JPH06511154A - Method for producing recombinant Borrelia protein - 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] Technical field of production method of recombinant Borrelia protein The present invention relates to a method for producing recombinant Borrelia lipoproteins, particularly for the pathogenesis of Lyme disease. The outer surface of the spirochete orrelia b soil L1 soil yue. The present invention relates to a method for producing OspA (OspA).

Related applications This application is filed in U.S. Patent Application No. 8, which is a continuation of U.S. Patent Application No. 779,048. This is a continuation application of No. 88,765.

Background technology Lyme disease is caused by the spirochete Borrelia bur, which is transmitted by ticks. It is an animal pathogenic infection caused by P. dorferi. Hotels infected with this spirochete The patient exhibits severe pathological symptoms affecting the skin, joints, nerves, and other areas. Lyme disease recently It is a serious epidemiological problem, particularly in North America and other parts of the world.

A vaccine that is effective against this disease and a vaccine that is effective for detecting antibodies against pathogenic spirochetes. Attempts are being made to develop immunodiagnostic reagents. The main outer surface protein 0spA has attracted attention. , whose antibodies have been demonstrated to protect mice from pathogenic spirochete attack. (References 1 and 2; a list of references is provided at the end of the disclosure).

Purify Borrelia soluble lipoproteins by purifying the exudate; Attempts have been made to isolate it. However, the culture of Borrelia and subsequent Purification of proteins from crude cell extracts is extremely complicated and requires enormous costs. Ta.

0spA and its inducers have the potential to produce large amounts of purified protein. Genetic recombination techniques have been proposed to produce conductors. These techniques are Once the orrelia gene is expressed in a suitable host/vector expression system such as It is something that makes you

PTC patent application number W0 90104411 includes B.

Os of burgdorferi New York stock B51 (ATCC35210) A DNA fragment encoding the pA protein and containing the ospA gene has been described. has been done. This PTC application encodes the full-length wild-type OspA. s The nucleotide sequence of the pA gene is described along with its amino acid sequence.

Dunn et al. reported that, although soluble, wild-type B. burgdorferi 0sp. Regarding the production of modified 0spA protein that retains specific reactivity to antibody A (Reference document 3). In this paper, two types of plasmid and DE T9-O8pA and pET9-preosoA have been described, but The authors performed polymerase chain reaction (PCR) encoding the recombinant OspA. ) containing an amplified DNA sequence, the latter of which is a PC that converts the full-length wild-type OspA into Contains R amplified DNA sequences. Both sequences are derived from bacteriophage T7φ10 protein. expressed from the lomotor.

The primary translation product of a full-length gene contains a hydrophobic N-terminal leader sequence, which substrate when a lipid moiety binds to the sulfhydryl side chain of the adjacent cysteine residue. Become. This binding is followed by cleavage by signal peptidase H and the lipid moiety Coupling to a new N-terminal occurs. On the other hand, the protein translated from the cut gene Paku is not lipidated and is dissolved in an aqueous solution. Some related to the representation of 0 It is said to solve problems. In other words, proteins interact with host external cells during the process of expression. Because it binds to the cell membrane, its solubility decreases, requiring the use of detergents for protein separation. However, purification becomes difficult.

Chromoseme replication and low levels of T7 RNA polymerase gene under control The pACYC184-derived plasmid pLysS produces T7 lysosomes. The plasmid has been inserted into the host strain. Even if induced, plasmid pET 9 preospA is highly induced compared to plasmid pET9-OspA. It is known that the amount of protein produced is small. The latter has no surfactant. The former requires the surfactant Triton X-100 for solubilization. It is known to do. The report by Dunn et al. The subsequent purification and isolation of the 0spA protein is not described.

The production capacity of recombinant full-length OspA is lower than that of soluble cut 0spA, and Surfactants must be used to recover long proteins (lipoproteins cannot be peptides in exactly the same way (treatment with chemical agents often impairs reactivity). (ref. 7), which is covalently bound to the N-terminus of B phosphorylated cells (ref. 7). Both ref. 4.5) and cytotoxic T-phospho8 cells (ref. 6) were stimulated. It is thought that the immunoreactivity is higher than that of cut proteins due to the , recombinant wild-type protein is considered desirable.

Long-term protection against spirochete infection by immunizing mice with 0spA protective immunity has been obtained (Reference 1.8). Antigens used in this test contains glutathione-5-transferase at the amino acid terminus of OspA. It is a fused protein containing large domains. This molten protein is a posttranslational lipid binder. It is unlikely that it will serve as a substrate for ts association. In this test, the ability of this antigen to induce a protective response is complete Freund's adjuvant followed by incomplete Freund's adjuvant. This is thought to be due to the administration of the antigen at point 9). This previous research See also PCTWO92100055.

As is clear here, the recombinant protein is converted into a =- code by the full-length ospA gene. similar compounds lacking bound lipids in the absence of adjuvant or in the form of alum adsorption. It exhibits immunogenicity that is not seen with recombinant OspA protein. This time it was approved. The present invention demonstrates that the immunogenicity of large protein antigens is similar to that of bacterial lipoproteins. It is disclosed for the first time that the expression increases with the expression .

Summary of the invention Coated with highly purified immunologically effective recombinant proteins and corresponding genes. A novel, easy, and and to provide an effective manufacturing method. This recombinant protein is a natural protein. Shows immunogenicity equivalent to that of

The present invention uses specific detergents to transfer recombinant lipoproteins to other cellular components. A method is used to selectively separate the components. This procedure of the present invention cannot be solved by the prior art. At the same time, we solved the problem related to the production of full-length OspA that could not be determined. The aim is to provide highly immunogenic products.

Altitudes produced by genetic engineering from host organisms transformed by plasmids It is the first to provide a highly purified and immunologically effective recombinant protein. Book The second object of the invention is such a novel tanno(ri). Several novel plasmids useful for such transformations are also provided.

Vaccines consisting of such proteins constitute an object of the present invention.

Furthermore, it is an object of the present invention to provide an immunologically effective amount of tan/comb.

As an immunodiagnostic reagent for detecting antibodies in the host and diagnosing the presence or absence of infection. can be used.

