Hybrid expression of neisseria proteins

FIELD: biotechnology.

SUBSTANCE: invention relates to method for heterological expression of Neisseria meningitidis protein in E. coli. Expressed protein has immunogenic activity in relation to Neisseria meningitidis.

EFFECT: immunogenic proteins of high effectiveness.

9 cl, 26 dwg, 1 tbl, 23 ex

 

The technical FIELD

This invention relates to the field of protein expression. In particular, it refers to the heterologous expression of proteins from Neisseria (e.g., N. gonorrhoeae, or preferably N. meningitidis).

BACKGROUND of the INVENTION

International patent application WO 99/24578, WO 99/36544, WO 99/57280 and WO 00/22430 describe proteins of Neisseria meningitidis and Neisseria gonorrhoeae. These proteins are usually described as expressed in E. coli (i.e. heterologous expression) in the form of either N-terminal GST-hybrids, or C-terminal His-tag-hybrids, although described other expression systems, including expression in native Neisseria.

The purpose of this invention is to provide alternative and improved approaches for heterologous expression of these proteins. These approaches usually affect the level of expression, ease of cleaning, cellular localization, expression and/or immunological properties of the expressed protein.

Description of the INVENTION

In accordance with this invention, two or more (for example, 3, 4, 5, 6 or more) proteins of this invention are expressed in the form of a single hybrid protein. It is preferable that was not used hybrid partner, not related to Neisseria (e.g., GST or poly-His).

This gives two advantages. First, a protein that may be unstable or poorly expressed separately, maybe in order to overcome these disadvantages by adding a suitable hybrid partner, which overcomes this problem. Secondly, simplified commercial receipt - you use only one expression and purification to obtain two separately applicable proteins.

Thus, this invention provides a method of simultaneous heterologous expression of two or more proteins of the present invention, which hybridize the two or more proteins of the present invention (i.e., they are translated as a single polypeptide chain).

The method usually involves the following stages: obtaining a first nucleic acid that encodes a first protein of the present invention; obtaining a second nucleic acid that encodes a second protein of the present invention; ligating the first and second nucleic acids. The obtained nucleic acid may be embedded in expressing vector or may already be part of an expressing vector.

When connecting only two proteins hybrid protein can be represented simply by the formula NH2-A-B-COOH. A and b each may be selected from any of the proteins of Neisseria and, in particular, proteins represented by SEQ ID NO: 1-4326. This method is convenient for the expression of protein orf1, orf4, orf25, orf40, orf46/46.1, orf83, 233, 287, 292L, 564, 687, 741, 907, 919, 953, 961 and 983.

Preferred are 42 hybrid specified "X" in the following table, the formula NH2-A-B-COOH:

↓→ORF46.1287741919953961983
ORF46.1XXXXXX
287XXXXXX
741XXXXXX
919XXXXXX
953XXXXXX
961XXXXXX
983XXXXXX

Thus, the preferred proteins for expression as hybrids are ORF46.1, 287, 741, 919, 953, 961 and 983. They can be used in their essentially full form or can be used to form polyglycerol deletion (Δ (G) (for example, 4 G-287, ΔGTbp2, ΔG741, ΔG983 etc) or can be used shortened forms (for example, Δ1-287, Δ2-287 etc) or a version with deleteregvalue domains (for example, W, S, WS, ORF461-433, ORF433-608, ORF46, s etc) and so on

Especially preferred are: (a) a hybrid protein containing 919 287; (b) a hybrid protein containing 953 287; (C) a hybrid protein containing 287 and ORF46.1; (d) a hybrid protein containing ORF1 and ORF46.1; (e) a hybrid protein containing 919 and ORF46.1; (f) a hybrid protein containing ORF46.1 and 919; (g) a hybrid protein containing ORF46.1, 287 and 919; (h) a hybrid protein containing 919 and 519; and (i) a hybrid protein containing ORF97 and 225.

The following options are shown on the drawings and include ΔG287-919, ΔG287-953, ΔG287-961, ΔG983-ORF46.1, ΔG983-741, ΔG983-961, ΔG983-961C, ΔG741-961, ΔG741-961C, ΔG741-983, ΔG741-ORF46.1, ORF46.1-741, ORF46.1-961, ORF46.1-S, 961-ORF46.1, 961-741, 961-983, 961C-ORF46.1, S-741, S-983, 961CL-ORF46.1, 961CL-741 and 961CL-983.

When applying 287 it is preferably located at the C-terminal side of the hybrid; if it should be used at the N end, it is preferable to use the form ΔG287 (for example, in the form of N-end hybrid with ORF46.1, 919, 953 or 961).

When applying 287 it is preferably from strain 2996 or from strain 394/98.

When applying 961 it is preferably at N-end. Can be used to form domains 961.

Mapping polymorphism ORF46, 287, and 953 919 described in WO 00/66741. Any of these polymorphic forms can be used in accordance with this invention.

Preferably the components of proteins (a and b) in a hybrid protein in accordance with this invention originate from the same strain.

Hybrid proteins in hybrid can be connected directly or can be linked via a linker peptide, for example, through polyglycine linker (i.e. Gnwhere n=3, 4, 5, 6, 7, 8, 9, 10 or more) or via a short peptide sequence that facilitates cloning. Obviously, it is preferable not to join ΔG-protein to the C-end polyglycine linker.

