal rotor (Sorvall Centrifuge for 20 minutes at 4°C at 10,000 revolutions/min using Ru. Collect the supernatant and equilibrate the glutathione-agarose with the above buffer. Ram (Sigma Chemical Co, Sigma Chemical Co, Apply to St. Louis, 5i, Louis SMo). Strong with the same buffer After thorough washing, the bound fraction was added to elution buffer (50mM Tris-HCl, pH 9,1 , 7mM reduced glutathione and 0.1mM dithiothreitol). Elute.

Next, the eluted fraction was neutralized using 0.5M potassium phosphate buffer (pH 7). , after dialysis against 10mM potassium phosphate (pH 7), aquacyto■ ( aquacide II) (Calbiohemubering Corporation, (Calbiochem-Behring Cotp, CA)) and concentrate.

The total glutathione S-transferase of Schistosoma mansoni purified in this way was , by Laemmli et al. ure) (1970), 227+680. Preparative polyacrylamide gel (13%) fractionate. After staining with Coomassie blue, 26 The band migrating to the kDa position is collected. Ballo et al. ul ef al, , Mol, Biochem, Parasilol, , (1985 JH 105). ctro-elujed). Dialyze it against water and use Aquacyto■ and concentrate.

Complete Freund's adjuvant (rabbits) Purified Sm26 with a total volume of 1 ml in the presence of complete adiuvan Administer 100 μg by multiple (40 points) intradermal injection. Freund after 2 weeks Freund's incomplete adjuvant 20 μg of Sm26 is injected subclavian in the presence of nr>.

1B, Complementary DNA library preparation Total RNA of adult Schistosoma was chilled. Biochemistry (Biochemistry) by Chirgwinet al. Chemistry (1979) 18:5294) and Gauss et al. A more modified method (Gausx et at, Mol, Biochem , ParasNol,.

(1983) 71293). Oligo(dT)1□-18-se Rulose Column (Collaborative Research, Lexington, Mny (Co) llaborajive Re5search.

Affinity chromatography using Lexingfon, MA) Thus, polyA RNA is obtained. Lambda GT11 vector (la Complementary DNA library expressed in mbda gj 11 vecjot) using the Amersham Kit (AmershamkN) by the following method. Build it. Using purified polyA RNAs as a starting material, complementary DNA5 (cDNA) by Gtibler and Hoffman Synthesized according to the method of Gene (1983) 25+263) do. After protecting the inner E cDNA site, E CoRI adapters are ligated to the cDNA fragment.

It was cleaved with EcoRI and excess adapter was removed by column chromatography. leave

These cDNA fragments were transformed into phage lambda G-T11 (phag e lambda gj]1) (Young and Davies d into the EcoRI site of Davis (1983). Like this Ec oRI inserts with the sequence encoding β-galactosidase , resulting in the lactose promoter r) be under the control of This recombinant phage is packaged in vitro. packaged.

IC, 1.5 of a selected complementary DNA library of cDNA encoding Sm26 ．． 106 independent phages were generated by Huini et al. DNA cloning (DNA cloning), a practical approach (Practical Approach) (1985), Grover Described in Glover D.M., ed., 1:49). The method described above was modified and tested as follows. That is, this lambda Samples from 11 libraries were collected at 5,000 frames per 85 mm diameter dish. Inoculate E. coli strain Y1090 at a dilution rate that will result in a large Bate.

The plaques were treated with 10 mM isopropyl β-lowthiogalactopyranoside (I Nitrocellulose filter pre-impregnated with PTG) 3 on Schleicher and 5chuell) Transfer for 5 hours at 7°C. IPTG is under the control of the lac promoter A certain fusion ■ Induces the expression of white. The filter was filtered (preliminarily with E. coli antigen extract). Incubate in the presence of rabbit anti-p26 antiserum (adsorbed on top). combined The antibody was incubated with biotin-labeled rabbit anti-1 gG secondary antibody (second, biotin -labeled rabbit anti-IgG antibody) i.e. then streptavidin-peroxidase (sjre ptavidin-peroxidase) binds to biotin, and this complex The rHRP Color Development Lige sold by Bio-Rad (IIRP color development reagen+) Peroxidizer with a staining reagent called J Develops color by reaction.

This detection using antibodies ensures that the cDNA insert is isolated from the specific anti-8m26 antibody. Synthesis of proteins or protein fractions with epitopes recognized by the body This allows for the identification of recombinant phages that direct.