- produced a purified, immunologically effective recombinant protein. Also, special In most cases, the nucleotide sequence differs from that of a given gene in some nucleic acids; Produced by the osoA gene of the actual 831, ACAI and Ip90 strains The complete gene product that differs from the one in most cases by a few amino acids A full-length 08pA gene is also present. Such genes and gene products are So Ba1. It was considered to be a member of the ACAI and ED90 strains.

Brief description of the drawing PCR oligonucleotides used for cloning into T9a expression vector Indicates the mode.

Figure 2 shows that pOAl is formed from the Ba1 gene and pOA9 is formed from the ACAI gene. In order to form pOAIO from the Ip90 gene, the full-length os The pA gene was inserted into the pET9a expression vector, and the 0SpA gene was inserted into the pET9a expression vector. The cloning procedure for placing the 7dlO promoter under control is shown.

Figure 3 shows the predicted restriction map of plasmid pOA1.

Figure 4 shows all 1,000! 1st place (M!\-car (digested with HasIII. l, X+7 DNA of 4); B-BamHl; E=Eco RIiH-Hind III; N Results of various restriction digestions of plasmid pOAI demonstrating the presence of =NdeI) Show results.

Figure 5 shows the completion of burgdorferi's Ba1, ACAL and ID90. Used when cloning full-length 08DA gene into DcMBI expression vector and j'-PCR nucleotides are shown.

Figure 6 shows that p05 is formed from the Baill gene and pOA7 is formed from the ACAI gene. In order to form pOA8 from the Ip90 gene, the full-length os Cloning to place the pA gene under the control of the T rc promoter The procedure is shown below.

Figures 7A, 7B and 7C show two types of hosts containing oOA1 (Figure 7A) and in one type of host, including pOA5 (Figure 7B) and pOA6 (Figure 7G). The time course of induction of OspA by ITPG is shown.

Figures 8A, 8B and 8C depict recombinant cells from E. coli according to embodiments of the invention. Cell culture and fusion used for production and purification of full-length 0spA, respectively. , is a flowchart showing extraction and purification steps using a surfactant.

Figure 9 shows various surfactants CDeo ÷ sodium deoxylate + E tn p” empigen; 5nrc=sodium lauryl sarcosine; 5DS-dodecy complete cell lysis with sodium sulfate; TX114=TritonX-114) Indicates protein elution into the solution, where M of lane = v-car (Bio-R low molecular weight standard material manufactured by ad); W-total melt; S = soluble fraction; P = pellet; A = water-soluble fraction; D = m surfactant.

Figure 10 shows Western plots using anti-0spA monoclonal antibody H5332. It is. Here, the substance with Dalton at 31 in the surfactant phase has 0 spA, Wh-total Cell molten solution, Ag = aqueous phase after Triton X-114 extraction, Dt = surfactant phase, Bx = aqueous phase extracted again with Triton X-114, PI = insoluble pellet .

Figure 11 shows pOAl or OspA nonlipoprotein expressing OspA lipoprotein. Cells containing pOA2 expressing protein were cultured until the peak of cell division and ITP G T o OAl was induced for 2 hours and DOA2 was induced for 4 hours, pOAI and lipoprotein phase partitioned with Tr on X-1+4 Shows DOA2. After centrifugation, cells were resuspended and thawed by freeze/thaw. 6 Tri+on

Equal amounts were taken from both sides and electrophoresed on a 4-20% SDS-Page gel. Ta. Gels were stained with Coomassie brilliant blue.

Both oOA1 and oOA2 have molecular weight markers (M) as shown. , total melt (W), aqueous phase (A), surfactant phase [1]), and insoluble pellets It is.

Figure 12A, +213 and 12C represent pOA1 (Figure 12A), oOA9 and p OAIo (Figure 12B) and pOA5, pOA7 and pOA8 (Figure 12G) This shows a method for purifying 05DA lipoprotein produced from.

Here, a portion of each portion was taken and electrophoresed on a 4-20% SDS-Page gel. The gel was stained with Coomassie brilliant blue. Lane 1 in Figure 12A is low molecular weight marker, lane 2 is total melt, lane 3 is surfactant phase, lane 4 is DEAE Sepharose, lane 5 is S-Sepharose eluate. Figure 128 P = pre-stained low molecular weight markers (106 kDa, 80 kDa, 49 ．． 5kDa, 32.5kDa, 27.5kDa, 18.5kDa), CL = full size cell melt, D = Triton X = 114 surfactant phase, D e = D E A　E Sepharose passage fraction, S-Sepharose pH 5,7 eluate (purified Os pA-2). Figure 12C: P-prestained low molecular weight marker (106kD a, 80kDa, 49.5kDa, 32.5kDa, 27.5 kDa, 18.5 kDa), 0 = pOA1-derived B purified with S-Sepharose 310spA-L (Fig. 12A), CL = whole cell lysate, D = T r i t o n X = + 14 surfactant phase, De = DEAE sepharose passage fraction .

Figure 13 shows the immunogenicity of purified full-length OspA in B o rrelia bur. Three types of tests used to compare the immunogenicity of T. gdorferi This is a silver-stained gel of the vaccine (M=mama--:OA-purified recombinant OspA; E = Fraction E of Dan burgdorferi; SA = Calf serum albumin).

FIG. 14 shows purified full-length OspA and I3. burgdorf I of mice pre-injected with 10 ug of eri fraction followed by booster immunization for 4 weeks. gG serum titer is shown. The IgG serum titer was determined using mouse serum as the first antibody and mouse serum as the second antibody. Goat anti-mouse IgG conjugated to alkaline phosphatase, purified Osp It was measured by the EL I SA method using a solid phase containing 0 μg of AI.

15A-151 are graphs showing the dose response of mice to lipoproteins. be. Figure 15A shows the effects of C3H/He strain mice on non-adsorbed lipoproteins. Shows a dose response. FIG. 15B shows B A L B / for non-adsorbed lipoproteins. The dose response of e strain mice is shown. Figure 15C shows the results for alum-adsorbed lipoproteins. Dose response of C3H/He strain mice is shown. Figure +5D shows non-adsorbed lipoprotein Figure 2 shows the dose-response of CH3/He strain mice to chlorine.