Hybrid proteins can be deprived of its leader peptide or may include the sequence of the leader peptide N-terminal hybrid partner.

The owner

Preferably the heterologous host. Heterologous host may be prokaryotic or eukaryotic. Preferably it is E. coli, but other suitable hosts include Bacillus subtilis. Vibrio cholerae. Salmonella typhi, Salmonella typhimurium, Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea, Mycobacteria (e.g., M. tuberculosis), yeast, etc.

Vectors, hosts, etc.

In addition to the above-described methods, the invention provides (a) a nucleic acid and vectors applicable in these ways; (b) cells chose the VA, containing these vectors; (C) proteins are expressed or can be expressed in these ways; (d) compositions containing these proteins that may be suitable, for example, as vaccines or as diagnostic reagents or as immunogenic compositions; (e) the composition for use as a drug (such as vaccines) or as diagnostic reagents; (f) the use of these compositions in the preparation of (1) a medicinal product for the treatment or prophylaxis of infections caused by bacteria Neisseria; (2) a diagnostic reagent for detecting the presence of bacteria Neisseria or antibodies induced against bacteria Neisseria, and/or (3) reagent, which can induce antibodies against the bacteria Neisseria; and (g) a method of treating a patient comprising the administration to a patient a therapeutically effective amount of songs.

Sequence

This invention also provides a protein or nucleic acid having any of the sequences disclosed in the following examples. It also provides proteins and nucleic acids having sequence identity to these sequences. The degree of sequence identity" is preferably higher than 50% (e.g., 60%, 70%, 80%, 90%, 95%, 99 or more).

The nomenclature used here

2166 sequences of proteins described in WO 99/24578, WO 99/36544 and WO 99/57280, called here the following SEQ ID NO:-rooms:

ApplicationSequences of proteinsSEQ ID NO: here
WO 99/24578The even-numbered SEQ ID 2-892SEQ ID NO:1-446
WO 99/36544The even-numbered SEQ ID 2-90SEQ ID NO:447-491
WO 99/57280The even-numbered SEQ ID 2-3020SEQ ID NO:492-2001
The even-numbered SEQ ID 3040-3114SEQ ID NO:2002-2039
SEQ ID 3115-3241SEQ ID NO:2040-2166

In addition to this SEQ ID NO: - numbering, we use the standard names used in WO 99/24578, WO 99/36544 and WO 99/57280 (for example, 'ORF4', 'ORF40', 'ORF40-1', etc. that are used in WO 99/24578 and WO 99/36544; 'm919', 'g919' and 'a', etc. that are used in WO 99/57280).

2160 proteins NMB0001-NMB2160 of Tettelin et al. [Science (2000) 287:1809-1815] are referred to here as SEQ ID NO:2167-4326 [see also WO 00/66791].

The term "protein of this invention" as applied here refers to a protein containing

(a) one of the sequences SEQ ID NO:1-4326; or

(b) a sequence having sequence identity relative to one of SEQ ID NO:1-4326;

or

(c) a fragment of one of SEQ ID NO: 1-4326.

The degree of sequence identity is, referred to in (b)is preferably higher than 50% (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more). This includes mutants and allelic variants (for example, see WO 00/66741). The identity is preferably determined by the search algorithm homology Smith-Waterman available in the MPSRCH program (Oxford Molecular), using search affine gap with the parameters of the fine opening gap=12 and fine extension gap=1. Usually 50%or higher identity between the two proteins is considered an indication of functional equivalence.

"Fragment", as referred to in (C), must contain at least n consecutive amino acids of one of SEQ ID NO:1-4326 and, depending on the particular sequence, n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more). A preferred fragment contains the epitope of one of SEQ ID NO:1-4326. Preferred fragments are those fragments described in WO 00/71574 and WO 01/04316.

Preferred proteins of the present invention found in the serological group b N. meningitidis.

Preferred proteins for use in accordance with this invention are proteins of strain or strain 2996 394/98 (new Zealand strain) N. meningitidis sero-group C. If not otherwise mentioned herein proteins are proteins from strain 2996 N. meningitidis. However, it should be clear that this izobreteny is in General not limited to the strain. References to specific protein (e.g., "287", "919", etc. can be aware of this protein from any strain.

It should be clear that the reference to "a nucleic acid" includes DNA and RNA, as well as their analogues, such as containing modified skeletons, and peptidoglicanova acid (NCP), etc.

BRIEF DESCRIPTION of DRAWINGS

Figure 1-26 shows the hybrid proteins of the present invention.

WAYS of carrying out the INVENTION

Example 1 hybrids ORF46

Complete protein ORF46 from N. meningitidis (serological group b strain 2996) has the following sequence:

Leader peptide is underlined.

Sequence ORF46 from other strains can be found in WO 00/66741.

ORF46 hybridized at its C-end and N-end 287, 919 and ORF1. These hybrid proteins were generally insoluble, but gave some good results ELISA and bactericidal results (against the homologous strain 2996):

ProteinELISABactericidal Ab
Qrf1-Orf46.1-His850256
919-Orf46.1-His12900512
919-287-Orf46-Hisnot ODA.not ODA.
Orf46.1-287His1508192
Orf46.1-919His 28002048
Orf46.1-287-919His320016384

For comparison, designed "triple" hybrids ORF46.1, 287 (either in the form of GST-hybrid or in the form of ΔG287) and 919 and tested them against different strains (including the homologous strain 2996) compared with a simple mixture of these three antigens. FCA (full beta-blockers) was used as adjuvant:

2996BZ232MSNGH38F6124BZ133
Mixture81922565121024>2048>2048
ORF46.1-287-919his163842564096819281928192
ΔG287-919-ORF46.1his81926440968192819216384
ΔG287-ORF46.1-919his409612825681925121024

And in this case, these hybrids find equivalent or superior immunological activity.