Four phage clones capable of expressing proteins that react with anti-3m26 antibody Collect the parts separately. The DNA is purified and digested with EcoRI. the The fragments obtained from the digest were then separated by agarose gel electrophoresis. do. One of the cDNA inserts large enough to encode the complete Sm26. One scene clean method (Geneclean Melhod) (Bye No. 101 1 nc, fBio 1011 nc,) La Jolla, Si Electro-elution (d) by electro-elution (CA) , M13TG130 vector (Kieney et al., ) Gene, (1983) 26:91) multiple insertion region (muHipl) Subclonin at the EcoRI site of e1nsertion region) Google. The nucleotide sequence of this cDNA was determined using the method of Sanger et al. 1977) 745463). This identification was determined by sequence studies. cDNA effectively encodes a 26Kd protein. Ru. This cDNA does not have the A base of the start codon ATG, so the start codon Therefore, t-F must be added. Additionally, the EcoRI site was replaced with Bgl It is desirable to replace it with the II site. These two processes mean that the array is 5’-GCTCTCCCGGAGATCTATGGCACCTAAG? j’ Directed mutagenesis (di rected mujagenesig) at the same time.

Vector M13TG4116 is obtained in this way.

The cDNA sequence is an identifier sequence (identifier sequence). nce).

Example 2: Production and purification of Sm26 The M13TG4116 vector was digested with EcoRI and BglII, and the DNA fragment was The fragments are separated by agarose gel electrophoresis. Contains 5M26 cDNA A 0.75 Kb fragment is eluted using the scene clean method. in parallel, The pTG959 vector described in EP-A292404 and represented diagrammatically in FIG. The vector is digested with EcoRI and BglII. With these cuts, 1acZ The fragment containing the 5'' portion of the gene is lost.

The 0.75 Kb fragment was inserted into the pTG959 vector prepared above. ligafe to obtain pT04170 vector. This pTG4 In constructing the 170 vector, the resulting DNA flag encoding Sm26 The protein is located downstream of the pL promoter of lambda phage that is induced at 42°C. and be controlled by it.

E. coli TGE901 (F- sup-his ilv bio (lambda-cl 857delta- Ban deNa41)) was added to 1 liter of LB-ampicillin medium (LB- ampicillin medium). Initially, 0D6oo was cultured. The temperature is maintained at 30°C until 0.2, and the second stage is maintained at 42°C for 5 hours.

After culturing, cells were harvested by centrifugation, sonicated, and formulated as follows. Dissolve with simultaneous enzyme treatment in equilibration buffer: 100 μg/ml Lysozyme and 0.1% Triton ．． 10mM potassium phosphate pH 7,1,1 further containing Sigma) 5% w/v potassium chloride, 0.5mM phenylmethylsulfonyl fluoride IITIM EDTAo then lysate at 4°C at 110,000 revolutions/min. Centrifuge for 20 minutes. Collect the supernatant and equilibrate with the above equilibration buffer in advance. Glutathione-agarose column (Sigma Chemical Company, S igma Chemical Co, St. Louis, Louis, M o) Load with continuous flow (30 ml/hour) on top. Wash thoroughly with the same buffer. After cleaning, the bound fraction was treated with the following formulation: 50mM Tris-HCl (pH 9,1), 7 Elution buffer with mM reduced glutathione and 0.1 mM dithiothreitol. Elute with 10ml of fur.

The total elution fraction was collected and purified using 065M potassium phosphate buffer (pH 7). Neutralize at 4°C overnight Prescription: 10mM potassium phosphate (pH 7), Dialyze against 1mM EDTA buffer. The liquid volume of the last dialyzed substance is 2 Aquacide II (Calbioheme) until ml. -Behring Corporation, Calbiochem-Behr ing Carp, CA)).

The purity of the purified product obtained in this way was determined by polyacrydamide gel electrophoresis of the sample. I'll check later. As shown in Figure 2, the presence of a single band of 26kd indicates that Sm2 6 is obtained in substantially purified form.

Furthermore, the glutathione S-transferase activity of preparations purified in this manner Check as follows: 10 μl of the preparation is diluted with 4.7 mM reduced Added glutathione and 360 μM 1-chloro-2,4-dinitrobenzene. 50mM potassium phosphate buffer solution (pH 6.5) containing 820μm Add. Run a negative control in parallel. Appearance of reaction products (yellow coloring) ) is monitored at 340 nm until complete conversion of the substrate.

The activity measured is on the order of 30-50 μM/min/mg.

Purification of 1 to 2 mg from 1 liter of culture using the above-mentioned method for preparing Sm26 The protein is obtained.

Example 3: Proof of the immunoprotective effect of Sm26 3A, actual, experiment N001 8 week old male fisher rats (Iffer Credo (Iffa-Credo) was administered to five rats of four to seven rats each. Divide into groups. 25 μg (25 μg) of Sm26 obtained in Example 2 was subcutaneously administered to rats. Group 1), 25 μg of Sm28 purified from adult protrusions (Group 2), Administer 25 μg of bovine serum albumin (group 3). Each antigen is 1. 25mg of aluminum hydroxide auxiliary (alun gel) gel), Serva) in a volume of 1 ml. parallel Group 4 rats received the same volume of 1.25 mg aluminum hydroxyl. Give doorshwand. Finally, rats in group 5 are given nothing. glue For rats in groups 1 to 4, the corresponding treatment is repeated 15 days after the first injection.