Figure 15B shows the dose response of CH3/He strain mice to nonlipidated proteins. show. Mice received the indicated amount of protein (suspended in PBS unless otherwise specified) on week 0. The mice were immunized and boosted with the same vaccine at 3 weeks.

Serum IgG titer at 4 weeks was measured using ELrSA method using 0spA lipoprotein as antigen. It was measured with

FIG. 16 is a graph showing the antigenicity of lilysomes and non-lipoproteins.

Figure 17 shows the production of DCMBI and pCMB2 plasmids.

System of identification name of ospA gene including plasmid p OA I B 31 pE T 9 aDOA2ma B31 pET9a DOA5 B31 DCMBI DOA6 B31 DCMB2 oOA7 ACAI DCMBI pOA 8 1 p 90 D CM B Io OA 9 A CA 1 p ET 90 apOA 10 1 o 90 p ET 90 a Invention description about B, Cloned ospA gene of burgdorferi strain B31 (as described in WO 90104411) (N-terminal region, SEQ ID NO: l; C-terminal region: sequence Column identification number 2; other sequences are the same as WO90104411) was added to the template (pTRH 44) and used in the polymerase chain reaction as shown in Figure 1. Specially designed oligonucleotides to amplify the entire native ospA gene Primers (PET-IN[CO1] (sequence identification number 3) and PET-2 73C[CO3] (sequence identification number 4) was used.

Similarly, oligonucleotide primers at the N- and C-termini, respectively Pair (a) OspN2 (sequence identification number 7) and BZI (sequence identification number 8) and (b) Osc+N2 (sequence identification number 7) and DK4 (sequence identification number 9). For the PCR reaction, B. burgdorferi ACA I and Ip90 strains were used. The cloned ospA gene (see reference 10; ACAl and I p90 N-terminal region of; Sequence identification number 1: ACAI (7) C-terminal region: Sequence identification number 5; C-terminal region of Ip90: using SEQ ID NO. 6), as shown in FIG. Amplified fragments were formed.

In general, the basic amplification method of target nucleic acids using oligonucleotide primers is It is known from U.S. Pat. Nos. 4,683,202 and 4. 800. 1 Since it is described in No. 59, the description of the technology to be used will be omitted.

The obtained fragment was inserted into the Nde+ and 13a of plasmid vector DET9. mH1 site and the T7 promoter control as shown in Figure 2. The ospA gene under the molecule and an efficient translation initiation system from bacteriophage T7. Introduced Gunar. pET9 and pLysS plasmids for colonization host bacteria, used for expression of cloned genes by T7 RNA polymerase. The culture medium and method have already been described in US Pat. No. 4,952,496 Therefore, detailed description will be omitted here. The T7 promoter system is preferred in the present invention. Although it is a desirable expression system, the expression of the full-length ospA gene is as described below. This can also be achieved using other expression systems that are compatible with the host organism.

DET9 uses the kan gene rather than the bla gene as its selective marker. I have it, so I used it. Therefore, using ampicillin in cell culture Since there was no need to There is no possibility of the formation of a conjugated bond. Such a conjugate bond is It is thought to be a major antigenic determinant in allergy and complicates immunological testing. Ru.

The resulting plasmid was B, burgdorferl Ba1. ACAl and IE) Contains the ospA gene from 90 strains, pOAI, pOA9 and and pOA10. The pOA1 plasmid was developed by Dunn et al. (supra). The pET9-p r e Os p A plasmid described as Although they are the same, the oligonucleotides used in the PCR reaction are different between the two. plastic Figure 3 shows the predicted limit map for Sumitomo DOA 1. The results of various restriction digestions As is clear from FIG. 4, it shows that all expected parts are present.

In order to produce proteins, plasmids pOA I, pOA 9 and DO AIO was transformed into a phenotypic strain of E. coli or other suitable host organism. . E.

coli strains as described in the aforementioned U.S. Pat. No. 4,952,496. T7 expressing strains of E. coli are preferred. In particular, the strain is as described above. It may also be a BL21 (DE3) (pLysS) expressing strain of E. coli. , E, Co11 strain 0MS174 (DE3) (pLyss).

The transformed host was cultured, and isopropyl-β-D-thiogalactoside (IP The protein was induced with TG). ■pOA after addition of PTG from 1 plasmid The induction time of OspA is shown in FIG. 7A. Using pOA9 and pOAlo In this case, similar results were obtained as in the case of c+OAI. Is it DOAI Plasmid? Synthesis of OspA protein in E. and coli stopped within 1 hour of induction. This indicates that there is some degree of protein toxicity. Nevertheless, Protein production was approximately 10 mg/L per medium, which was at an acceptable level. .

Plasmid pOA1. pOA9 and pOA10 and T7 promoter In addition to the expression of 0spA lipoprotein in E. coli using The full-length B31. ACAI and Ip900SDA genes A plasmid containing the protein was constructed to express lipoproteins. Regarding this, Ba1? The PCR-amplified fragment of ospA was inserted into the plasmid expression vector pcMnt. and cloned into the Ncol and BamH1 sites of pCMB2. Plasmids DOA5 and pOA6 were prepared, while plasmids DOA7 and pOA6 were prepared. poAa is os from ACA 1 strain (pOA7) and 1p90 strain (pOA8) Ncof and Ba of the vector pCMBI expressing the pcR amplified fragment of pA Prepared by cloning into mHI site (pOA5, pOA7 and pOA8) (See Figure 6).

As is clear from Figure 5, B, burgdorferi's Ba1, ACAl and The cloned 08OA gene of the IC90 strain was prepared using a pair of oligonucleotide primers. (a) PX3 (sequence identification number to) and C03 (sequence identification number 3), (b ) PX3 (sequence identification number 10) and BZI (sequence identification number 8) and (c ) PC using PX3 (sequence identification number 10) and PX4 (sequence identification number 9) The N-terminus and C-terminus of the corresponding genes are amplified by the R reaction, respectively. , formed the corresponding amplified fragment.