Hybrids of the two proteins (strain 2996) was compared with individual proteins against heterologous strains

1000MSF6124 (MenA)
ORF46.1.His<44096<4
ORF1-His8256128
ORF1-ORF46.1-His10245121024

Again, hybrids find equivalent or superior immunological activity.

Example 2 hybrids ΔG287

It was found that the deletion of (Gly)6sequences 287 has a strong effect on the expression of the protein. Protein lacking N-terminal amino acids up to GGGGGG, called "ΔG287". In the strain MS its main sequence (leader peptide is underlined) below:

ΔG287, with His-tagged or without His-tag (″ΔG287-His" and "ΔG287K", respectively), is expressed at very good levels in comparison with "287-His" or "287its".

On the basis of data on the variability of genes, variants ΔG287-His expressed in E. coli from a number of MenB strains, in particular from strain 2996, MS, 1000 and BZ232. The results were also good: each of them gave high titers by ELISA, and serum bactericidal titers >8192. ΔSC expressed from the plasmid pet-24b, gave excellent titles in the Largest serum bactericidal assay.

Deletion poly-Cly-sequences are also applicable to Tbp2 (NMB0460), 741 (NMB 1870) and 983 (NMB1969). When cloning into the vector pet and expression in E. coli without sequence that encodes their leader peptides and without poly-Gly (i.e. in the form "ΔG-forms"), observed the same action - the expression was good in clones carrying a deletion polyglycidol cut, and weak or absent, if glycine was present in the expressed protein.

ΔG287 hybridized directly into the frame of the reading progress against transcription (left) 919, 953, 961 (sequences shown below) and ORF46.1:

ELISABactericidal test
ΔG287-953-His383465536
ΔG287-961-ffis10862765536

Bactericidal efficiency (homologous strain) antibodies induced against hybrid proteins, compared with antibodies induced against simple mixtures of components antigens (using 287-GST) to 919 and ORF46.1:

The mixture 287Hybrid ΔG287
91932000 128000
ORF46.112816000

Received data bactericidal activity against heterologous strains of MenB and against serotypes a and C:

919ORF46.1
StrainMixtureHybridMixtureHybrid
NGH38102432000-16384
MS5128192-512
BZ232512512--
MenA (F6124)51232000-8192
MenC (C11)>2048>2048--
MenC (BZ133)>409664000-8192

Thus, the hybrid proteins with ΔG287 on the N-end outperform immunological simple mixture, and ΔS-ORF46.1 is particularly effective, even against heterologous strains. ΔG287-ORF46.1 can be expressed in the pet-24b.

Such hybrid proteins were obtained using the new Zealand strain 394/98, not 2996:

Example 5 hybrids 287

Expression of 287 as full-length protein with C-terminal His-tag or without leader peptide, but with C-terminal His-tag gives a fairly low levels of expression. The best expression is achieved using N-terminal GST-hybrid. As an alternative to using GST as N-terminal hybrid partner, 287 were placed on the C-end of the protein 919 ("919-287"), protein 953 ("953-287") and proteins ORF46.1 ("ORF46.1-287"). In both cases, the leader peptides were delegated and hybrids were direct hybrids in reading frame.

To generate hybrid 953-287 leader peptides of these two proteins were removed by constructing a direct primer during transcription (right) from the leader of each sequence; the sequence of the stop codon was removed in the reverse primer 953, but included in the reverse primer 287. For gene 953 5' and 3' primers used for amplification included the restriction sites Ndel and BamHI, respectively, then to the to for amplification of gene 287 5' and 3' primers included the restriction sites BamHI and XhoI, respectively. This way could be achieved consistent directional cloning of these two genes in 21b+using NdeI-BamHI (for cloning of the first gene) and then BamHI-XhoI (for cloning of the second gene).

Hybrid 919-287 was obtained by cloning of the sequence encoding the Mature part 287 in the XhoI site at the 3'-end 919-His-clone in 21b+. The primers used for amplification of gene 287, designed by the introduction of a Sail restriction site at the 5'-and XhoI site at the 3'end of the PCR fragment. Because the sticky ends produced by restrictase SalI and XhoI, are compatible PCR product 287, split SalI-XhoI, could be built into the clone 21b-919, split XhoI.

Hybrid ORF46.1-287 was obtained in a similar manner.

Bactericidal activity against the homologous strain) antibodies induced against hybrid proteins, compared with antibodies induced against simple mixtures of components antigens:

The mixture 287Hybrid 287
9193200016000
95381928192
ORF46.11288192

Received data bactericidal activity against heterologous strains of MenB and against serotypes a and C for 919-287 and 953-287:/p>

919953ORF46.1
StrainMixtureHybridMixtureHybridMixtureHybrid
MS51210245121024-1024
NGH381024204820484096-4096
BZ232512128102416--
MenA (F6124)5122048204832-1024
MenC (C11)>2048the".>2048not ODA.-not ODA.
MenC (BZ133)>4096>8192>4096<16-2048

Designed hybrids ORF46.1 and 919. The best results (four times higher titers) were achieved with 919 N-end.