Thirty days after the second injection, 1000 rats in each group were administered percutaneously after anesthesia. cercariae. Liver ligation 21 days after infection Physiological saline containing 1% heparin and 0.1% Tween Irrigate with water. This collects the protrusion and converts the protrusion into a binocular lens. Count under 1 ens). The results are shown in the table below.

Group No. 2 (positive control) and Group No. 1 are Compared to Group No. 4 (adjuvant control), the contribution was 45% and 16%, respectively. A decrease in the amount of live worms (paras.e) is observed. The results of group No. 1 are: lower than that of group N092, but not fully significant considering the low standard deviation. Yes (p・0.03).

3B, Experiment No. 2 Independently conduct a new experiment in which the experimental conditions were the same as those shown in 3A in each detail. conduct. Rat identifier sequence immunized with Sm26 (IDENTIFIER5EQtlENCE) Subject (suJect) : 26Kd of Schistosoma mansoni (S. mansoni) called Sm26 The amino acid sequence of glutathione S-transferase' and the above-mentioned protein were Nucleic acid sequence of complementary DNA fragment coded ATT TCG ATCCTT GAA CGA GCG GTT TTG G AT ATT AGG ATG GGT GTT 315'fTA AGA A TCGCA TACAAT AAG GAA TAT CAA ACCCTCM A GTT GAT 360TTT CTCAACAAA CTT CCT G GG AGG Enter CT Enter A Enter TG TTCCAA CAT CGT 40 5TTG TCT MCAAAA ACT TAT TTG MCGGT MT TGT GTA ACT CAT CCT 450Lau Ser ksn L ye Thr T’yr Leu Asn Gly Asn Cys Val Thr　His　Pr.

Cys engineering 1a lu A! lp Lau Pro Gin l Asn Tyr Leu Asn Ser 5er3133 AarI Engineering hoI 7B FXCA positive E2 5j26 5PILGYWK KGLVQPTRLLEYLEEKYEEHL YERDEGDKWRNKKFELGLEFPNLPYYIOGOVKLTQ5 MA11RYIADKIJJMLGGCPKERA;==:=:==+-=== Tomi = Tomi ==============x=====2zzE Engineering 5ML EGAVLDIRYGV5RIAYSKDFETLKVC)FL3KLPEXL KMFEDRLCl-IKTYLNGOHVTI-IPDFMLYDALOWL YMDPMCLC) AFPKLVCFKKCIEDLPQIKNYLNSiSR Yl to WPLQGWOATFGGGC) TPF' to R1εAIFQIDKYL 55KYIAWPLQGWOATFG3GI) HPPK summary The present invention is directed to the use of S. mansoni with a molecular weight of 26 Kd, known as p26 or Sm26. Glutathione-S of Schistosoma ma++5oni - transferases and methods of producing said proteins in substantially purified form; related.

international search report 1ms+Aalle++al AnatM&bn No, “TE//F’R911 00091 International Search Report

Claims (10)

[Claims] 1. The amino acid sequence starts with an alanine residue at position 1 and ends with a lysine residue at position 217. In the identifier sequence a substantially purified form having at least 90% homology with the sequence shown in protein in the state of 2. The amino acid sequence starts with an alanine residue at position 1 and ends with a lysine residue at position 217. protein in a substantially purified form as shown in the relevant identifier sequence. white matter, or a fragment of the protein containing at least 22 amino acids. 3. The protein according to claim 1 or the protein or fragment according to claim 2 A DNA fragment that encodes. 4. The DNA fragment according to claim 3 and in a host cell. An expression cassette (express) containing the elements necessary for the expression of the DNA fragment. cassette). 5. A cell transformed with an expression cassette according to claim 4. 6. The cell according to claim 5, which is a species of Escherichia coli. . 7. The cells according to claim 5 or 6 are cultured, and the protein according to claim 1 or 2 is obtained from the culture. The method for producing a protein according to claim 1 or 2, which includes an act of collecting white matter. 8. Claim 1 or 2 as a drug having glutathione transferase activity Application of the proteins described in . 9. As a therapeutic agent, the amino acid sequence starts with an alanine residue at position 1 and begins at position 217. is shown in the identifier sequence ending in a lysine residue, and less A predictor of schistosomiasis containing at least one protein with 90% homology with both Preventive or therapeutic pharmacological composition. 10. As a therapeutic agent, the amino acid sequence starts with an alanine residue at position 1, The sequence shown in the identifier sequence ends with a lysine residue. at least one protein or a fragment of said protein containing at least 22 amino acids A pharmacological composition for the prevention or treatment of schistosomiasis, comprising a drug.