Plasmids DCMBI and pCMB2 are derived from plasmid pTrc99a ( Pharmacia catalog number 27-5007-01) and kanamycin resistant Antibiotic gene (Pharmacia catalog number 27-4897-01) was digested. , separate the fragments by gene purification method and ligate the fragments to each other. (Figure 17). Plasmid p T rc99a (4197 bp) has a strong promoter adjacent to the multiple colonization site; It has a strong transcription aggregation signal (rrnB). Expression of target traits requires R of host cells. Because it uses NA polymerase, it can be used with a wide range of E. coli strains. can.

Expression is achieved by the lactose suppressor gene (lac[g) contained in the vector. is controlled. Lactose repressor protein is an inducer [PTG In the absence of F, transcription of the target gene f is inhibited. The kanamycin resistance gene is Contains the gene of transposon Tn903 flanked by restriction enzyme sites. It is a linear double-stranded DNA fragment (12a2bp) containing kanamycin and Aminoglycoside 3 with a combined effect on resistance to neomycin and neomycin - Encodes a phosphotransferase enzyme.

DCMBI (5.5 kb) is transcriptionally derived from the Trc promoter. contains the kanamycin resistance gene oriented in the same direction as DCM B2 contains the kanamycin resistance gene located in the opposite direction, making it resistant. genes and specific genes under the control of the T　r　c promoter. The transfer results in a width ring transfer. 3ma 1. Hi nd T I I and Limit o CM B I and p CM B 2 by Bam1ll+Ncol Enzymatic digestion yielded fragments of exactly the same size as expected.

Plasmids pOA5 and oOA6 were transformed into E, co+1 or other suitable organisms. , preferably into a expressing strain of DH5α Humbonen cells. transformation The transformed host was cultured, and the protein was induced with [PTG].

o The induction time course with OA 5 (Figure 7B) is similar to DOA 1 (Figure 7A). However, the level of OspA produced by DOA6 was several times lower ( Figure 70).

Similar results to pOA5 were obtained when oOA7 and DOA8 were used. It was.

The production and purification process of recombinant full-length OspA is schematically shown in Figures 8A and 8C. show. The specific conditions for the process are defined in the Examples described below, but are summarized below.

Following cell culture and protein induction (Figure 8A), cells were frozen and thawed. Melted. 0 spA protein than other bacterial proteins present in the melt. The melt is treated with a surfactant that selectively solubilizes. Using various surfactants A series of experiments were conducted to determine which surfactant to use for the OspA protein. I considered whether it has a high degree of sex. Among the surfactants tested, Triton X-114 was 3 The Dalton protein was selectively solubilized in 1 (Figure 9), and this protein was It was confirmed by lotting that it was OspA (Figure 10).

The present invention is not limited to the use of Triton under mild conditions as described below. Other materials may be used as long as they have phase separation properties.

Following the addition of the selective surfactant, gradually increase the temperature to approximately 35°C to 40°C and mix. Warm the mixture. During this time, phase separation occurs and the solution becomes cloudy. Refining method of the present invention In order to avoid protein denaturation or damage to immunological properties, this phase separation is Perform under mild conditions. (For example, TritonX- described by Dunn et al. 100 (Ref. 3) as a surfactant, the selection of 0spA proteins It is effective for phase separation, but heating to about 60-65℃ is necessary to cause phase separation. (This is extremely disadvantageous when it comes to product usage.)

When a turbid mixture is centrifuged, the mixture has three phases, i.e. 50% or more. A surfactant containing 0spA protein and a small amount (approximately 5% by weight) of bacterial protein. , separates into an aqueous phase containing the remaining JllIl bacterial proteins and a solid pellet of cell debris. . Separate the surfactant from the aqueous phase and solid pellet.

Treatment with surfactants selective for 0 spA and subsequent phase separation for surfactant release In this step, OspA can be almost completely separated from IIa idle protein. (Figure 12). The final purification step removes the *S protein present in surface activity. OspA is separated from the OspA.

The final protein purification selectively adsorbs S+@protein and does not adsorb 05DA. chromatography columns, especially DEAE-Sephacel, DEAE-Sephalo This is done using chromatography materials such as a base or its base. Surface active application Collect the flow-through containing all of the purified OspA protein. Ru. The adsorbed fraction contains all of the mm protein that was included in the surfactant. S- Further purification using Sepharose or equivalent chromatography column. As a result, not only the mixed proteins but also the pimples were detected in the Limulus cell lysate (LAL) test. Since rhodiene properties were not observed, the content of ribopolysaccharide (LPS) is also likely. A low throughput can be obtained. Highly purified 0spA solutions can be lyophilized or If there is none, we will process it.

The above procedure is particularly useful for producing highly purified recombinant OspA proteins. As mentioned above, this procedure and technique requires the proper loning of the appropriate gene, Construction of a suitable plasmid vector containing the gene; and by transformation of suitable hosts and production and purification of lipoproteins. By appropriate selection of selective surfactants, other highly purified It can also be easily applied to the production of recombinant Borrelia lipoproteins.

Example Example 1 Using plasmid DOA 1 prepared as above, E. coli B. L21 (DE3) (pLyss) (pOA+) strain and IIMS 174 (DE3) CoLyss) (pOAI) strain was transformed. Transformation E, E. coli was grown in sulfuric acid to obtain 12 ml of preculture per IL of full-scale culture medium. Kanamycin 25 μg/m I, chloramphenicol 25 μg/m The cells were inoculated into LD medium containing l. The cells were precultured at approximately 37° C. overnight with shaking.

The next morning, 10ml of the overnight culture medium was treated with 25μg/ml of kanamycin sulfate. Inoculate LB medium IL containing 0D=0. 6 (01) = 1. Cultivate up to 5 The cells were cultured at about 37° C. for about 3 hours until they reached a normal temperature. inpropyl thiogara Add ectoside (IPTG) to the medium to a final concentration of 0.5mM, and The cells were further cultured at 37°C for 2 hours. After culturing, the medium was cooled to about 4°C and incubated at 1000 °C. Centrifuged at 0xg for 10 minutes. Discard the supernatant and add the cell pellet to 10 times the volume of PB. It was suspended in S.

Cell suspensions were frozen in liquid nitrogen. The frozen cells can be frozen at -70°C if necessary. can be stored indefinitely.