Experienced hybrids 919-519His, ORF97-225His and 225-ORF97His. They gave moderate titers of ELISA and moderate reactions in the form of bactericidal antibodies

As hybrids of the two proteins a and b can be either NH2-A-B-COOH or NH2-In-A-COOH was also "converted" hybrids with 287 at N-end, but using ΔG287. Used a panel of strains, including homologous strain 2996. FCA was used as adjuvant:

287 and 919287 and 953287 and ORF46.1
StrainΔS-919919-287ΔG287-953953-287ΔG287-46.146.1-287
299612800016000655368192163848192
BZ232256128128<4<4<4
10002048<4<4<4<4<4
MS81921024163841024512128
NGH38320002048>20484096163844096
394/98409632256128128 16
MenA (F6124)320002048>20483281921024
MenC (BZ133)64000>8192>8192<1681922048

The best bactericidal titers usually observed with 287 at N-end.

When hybridization with protein 961 (NH2-ΔG287-961-COOH sequence shown above) obtained protein is insoluble and must denaturirate and reatuarants for cleaning. It was found that after renaturation about 50% of this protein remained insoluble. Soluble and insoluble proteins were compared, and a much better bactericidal titers were obtained with soluble protein (using FCA adjuvant):

2996BZ232MSNGH38F6124BZ133
Instant655361284096>2048>20484096
Insoluble8192<4<416not ODA.not ODA.

However, the titles with an insoluble form was improved with the use of alum as the e adjuvant:

Insoluble327681284096>2048>20482048

S also used in hybrid proteins (see above). Because 961 and its "domain" options directs efficient expression, they are ideally suitable as N-terminal part of the hybrid protein.

Example 23 - other hybrids

Other hybrid proteins of the present invention shown in the drawings and are presented below. They have advantages in comparison with individual proteins:

It should be clear that the invention was described only as examples, and can be produced modifications without deviating from the idea and scope of this invention. For example, assuming proteins from other strains (for example, see WO 00/66741 against polymorphic sequences for ORF4, ORF40, ORF46, 225, 235, 287, 519, 726, 919 and 953).

EXPERIMENTAL DETAILS

Strategist who I cloning and construction of oligonucleotides

Genes encoding interest antigens, amplified using PCR using oligonucleotides designed based on the genomic sequence of N. meningitidis IN MS. Genomic DNA from strain 2996 always used as template in PCR reactions, unless otherwise noted, and amplificatoare fragments cloned in expressing vector 21b+ (Novagen) for expression of the protein in the form of His-tagged at the C-end product or in pET-24b+ (Novagen) for expression "its" form (for example, ΔG287K).

When the protein is expressed without hybrid partner and with its own leader peptide (if present)was performed to amplify open reading frame (ATG codons STOP-codon).

When the protein is expressed in its" form, the leader peptide was removed by the construction of the 5'-end primer amplification to the right of the predicted leader sequence.

The melting temperature of the primers used in PCR, depended on the number and type of hybridization of nucleotides in the primer, and it was determined using the formula:

TPL=4(G+C)+2(A+T)(tail excluded)
TPT2=64,9+of 0.41(%GC)-600/N(full primer)

The melting temperature of the selected oligonucleotides was is usually 65-70° For full of the oligonucleotide and 50-60°only for hybridization of the site.

Oligonucleotides were synthesized using a DNA/RNA synthesizer Perkin Elmer 394, suirable from the speakers in 2.0 ml of NH40H and freed from the protective groups 5-hour incubation at 56°C. the Oligonucleotides were precipitated with addition of 0.3 M Na-acetate and 2 volumes of ethanol. Samples were centrifuged and precipitation resuspendable in the water.

Direct primers used in combination with Reverse primer 287-His.

NB - All PCR reactions used the strain 2996, if there are no other indications (for example, strain MS).

In all constructions, starting with ATG, not accompanied by a unique NheI site, codon ATG is part of the NdeI site used for cloning. Designs made with NheI as cloning site at the 5'-end (for example, all of the constructs containing 287 N-end), have two additional codon (GCT AGC), hybridisable with the coding sequence of the antigen.

Obtaining matrices chromosomal DNA

Strains of N. meningitidis 2996, MS, 394.98, 1000 and BZ232 (and others) were grown to exponential phase in 100 ml of GC-medium, collected by centrifugation and resuspendable in 5 ml buffer (20% weight/volume sucrose, 50 mm Tris-HCl, 50 mm EDTA, pH 8). the donkey 10 minutes incubation on ice, the bacteria were literally by adding 10 ml lisanova solution (50 mm NaCI, 1% Na-sarkosyl, 50 µg/ml Proteinase K) and the suspension was incubated at 37°C for 2 hours. Had two extraction with phenol (balanced to pH 8) and extracted with a mixture of CHCl3/isoamyl alcohol (24:1). DNA precipitated with addition of 0.3 M sodium acetate and 2 volumes of ethanol and collected by centrifugation. The precipitate was washed once with 70% (vol./about.) ethanol and pererestorani 4.0 ml TE-buffer (10 mm Tris-HCl, 1 mm EDTA, pH 8.0). The DNA concentration was measured by OD reading260.