Following freezing of the cell suspension, the cells were thawed at room temperature (approximately 20-25°C). This passing At this point, cell lysis occurs.

Add DNase I to the thawed cell suspension at a concentration of 1μg/mI. was added and incubated for 30 minutes at room temperature. During this time, the viscosity of the solution decreases. do.

After the incubation, the cell suspension was cooled to below 10°C on ice and Add a 10% stock solution of ton X-114 to a final concentration of 0.3 to 1% by weight. added to. This mixture was kept on ice for 20 minutes.

The cooled mixture was then warmed to approximately 37° C. and held at that temperature for 10 minutes.

As phase separation occurs, the liquid becomes cloudy. This turbid liquid was heated to about 20°C, 12. ooooo After centrifugation for 10 minutes at and solid pellets. Use surfactant to avoid disturbing the pellet. It was separated from other phases and cooled to 4°C. Buffer A, i.e. 50mM Tris (pH 7,5), 2mM EDTA and 10mM Nac+ in a cooled surfactant solution. 1/3 of the original volume. The resulting solution was frozen and stored. After that, it may be processed as described below, or it may be processed immediately. good.

1 ml of DEAE-Sepharose CL-6B column per 10 ml of surfactant Prepare twice the volume of buffer C1, that is, 50mM Tris (DH7,5 ), 2mM EDTA, IM NaCl 1,0,3 wt% Triton X-1 00 and then buffer B1, 50mM Tris (pH 7, 5) Washed with 2mM EDTA, 0.3tlL% TriLon X-100 .

Next, a surfactant was placed on the column, and the flow-through containing OsoA was collected. Sara The column was washed with an equal volume of Buffer B, and this flow-through was also collected. Purified Osp Combine the flow-through containing A and store frozen.

The column was eluted with twice the volume of buffer C to remove bacterial proteins. Can be reused.

In addition, the pass-through from the DEAE-Sepharose column is transferred to the S-Sepharose column. Final purification was carried out by tography. First, DEAE-Sepharose Scalar The filtrate was made into I)H4,2 with 0.1M citric acid.

After washing the S-Sepharose column repeatedly with buffer C, the pH was adjusted to 4.0 with citric acid. It was set as 2.

Buffer C was used to elute highly purified OSOA. Eluate p1 1 was 5.7 because it was citric acid. Immediately pH 7.6 with 2M Tris base Adjusted to.

The highly purified 0 spA aqueous solution obtained in both chromatography steps was Contains highly purified 0soA, analyzed on a SEA-stained gel (Fig. 12A). I was sure that. The purity of the product obtained in the latter chromatographic step is endotoxic It was confirmed that the level of oxidation was extremely low.

Example 2 The procedure of Example 1 was repeated. However, Trit.

Surfactant applications were obtained using not only nX-114 but also other surfactants. profit The results are shown in Figure 9.

As is clear from Figure 9, it can be purified directly by column chromatography. Of the surfactants used, Trit was able to selectively solubilize 0spA. It was only onX-114.

Example 3 Plasmids pOA5 and DOA6 were prepared as described above and incubated with E. coli. Proteins were expressed using the same procedure as in Example 1 used to transform the DH5α strain. I set it. The temporal expression of OS pA lipoprotein by DOA5 is It was the same as that recognized in

On the other hand, the expression of 0spA by pOA6 is several times lower than that by pOA5. (Figures 7B and 7C). Purification of 0spA protein is expressed as pOA5. The experiment was performed only on the cells that had been subjected to this experiment.

In this way, the B310spA lipoprotein produced by the Trc expression system is , purified using the same procedure as in Example 1. However, the harvested cell pellets have 0. 1 Add mg/m + Norizozyme and incubate for 30 min at room temperature before freezing the cells. suspended for a while.

DEAR-Sepharose column AT& is highly purified Osp A lipoprotein was included (Fig. 9G).

Example 4 B. Cloning of the Asian strain of B. burgdorferi (Ip90) (ATCC) did. The soA gene was introduced into the plasmid DOAI (oET) using the same procedure as above. promoter) and pOA5 (TRC promoter), and Plasmids containing pOA8 (Trc promoter) and oOA10 (p ET promoter) was formed. Restriction digestion was performed on these plasmids. When tested, it was confirmed that all the expected parts were present.

In addition, we enclosed the spA gene of the Yottuba strain of E. burgdorferi and A plasmid (ET promoter) containing the gene was formed. these pluses When we performed restriction digestion on Mitsudo, we found that all the expected sites were present. Admitted.

Cultivation of pOA7 and pOA8 expressing strains using exactly the same procedure as for the DOAS strain (Example 3) and induction.

On the other hand, the cultivation and induction of DOA9 and pOA10 expressing strains were performed using pOA1 strain (implemented). The same procedure as in Example 1) was carried out.

The 0spA lipoproteins of ACA I and I p90 obtained by these operations are , the same as the 0soA lipoprotein of B51 (pOAl) described in Example 1. Purified by procedure. The solution passing through the DEAE-Sepharose column contains highly purified O It contained spA lipoprotein (Figures 12B and 12C).

Example 5 Full-length recombinant O purified from E. coli according to the procedure described in Example 1. The immunogenicity of spA to mice was determined by B, bu, which has a high content of OspA. rgdorferi fraction E (described in the above PCT No. WO90104411 (prepared as described above) and calf serum albumin. C3f(/ 10 μg of each vaccine without adjuvant was injected into He mice. Ta. Four weeks after the first injection, a booster injection of the same antigen as the first injection was given. I shot it.

The direct value was measured by the EL ISA method using purified recombinant 0spA lipoprotein. (Figure 14).

As is clear from the figure, both antigens produce very similar responses, weak but detectable. showed a potent primary serum IgG response, with a 100-fold increase in serum titer after boosting.

For both immunogens, serum 1gG titers were stable for at least 6 weeks. In either case , no significant serum 1 gM reaction was observed.

It shows that it has a sexual nature.

As is clear from the following table, the immunological reliability of M-replaced Osc+A lipoprotein is as follows: Sera from mice immunized with recombinant Ributono (Ri) were tested in vitro. burgdorferi [331 Does it completely inhibit the growth of spirochetes? are also demonstrated.

As is clear from Table 1, the inhibitory activity of serum was It is proportional to the dose and agrees well with the results of anti-0spA IgG assay by ELISA method. Ta. Contains no detectable anti-0spA antibodies\immunizes 0spA non-lipoproteins No effect on the growth of spirochetes was observed in the serum of mice treated with the virus. It was.

Live results obtained from mice immunized with 2.5 μg of 0spA lipoprotein in PBS. The growth inhibition titer was determined by adding 20 μg of total protein of B. burgdorferi to Freund's Mice immunized with complete adjuvant and then boosted with whole protein in PBS. The results were comparable to or better than those obtained in the previous study. Recombinant Os　A lipoprotein spirochetes have the ability to cause a serum reaction that inhibits the growth of spirochetes. is an important protein protein that is expressed in E.coli and during the subsequent purification process. This demonstrates that protective epitopes are conserved.

Example 6 Plasmid oOA2 was prepared in the same manner as the plasmid DOA+ described above. Ta However, in the PCR reaction, a truncated ospA residue lacking the lipoprotein signal peptide is used. In order to amplify the gene, the following was used along with the C-terminal primer PET-273C. Primer PET-18N (Sequence ID No. 11) having the sequence was used.

5’CAT CAT ATG GCT AAG CAA AAT GTT AG C3' The underlined part of PET-18N is the coding list of the OspA gene. Identical to nucleotides 51-67 of Rand. Using plasmid pOA2 Dan.

colif) BL21 (DE3) (pLyss) (p.

A2) and HMS174 (DE3) (DLYSS) (DOA2) strains were transformed. As described in Example 1, cut Os .

A protein was expressed. Cell lysis and surfactant extraction as in Example 1 were performed. However, it was revealed that non-lipoproteins were present in the aqueous phase as expected. .

Example 7 The effect of lipid binding to 0spA protein on immunogenicity was investigated. fruit 0soA or D formed as described in Example 1 and purified or purified as described in Example 1. Non-lipoprotein type Os DA prepared by unn et al. /He and BALB strain mice were immunized and boosted 3 weeks later.

Serum was collected 1 to 2 weeks after the booster administration, and purified 0soA lipoprotein was used as the antigen. It was tested using the EL ISA method.

Figures 15A and 15B show that 0spA lipoprotein administered in PBS is e (Fig. 15A) and BALB/c mouse (Fig. 15B). However, we show that 1gG induces a strong, dose-dependent secondary 1gG response. figure As is clear from 15C, protein: aluminum is added to aluminum hydroxide. When adsorbed and administered at a leek ratio of 1:5 (weight), the increase in immunogenicity was Even if it was recognized, it was rare.

A reaction was observed with 0spA lipoprotein, whereas a reaction was observed with non-lipoprotein. As shown in Figure 15B, anti-0s was detectable even at the highest dose of 2.5 μg/mouse. No induction of oA antibodies was observed. Arum-absorbing non-lipoproteins also induce an immune response. I don't think it will guide you.

The deduced amino acid sequences of 0spA lipoproteins and non-lipoproteins are They are the same except for the residue.

The terminal amino acid residue of lipoproteins is a modified cysteine; The amino acid terminal residues are methionine-alanine. Therefore, the immunogenic This strongly suggests that the difference is due to the presence of lipid moieties.

Example 8 Whether the difference in immunogenicity observed in Example 7 is due to an essential difference in antigenicity or Distinguish between two types of proteins and clarify whether they differ in their responses to them. In order to The origin was investigated.

As shown in Figure 16, serum from mice immunized with lipoproteins was Even when using 0spA as the test antigen, the ELISA method showed a positive signal. did. On the other hand, serum from mice immunized with non-lipoproteins did not react with either antigen. Didn't react. Therefore, there is no difference in the antigenicity of both 0spA, and Ributon/< Lipoproteins are well recognized by the immune system, whereas non-lipoproteins are not. .

Example 9 Differences in immunogenicity depending on the origin of the 0spA protein were investigated. I) OAl, respectively; Purified 0spA lipoprotein B31. derived from DOA9 and pOAIO. A Mice were immunized with CA1 and Ip90. 2.5 μg protein at week 0 The same dose was given as a booster at 3 weeks, and blood was collected from the mice at 4 weeks. Ta.

Purified OsoA lipoprotein B31. as an antigen. Use ACAl or l990 Then, the blood mTgG titer was measured at 4 weeks by ELISA method. The titer is goat anti-coagulant. It was measured using a mouse IgG secondary antibody.

The results obtained in these experiments are shown in Figure 15D. As is clear from this data Both strains of recombinant OspA tested were immunogenic in mice; Ronal serum showed partial cross-reactivity.

Disclosure summary To summarize the present disclosure, the present invention provides methods for selectively extracting Ributanum from a host. 1, then column chromatography of the surfactant solution. 1 lipoprotein gene, especially the 05DA gene of B. burgdorferi A novel, highly purified, immunologically effective recombinant protein , provide a simple manufacturing method. Improvements are possible within the scope of the invention.

References L Fikrig! ,, S, W, Barthold, F, S,)C antor, and R,A.

Flavall (1990), Protection of Mica A gainst the Lym@Disease Agent by engineering mmu nizing with Racombinant 0spA.

a discussion, 250 = 553-556 2.Simon, M.M., ,U.E., 5chaibla, M.D., Kramar. ,C.

Eck@rskorn, C, Mus@taanu, H-ni, Mullar - Harmalin) c, and R, Wallich (1991), Ra combination 0uter 5surface ProteinA fro m Borr@lia burgdorferi engineering nduces Antib odi@g3, Dunn, J, J,, B, N, IJd@, and A , G., Barbour (1990).

Out@r 5urfaca Protein A (OspA) from the Lyme Disaas@5pirochet@, Borralia burgdorfari: High Lav@IExpression an d Purification of 5 olubla Racombinan tForm of ospA-′・1:159-168゜ 4, Ba5slar, -W, G,, a, 5uhr, H, -+:r, Bu hring, C.P., Muller.

K,-H, Wiagmullar, G, Bac)car, and C,J ung (1985).