PCR amplification

The standard PCR Protocol was as follows: 200 ng of genomic DNA from strain 2996, MS, 1000 or BZ232 or 10 ng of the preparation of plasmid DNA from recombinant clones was used as matrix in the presence of 40 μm of each oligonucleotide primer, 400-800 μm dNTP solution, 1 x PCR buffer (containing 1.5 mm MgCl2), 2.5 units of DNA polymerase TaqI (using a Perkin Elmer AmpliTaQ, Boehringer Mannheim Expand™ Long Template).

After a preliminary 3-minute incubation of the whole mixture at 95°C, each sample was subjected to two-stage amplification: the first 5 cycles were performed using the hybridization temperature, which excluded the tail restrictase primer (TPL). Followed by 30 cycles in accordance with the temperature of hybridization, designed for full-length oligonucleotides (TPT2). Elongation time performed at 68°72°With, in what was removals in accordance with the length of the Orf, subject to amplification. In the case of Orf1 elongation time, starting with 3 minutes, increased to 15 seconds with each cycle. Cycles ended a 10-minute stage of elongation at 72°C.

Amplified DNA was applied directly on a 1% agarose gel. The DNA fragment corresponding to the band of the correct size were purified from the gel using a kit for the extraction of gels Qiagen Gel Extraction Kit according to the manufacturer's Protocol.

Cleavage of the PCR fragments and cloning vectors

Purified DNA corresponding to amplificatoare fragment was digested with suitable restrictable for cloning into pet-21b+, 22b+ or pet-24b+. Splintered fragments were purified using the cleaning kit QIAquick PCR purification kit according to the manufacturer's instructions) and suirable or H2O, or 10 mm Tricom, pH 8.5. Plasmid vectors were digested with appropriate restrictase, inflicted by 1.0% agarose gel and the band corresponding to the cleaved vector was purified using a kit for the extraction of gels Qiagen QIAquick Gel Extraction Kit.

Cloning

The fragments corresponding to each gene, previously cleaved and purified, ligated in 21b+, 22b+ or pet-24b+. Used a molar ratio of 3:1 fragment/vector with DNA ligase T4 buffer for ligation, supplied by the manufacturer.

Recombinant plasmid transformation which was miraval in competent cells of E. coli DH5 or NW incubation solution ligase reaction and bacteria for 40 minutes on ice, then at 37°C for 3 minutes. Followed by adding 800 ál of LB-broth and incubation at 37°C for 20 minutes. The cells were centrifuged at maximum speed in microfuge Eppendorf, resuspendable in approximately 200 μl of the supernatant and were sown on containing LB-ampicillin (100 mg/ml) agar.

Screening for recombinant clones was performed by growing randomly selected colonies overnight at 37°With 4.0 ml of a mixture of LB-broth + 100 μg/ml ampicillin. Cells were besieged and plasmid DNA was extracted using a kit Qiagen QIAprep Spin Miniprep Kit according to the manufacturer's instructions. Approximately 1 µg of each individual minipreparation were digested with appropriate restrictase and the cleavage product was applied at 1-1 .5% agarose gel (depending on the expected size of the insert) along with molecular weight marker (1 TPN DNA ladder, GIBCO). Positive clones were selected on the basis of the size of the insert.

The expression

After cloning each gene in expressing vector recombinant plasmids were transformed into strains of E. coli, suitable for expression of the recombinant protein. 1 ál of each construct was used to transform E. coli BL21-DE3, as described above. Individual recombinant Colo the AI was inoculable in 2 ml of LB + Amp (100 μg/ml), incubated at 37°C overnight, then diluted 1:30 in 20 ml of LB + Amp (100 μg/ml) in flasks of 100 ml with obtaining OD600between 0.1 and 0.2. Flasks were incubated at 30°or at 37°a rotary shaker water bath up until OD600not indicated exponential growth, suitable for the induction of expression (0,4-0,8 OD). Expression of protein was induced by addition of 1.0 mm IPTG (isopropylthioxanthone). After 3 hours incubation at 30°or at 37°measured OD600and explored the expression. 1.0 ml of each sample was centrifuged in microfuge, sediment resuspendable in SFR and analyzed by electrophoresis in LTOs/SDS page and staining Kumasi blue.