5pacifice Antibodies Elicitad by Anti gan Coval@ntly5, Biesart, L,, W, 5che uar, and Ba5slar, 'W, G. (1987).

Engineering of Mitogonia Bacterial L ipoprotein and aSynthetic Analogue w ith Mousa Lymphocytas, -! 21 Peng-m■, 162: 651-657゜6, Dares, K,, H,5ehild, X, H, Wi@smullar, G, Jung, and H, G+Rammansa e (1989) + engineering n vivo Priming of Virus-8 pecific Cytotoxic T Lymphoeyt@s with 5ynthatic Lipopaptide Vaccine, apeng J342 : 561-564゜7, Brandt, M, E,, B, S, R11ey , J.D., Radolf, and M.V.

Norgard (1990), Engineering mmunog@nic Int・gral MambranaProt@ins of Borrtlia burgdo rfari Are Lipoproteins.

'e' ad u't 5B=98:]-991°a, Fikrig, Σ ,, S.W., Barthold, F.S.) Cantor, and R.A.

Flavall (1992), Long-turn protection of m1ca fromLym@disaase by vaccinati on with O! ! pA, engineering nfact, and engineering mmun, 60: 77 3-F7゜9, Fi) crig, E,, S, W, Barthold, F, 5. Kantor, and R.A.

Flav@ll (1991), Prot@ction of mic@fr om Lym@10+ Johnson, M,, L, Noppa, A+G, B arbour, and S.

B@rgstram 1992. Hataroganicity of Ou t@r　MambranaProt@ins　in Hiko March 1burgdor fari: Comparison of store-op@rons of thr e@1solates of different gaographicor igins, EngfaCt, Engmmun, 60:1845-1853-Table 1. C3H in serum of mice immunized with 0spA lipoprotein and non-spA lipoprotein. /He　spA　O<8 Lipoprotein 0.1 32 0.5 256 2.5 1024 0spA 0.1 <8 Non-lipoprotein 0.5 <8 2.5 <8 BALB/c 0spA O' <8 Lipoprotein 0.1 4 0spA　O,1<8 Non-lipoprotein 0.5 <8 Array audience 1j 1 N-terminal region of B31, ACAI and Ip90 strains Kachinichimen and PΔ 1.5 Area coding strand 3 O') fnu'yLt+de Primer PET- IN[CO3] 14 Oligonucleotide Primer PET-273C1, 5 [CO3] 5 Coding of C-terminal region of m from ACAI 1.5 strand 7 Oligonucleotide Primer 0spN2 18 Oligonucleotide Primer BZI 1.59 Oligonucleotide Primer PK4 1,5 10 oligonucleotide primer PK3 511 oligonucleotide primer Rymer pET-18N-800 list (1) ^171Je issue l information (i)＾J1荊性 (A) Length 27 salt pigs (B) Type 1w (C) Strand:” (D) Form: Straight line (ii) Molecular type: DNA (genome) (xi)! Description of gri Sequence identification number IATATATTATG AAAAAAT ATT TATTGGG (2) itlVIM and ii+) No. 8 2 (7) tf￥I a(i) Sequence characteristics (A) Length, Z7 salt nose 1 (B) Type: W (C) Strand:” (D) Form: Straight line (ii) Molecular type: DNA (genome) (xi) Explanation of 1 ri of chuu 1 ka U hat 2 GAGGAATAAA ATTTCG CAAA AATTAAA (3) icV1m+) No. 8 3m (i) West Jl Tower (A) Length: 26 bases x CB) type, PJ (C) Strand ° CD) Form 2 Fart 1 Silence (ii) Molecular type: DNA (genome) (xi)! Explanation of 5: Distribution wI: lJ No. 3GCACATATGA AAAA ATATTT ATTGGG (4) Information on 4-frame III No. 4 (i) Sequence temporality (A) Length °26 base pairs (B) type sound (C) Double strand (D) Form straight line (ii) Molecular type: DNA (genome) (xi) Sequence description: 1713m1 seeding 4CGGGGATCCCTCCTTAT TTT AAAGCG (5) West J1 Surihagi 1 Bango 5 information (i) 翫 11 荊性 (A) Length゛31 base pairs (B) Type planting (C) Strand:” (D) Form straight line (i) Molecular type DNA (genome) (xi) Explanation of fragments Table 1w31 No. 5 GAGGAATAAA TAAAGTTTCG CAAAAAAATTCA A (6)! ! iw3! Information on issue number 6 (A) Length゛27 base pairs (B) Type: w (C) Strand:” (D) Form: straight line (ii) Molecular type: DNA (genome) (xi) Explanation of the array° 1st lap 4g No. 6GAGGGATAAA ATTTCGT AGA AATTCAA (7) iBIJ [t) No. 1 7 (7) 1 m (i) ^ Late 1 荊 sex (A) Length: 30 bases (B) Type: woodcutter (C) Strand” (D) Form: Straight line (ii) Molecular type: DNA (genome) (xi) Sequence description, sequence identification number 7 GGCCGCACAT ATGAAAAAAATATTTATTGG (8) Nishiki Evening angle J Anchor Shig1 No.8σ) Ya (A) Length: 28" Luna (B) type bond (C) Strand double (D) Form straight line (ii) Molecular type: DNA (genome) (xi) Array explanation:! ! #1m hat number 8CGGGGATCCCCTTATTTA TT TCAAAGCG (9) Distribution 4-way number 9 information (i) 911 lung (A) Length: 24 base pairs (B) Type: woodcutter (C) Strand:” (D) Form: Straight line (ii) Molecular type: DNA (genome) (xi) Explanation of Nishikori: Drunkenness 1 wetness U1 number 9CGGGGATCCCCTATTTTA AAG CATC (10) W dark 11iZIJ number IO information (i) Array temporality (A) Length = 25 base pairs G (B) type stirring (C) Strand:” CD) form゛straight line (ii) Molecular type: DNA (genome) (xi) Explanation of kansori, kanmantohahako No. 10CGGCCATGGA AAAATA TTTA TTGGG (11) Information on sequence identification number 11 (i) Sequence temporality (A) Length °27 Shio A Shioji (B) type; sound (C) Strand (D) Form, straight line (ii) Molecular type: DNA (genome) (xi) Array explanation: Kanso Sho Shiman No. 11CAGCATATGG ATAAGCA AAA TGTTAGC-N V CO emHI FIG, 2゜ T3 diagram γφ out 68mHI FIG, 6° OS, A Sojochosuzaku I agree a lot Funeral diagram 13 E. coli 0spA Borrelia FracVACCINE FIG. 14- SERLIM DIL SERUM DIL SERUM 0IL FIGj5C.

FIG, 17° Procedural amendment June 21, 1994

Claims (1)

[Claims] Claim 1. Encoding a full-length wild-type Borrelia ribon protein gene, highly recombinantly formed host organisms transformed with plasmids containing offspring. Purified and immunologically effective protein. Claim 2. Full-length wild type B. The signal encoded by the burgdorferiospA gene The protein according to claim 1. Claim 3. Claim 2, characterized in that said plasmid is plasmid pOA1. Proteins listed. Claim 4. Claim 2, characterized in that said plasmid is plasmid pOA5. Proteins listed. Claim 5. Claim 2, characterized in that said plasmid is plasmid pOA6. Proteins listed. Claim 6. Claim 2, characterized in that said plasmid is plasmid pOA7. Proteins listed. Claim 7. Claim 2, characterized in that said plasmid is plasmid pOA8. Proteins listed. Claim 8. Claim 2, characterized in that said plasmid is plasmid pOA9. Proteins listed. Billing 9. Claim 2, wherein the plasmid is plasmid pOA10. Proteins listed in. Claim 10. The ospA gene described above is derived from B. isolated from the B31 strain of S. burgdorferi The protein according to claim 2, which is a gene. Claim 11. The ospA gene described above is derived from B. isolated from the Ip90 line of S. burgdorferi. 3. The protein according to claim 2, wherein the protein is a gene derived from a protein. Claim 12. The ospA gene described above is derived from B. isolated from the ACA1 strain of P. burgdorferi. 3. The protein according to claim 2, wherein the protein is a gene derived from a protein. Claim 13. The host organism is E. The protein according to claim 1, which is E. coli. nine. Claim 14. The above plasmid consists of plasmid vector pET9, and the gene is T 7 promoter and effective translation initiation signals from bacteriophage T7 The protein according to claim 2, characterized in that it is under the control of. Claim 15. The host organism is E. Claim 14, characterized in that the strain is a T7 expressing strain of E.coli. Proteins listed in. Claim 16. The host organism is E. E.coli BL21(DE3)(pLysS) or HM Claim 15, characterized in that the strain is an S174 (DE3) (psysS) expressing strain. Proteins listed. Claim 17. If the plasmid is from the plasmid vector pCMB1 or pCMB2. and the gene is under the control of the Trc promoter. The protein according to claim 2. Claim 18. According to claim 1, it contains almost no bacterial protein and lipopolysaccharide. protein. Claim 19. Protein encoded by the full-length wild-type Borrelia ribon protein gene A method for producing a host organism transformed with a plasmid containing the above gene. Induce Borrelia Ributonpaku from objects, lysing the cells of said host organism; Selective lysis of Borrelia ribon proteins from bacteria or other proteins molten cells with a surfactant that allows phase separation of the surfactant phase under mild conditions. process, A surfactant phase containing dissolved Borrelia ribbon protein and bacteria and their Phase separation is performed into an aqueous phase containing other proteins and a solid phase containing cell debris, and the solid recovering said surfactant phase from said phase and said aqueous phase; removing proteins other than Borrelia lipokunpaku from the surfactant phase; A manufacturing method consisting of refining. Claim 20. Protein is B. encoded by the ospA gene of E. burgdorferi 20. The method according to claim 19, wherein the OspA protein is an OspA protein. Claim 21. Claim 20, characterized in that the surfactant is Triton X-114. The manufacturing method described in. Claim 22. Said treatment of said molten cells is carried out at a temperature of about 0° to 10°, and the resulting mixture is The surfactant phase is separated by gradual heating to a temperature of about 35° to 40° and separating said surfactant phase from said aqueous phase and solid phase by centrifugation; The manufacturing method according to claim 21, characterized in that: Claim 23. Adsorbs proteins other than Borrelia ribon protein, and The surfactant phase is chromatographed under conditions that allow the protein to be recovered from the flow-through. Said purification of said surfactant phase is carried out by contacting said surfactant phase with a graphic column. The manufacturing method according to claim 22, characterized in that: Claim 24. Further, the column is contacted with a buffer solution to remove Borrelia from the column. The ribon protein is eluted while the other proteins are retained on the column and 24. The manufacturing method according to claim 23, wherein the eluate is collected. Claim 25. The method is characterized in that the above-mentioned thawing of the host organism is carried out by freezing and thawing the host organism. The manufacturing method according to claim 19. Claim 26. The host organism is E. Claim 25, characterized in that it is a strain of E. coli. manufacturing method. Claim 27. The plasmids are plasmids pOA1, pOA5, pOA7, pOA8, 27. The production method according to claim 26, wherein the pOA9 or pOA10 is pOA9. Claim 28. comprising an immunologically effective amount of the protein according to claim 1, Vaccine effective against Heta infection. Claim 29. The protein comprises an immunologically effective amount of the protein according to claim 1, and is effective against Lyme disease infection. vaccine. Claim 30. comprising an immunologically effective amount of the protein according to claim 1, An immunodiagnostic reagent used to detect antibodies against infection of the host by Heta. Claim 31. comprising an immunologically effective amount of the protein according to claim 2, Immunodiagnostic reagent used to detect antibodies against infection. Claim 32. A host comprising administering to a host an immunologically effective amount of the protein according to claim 1. A method of immunizing a primary mammal against Borrelia spirochetes. Claim 33. The aforementioned Borrelia spirochete is the causative agent of Lyme disease, and the aforementioned protein 33. The method according to claim 32, wherein is the protein according to claim 2. Claim 34. The host mammal is a human, and the protein is added to an immunogenicity-enhancing adjuvant. 34. The method according to claim 33, comprising administering in the presence of.