Purification of His-tagged proteins

Different forms 287 cloned from strain 2996 and MS. They were designed with His-tagged hybrid-end and they included the Mature form (amino acids 18-427), constructs with deletions (Δ1, Δ2, Δ3 and Δ4) and clones, consisting of either In-or out-domains. For each clone, purified as His-hybrid, a single colony was planted by the bar and were grown overnight at 37°With the Cup with agar LB/Amp (100 μg/ml). An isolated colony from this Cup was inoculable in 20 ml of liquid LB/Amp (100 μg/ml) and grown overnight at 37°when shaken. Night culture was diluted 1:30 in 1.0 l of a liquid medium LB-Amp (100 μg/ml) and expressed ivali at the optimum temperature (30 or 37° (C)until OD550he reached 0.6 to 0.8. The expression of recombinant protein was induced by adding IPTG (final concentration 1.0 mm) and the culture incubated for another 3 hours. Bacteria were collected by centrifugation at 8000 g for 15 minutes at 4°C. the Precipitate bacteria resuspendable 7.5 ml (i) of cold buffer A (300 mm NaCl, 50 mm phosphate buffer, 10 mm imidazole, pH 8.0) for soluble proteins, or (ii) buffer (10 mm Tris-HCl, 100 mm phosphate buffer, pH 8.8, and, optionally, 8 M urea) for insoluble proteins. Proteins purified in soluble form, included 287-His, Δ1, Δ2, Δ3 and Δ4287-His, Δ4287MC58-His, 287c-His and 287cMC58-His. Protein 287bMC58-His was insoluble and was purified accordingly. Cells were destroyed by sonication on ice four times for 30 seconds at 40 W using an ultrasonic disintegrator 450 Branson and was centrifuged at HD for 30 minutes at 4°C. For insoluble protein precipitation resuspendable in 2.0 ml of buffer (6 M guanidine hydrochloride, 100 mm phosphate buffer, 10 mm Tris-HCl, pH 7.5) and treated with 10 activations homogenizer of downs. The homogenate was centrifuged at 13000 g for 30 minutes and the supernatant retained. Supernatant as soluble or insoluble drugs were mixed with 150 ál Ni2+-resin (pre-equilibrated with buffer a or buffer B, the AK is required), and incubated at room temperature with careful stirring for 30 minutes. The chelating resin was Sepharose Fast Flow (Pharmacia), obtained in accordance with the Protocol of the manufacturer. Received periodically, the preparation was centrifuged at 700 g for 5 minutes at 4°C and supernatant was discarded. The resin was washed twice (if periodic) 10 ml buffer a or b for 10 minutes, resuspendable in 1.0 ml of buffer a or b and put on a disposable column. The resin continued to wash either (i) buffer And at 4°or (ii) a buffer at room temperature until OD280flowing solution is not reached 0,02-0,01. The resin was further washed either (i) cold buffer (300 mm NaCl, 50 mm phosphate buffer, 20 mm imidazole, pH 8.0), or (ii) buffer D (10 mm Tris-HCl, 100 mm phosphate buffer, pH 6.3, and, optionally, 8 M urea), while OD280flowing through the column of solution is not reached 0,02-0,01. His hybrid protein was suirable by adding 700 μl of either (i) cold buffer for elution And (300 mm NaCI, 50 mm phosphate buffer, 250 mm imidazole, pH 8.0), or (ii) buffer for elution (10 mm Tris-HCl, 100 mm phosphate buffer, pH 4.5, and, optionally, 8 M urea) and fractions were collected until OD280did not show that received the recombinant protein. Aliquots of 20 μl of each elution fractions were analyzed by electrophoresis in LTO-PAG. The concentration of protein was determined by the method of Bradford.

Resaturate denaturirovannykh His-g is brednich proteins

Denaturation was required to solubilize 287b8, so before immunization was used stage renaturation. To denatured fractions obtained as described above was added glycerin to obtain a final concentration of 10% (vol./vol.). Proteins were diluted to 200 μg/ml using dialysis buffer I (10% vol./about. glycerol, 0.5 M arginine, 50 mm phosphate buffer, 5.0 mm restored glutathione, 0.5 mm oxidized glutathione, 2.0 M urea, pH 8.8) and were dialyzed against the same buffer for 12-14 hours at 4°C. Additional dialysis was performed with buffer II (10% vol./about. glycerol, 0.5 M arginine, 50 mm phosphate buffer, 5.0 mm restored glutathione, 0.5 mm oxidized glutathione, pH 8.8) for 12-14 hours at 4°C. the protein Concentration was determined using the formula:

Protein (mg/ml)=(1,55×OD280)-(0,76 ×OD260)

Immunization

BALB/c mice were immunized with antigen on day 0, 21 and 35 and the serum was analyzed at day 49.

Analysis of sera by ELISA

Devoid of capsules MenB M7 and encapsulated strains were sown in a Cup with chocolate agar and incubated overnight at 37°With 5% CO2. Bacterial colonies were collected from cups with the agar using a sterile swab (dracon swab) and inoculable in broth Mueller-Hinton (Difco)containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by definition OD620. BA the bacteria were grown, while OD620reached a value of 0.4-0.5. The culture was centrifuged for 10 minutes at 4000 rpm, the Supernatant was discarded and the bacteria were washed twice SFR, resuspendable in SFR containing 0.025% formaldehyde, and incubated for 1 hour at 37°and then overnight at 4°under stirring. 100 µl of the bacterial cells was added to each well of 96-hole tablet Greiner and incubated over night at 4°C. Then the wells were washed three times SFR-buffer washes (0.1% tween-20 in SFR). 200 ál of buffer saturation (2,7% polyvinylpyrrolidone 10 in water) was added to each well and the plates were incubated for 2 hours at 37°C. the Wells were washed three times SFR. 200 ál of diluted sera (the dilution buffer: 1% BSA, 0.1% tween-20, 0.1% of NaN3in SFR) was added to each well and the plates were incubated for 2 hours at 37°C. the Wells were washed three times SFR. 100 μl of HRP-conjugated rabbit antimachine serum (Dako)diluted 1:2000 in buffer for dilution was added to each well and the plates were incubated for 90 minutes at 37°C. the Wells were washed three times SFR-buffer. 100 μl of substrate buffer for HRP (25 ml citrate buffer pH 5, 10 mg of O-phenyldiamine and 10 μl of N2About2) was added to each well and the tablets were left at room temperature for 20 minutes. 100 μl of 12.5% H2SO4was added to the introduced each well was determined by OD 490. The ELISA titers were calculated in relative units in the form of dilution of serum that gave an OD value4900,4 above pridumannyh sera. The ELISA was considered positive when the dilution of sera with OD4900,4 had more than 1:400.

Analysis of sera FACS-Scan analysis of the binding of bacteria

Devoid of capsules MenB strain M7 were sown in a Cup with chocolate agar and incubated overnight at 37°With 5% CO2. Bacterial colonies were collected from cups with the agar using a sterile swab (dracon swab) and inoculable 4 tubes containing, each, 8 ml of broth Mueller-Hinton (Difco)containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by definition OD620. Bacteria were grown until OD620reached a value of 0.35 to 0.5. The culture was centrifuged for 10 minutes at 4000 rpm, the Supernatant was discarded and the precipitate resuspendable in blocking buffer (1% BSA in STR, 0,4% NaN3) and centrifuged for 5 minutes at 4000 rpm Cells resuspendable in blocking buffer with obtaining OD6200,05. 100 µl of the bacterial cells was added to each well of 96-hole tablet Costar. 100 μl of the diluted(1:100, 1:200, 1:400) serum (blocking buffer) was added to each well and the plates were incubated for 2 hours at 4°C. the Cells were centrifuged for 5 minutes at 4000 rpm,the supernatant was aspirated and cells were washed by adding 200 μl per well of blocking buffer to each well. To each well was added 100 μl anywhereand with R-phycoerythrin goat F(ab)2against mouse antibodies diluted 1:100, and the plates were incubated for 1 hour at 4°C. the Cells were unscrewed by centrifugation at 4000 rpm for 5 minutes and washed by adding 200 μl per well of blocking buffer. The supernatant was aspirated and the cells resuspendable 200 µl per well SFR, 0.25% formaldehyde. The sample was transferred into a test tube FACScan and read performance. Conditions for installation FACScan (laser power of 15 mW) were as follows: FL2 incl.; the threshold FSC-H: 92; voltage FSC RMT: E 01; SSC RMT: 474; a gain of 6.1; FL-2 RMT: 586; the magnitude of compensation: 0.

Analysis of sera - bactericidal analysis

The N. meningitidis strain 2996 were grown over night at 37°on cups with chocolate agar (source material was taken from the frozen initial solution) with 5% CO2. Bacterial colonies were collected and used for inoculation of 7 ml of broth Mueller-Hinton containing 0.25% glucose, to obtain OD620of 0.05-0.08. The cultures were incubated for approximately 1.5 hours at 37°With shaking until the OD value620he reached 0,23-0,24. Bacteria were diluted in 50 mm phosphate buffer, pH of 7.2, containing 10 mm MgCl2, 10 mm CaCl2and 0.5% (weight/volume) BSA (buffer for analysis) at a working dilution 105CFU (colony forming units) per ml of Obioban reaction mixture was 50 μl with 25 μl of serial 2-fold dilution of the test serum, 12,5 µl of bacteria at a working dilution, and 12.5 μl of the complement of a baby rabbit (final concentration 25%).

Controls included bacteria, incubated with serum complement, immune serum, incubated with bacteria and complement inactivated by heating at 56°C for 30 minutes. Immediately after adding the complement of baby rabbit 10 µl controls were sown on plates with agar Mueller-Hinton method with slope (time 0). 96-well plate were incubated for 1 hour at 37°C with rotation. 7 μl of each sample were sown on plates with agar Mueller-Hinton in the form of spots, while 10 μl of controls were sown on plates with agar Mueller-Hinton method with slope (1). Cups with agar were incubated for 18 hours at 37°and believed the colonies corresponding to the time 0 and time 1.

Analysis of sera in Western blots

Purified proteins (500 ng per track), bubbles (vesicles) outer membranes (5 μg) and total cell extracts (25 μg)derived from MenB strain 2996, were applied to 12% of the LTO-polyacrylamide gel and transferred to nitrofurazone membrane. The transfer was performed for 2 hours at 150 mA at 4°using buffer for transfer (0,3% Tris-base, 1.44% glycine, 20% (vol./about.) methanol). The membrane was saturated by overnight incubation at 4°buffer for saturation (10% obese the counter milk, 0.1% Triton X-100 in SFR). The membrane was washed twice with wash buffer (3% skimmed milk, 0/1% Triton X-100 in SFR) and incubated for 2 hours at 37°With mouse sera diluted 1:200 in wash buffer. The membrane was washed twice and incubated for 90 minutes at a dilution of 1:2000 labeled with horseradish peroxidase antimisting Tg. The membrane was washed twice with 0.1% Triton X-100 in SFR and showed with the use of substrate kit Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.

OMV (vesicles of outer membrane) was prepared as follows: N. meningitidis strain 2996 were grown over night at 37°With 5% CO2on GC-cups, gathered loop and resuspendable in 10 ml of 20 mm Tris-HCl, pH 7.5, 2 mm EDTA.

Inactivation by heating was performed at 56°C for 45 minutes and the bacteria was destroyed by sonication for 5 minutes on ice (50% duty cycle, 50% yield, microeconomic 3 mm ultrasonic disintegrator Branson). Intact cells were removed by centrifugation at 5000 g for 10 minutes, the supernatant containing the total fraction of cell membranes, were removed and additionally centrifuged overnight at 50000 g at a temperature of 4°C. the Precipitate containing membranes, resuspendable 2% sarkosyl, 20 mm Tris-HCl, pH 7.5, 2 mm EDTA, and incubated at room temperature for 20 mins is to solubilize the inner membrane. The suspension was centrifuged at 10000 g for 10 min to remove aggregates, the supernatant was additionally centrifuged at 50000 g for 3 hours. The precipitate containing the outer membrane was washed in SFR and resuspendable in the same buffer. The protein concentration was measured according to the method of D.C. Bio-Rad Protein assay (modified Lowry method) using BSA as a standard.

Total cell extracts were obtained as follows: N. meningitidis strain 2996 were grown overnight on GC-Cup, collected by loop and resuspendable in 1 ml of 20 mm Tris-HCl. Inactivation by heating was performed at 56°C for 30 minutes.

1. The method of simultaneous heterologous expression in E. coli of the first protein and the second protein, where

(a) a first protein and the second protein are translated into a single polypeptide chain;

(b) a first protein contains

amino acid sequence AG287 from Neisseria meningitidis:

SPDVKSADTLSKPAAPVVSEKETEAKEDAPQAGSQGQGAPSAQGSQDMAAVSEENTGNGGAVTA

DNPKNEDEVAQNDMPQNAAGTDSSTPNHTPDPNMLAGNMENQATDAGESSQPANQPDMANAADG

MQGDDPSAGGQNAGNTAAQGANQAGNNQAAGSSDPIPASNPAPANGGSNFGRVDLANGVLIDGP

SQNITLTHCKGDSCSGNNFLDEEVQLKSEFEKLSDADKISNYKKDGKNDKFVGLVADSVQMKGI

NQYIIFYKPKPTSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGN

YRYLTYGAEKLPGGSYALRVQGEPAKGEMLAGAAVYNGEVLHFHTENGRPYPTRGRFAAKVDFG

SKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGSGDVSGKFYGPAGEEVAGKYSYRPTD

AEKGGFGVFAGKKEQD

or a sequence that more than 80% identical to this sequence is lesti and retains its function, or immunogenic fragment epitope specified protein sequence containing at least 7 consecutive amino acids; and

(C) a second protein contains the amino acid sequence 953 from Neisseria meningitidis

ATYKVDEYHANARFAIDHFHTSTNVGGFYGLTGSVEFDQAKRDGKIDITIPVANLQSGSQHFTD

HLKSADIFDAAQYPDIRFVSTKFNFNGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPMAKTE

VCGGDFSTTIDRTKWGVDYLVNVGMTKSVRIDIQIEAAKQ

or a sequence that more than 80% identical to the sequence and retains its function, or immunogenic fragment epitope specified protein sequence containing at least 7 consecutive amino acids.

2. The method according to claim 1, where the first protein is located before the second protein in the polypeptide chain.

3. The method according to claim 1, where the first protein is located after the second protein in the polypeptide chain.

4. A hybrid protein of the formula NH2-A - b-COOH, obtained by the method according to claim 1 and having immunological activity against Neisseria meningitidis, which contains

(i) the amino acid sequence of ΔG287 from Neisseria meningitidis

SPDVKSADTLSKPAAPVVSEKETEAKEDAPQAGSQGQGAPSAQGSQDMAAVSEENTGNGGAVTA

DNPKNEDEVAQNDMPQNAAGTDSSTPNHTPDPNMLAGNMENQATDAGESSQPANQPDMANAADG

MQGDDPSAGGQNAGNTAAQGANQAGNNQAAGSSDPIPASNPAPANGGSNFGRVDLANGVLIDGP

SQNITLTHCKGDSCSGNNFLDEEVQLKSEFEKLSDADKISNYKKDGKNDKFVGLVADSVQMKGI

NQYIIFYKPKPTSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGN

YRYLTYGAEKLPGGSYALRVQGEPAKGEMLAGAAVYNGEVLHFHTENGRPYPTRGRFAAKVDFG

SKSVDGIIDSGDDLHMGTQKFKAADGNGFKGTWTENGSGDVSGKFYGPAGEEVAGKYSYRPTD

AEKGGFGVFAGKKEQD

or a sequence that more than 80% identical to the sequence and retains its function, or immunogenic fragment epitope specified protein sequence containing at least 7 consecutive amino acids, and contains

(ii) the amino acid sequence 953 from Neisseria meningitidis

ATYKVDEYHANARFAIDHFHTSTNVGGFYGLTGSVEFDQAKRDGKIDITIPVANLQSGSQHFTD

HLKSADIFDAAQYPDIRFVSTKFNFNGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPMAKTE

VCGGDFSTTIDRTKWGVDYLVNVGMTKSVRIDIQIEAAKQ

or a sequence that more than 80% identical to the sequence and retains its function, or immunogenic fragment epitope specified protein sequence containing at least 7 consecutive amino acids.

5. The protein according to claim 4, where a and b are directly connected.

6. The protein according to claim 4, where a and b are connected via a linker peptide.

7. The protein according to claim 6, where the linker peptide is polyglycidyl the linker.

8. The protein according to claim 4, where the protein is expressed in the cell host E. coli.

9. Expression vector containing a DNA sequence which encodes a hybrid protein according to claim 4.



 

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