CN109705213B - Anti-staphylococcus aureus toxin antibody and application thereof - Google Patents

Anti-staphylococcus aureus toxin antibody and application thereof Download PDF

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CN109705213B
CN109705213B CN201811471780.2A CN201811471780A CN109705213B CN 109705213 B CN109705213 B CN 109705213B CN 201811471780 A CN201811471780 A CN 201811471780A CN 109705213 B CN109705213 B CN 109705213B
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邹最
侯炜彤
郭诗雨
陈思敏
田复波
邹泽强
安毛毛
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Shanghai Changzheng Hospital
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Abstract

The invention discloses an anti-staphylococcus aureus toxin antibody and application thereof, relating to the technical field of antibodies. An antibody or a fragment thereof capable of specifically binding staphylococcus aureus gamma-hemolysin H1gB component and generating cross reaction with other hemolysin components of the hemolysin; the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the following CDR combinations: VH-CDR1, VH-CDR2 and VH-CDR3; the light chain variable region (VL) comprises the following CDR combinations: VL-CDR1, VL-CDR2 and VL-CDR3. The invention can play a role in neutralizing at least two-component toxins containing neutralizing H1gB, thereby realizing the combined treatment of staphylococcus aureus infection by single or combined anti-staphylococcus aureus chemical drugs.

Description

Anti-staphylococcus aureus toxin antibody and application thereof
Technical Field
The invention relates to the technical field of antibodies, in particular to an antibody specifically binding to staphylococcus aureus toxin.
Background
Staphylococcus aureus (Staphylococcus aureus) belongs to Staphylococcus, is an important gram-positive pathogenic bacterium, and can cause local infection such as pyogenic infection, pneumonia, pseudomembranous enteritis, pericarditis, etc., and systemic infection such as septicemia, sepsis, etc. Staphylococcus aureus is primarily pathogenic by producing toxins and invasive enzymes including hemolysins, leukocidins, enterotoxins, coagulases, and the like. Hemolysin is one of the most important virulence factors secreted by staphylococcus aureus. Hemolysin can be divided into four types of alpha-hemolysin, beta-hemolysin, gamma-hemolysin and delta-hemolysin according to different antigens, wherein the role of the alpha-hemolysin in the field planting and pathogenic process of staphylococcus aureus is most important.
During infection with s.aureus, s.aureus is able to release cytotoxins to attack host neutrophils. Gamma hemolysin (Hlg) is ubiquitously expressed in all staphylococcus aureus. The gene product can produce two types of toxins: the pore-forming process of the HlgAB and HlgCB, gamma-lysin can be divided into the following steps: (1) the water-soluble F, S monomer secreted by the bacteria reaches the target cell; (2) binding of the monomer to a cell membrane forming heterodimer; (3) oligomerization to a cyclic structure of the prepore; (4) formation of transmembrane beta barrel hole. Gamma-lysins are highly effective in killing human immune cells including: neutrophils, lymphocytes and macrophages. And the HlgAB can efficiently destroy the red blood cells of the human body.
Leukocidin (PVL) is an important exotoxin produced by staphylococcus aureus and in recent years there has been an increased drive for mortality in patients infected with (PVL) -producing staphylococcus aureus. PVL is an extracellular toxin produced by staphylococcus aureus, belongs to the family of membrane-boring toxins, and consists of S and F proteins. These 2 proteins are encoded by the Luks-PV gene and the LukF-PV gene and have relative molecular weights of 33X 103 and 34X 103, respectively. The biological activity and function of S protein and F protein are different, and the Lusk-PV first binds to receptor on PMNs cell membrane with high specificity and affinity to human polymorphonuclear leukocytes (PMN) and macrophages. LukF-PV and Luks-PV are combined to form a dimer, and then the LukF-PV and the Luks-PV are combined to form a hetero-polymer in sequence. The heteropolymer is perpendicular to the plane of PMNs cell membranes, forms a transmembrane pore and only allows divalent cationsIons such as Ca 2+ And so on. The PMNs cell death depends on the concentration of PVL, and at low concentration, PVL is specifically combined with an undefined cell surface receptor to generate more heteromers, so that more PVL molecules can enter cells to form pore canals on the outer mitochondrial membrane, the internal environment of mitochondria is damaged, caspase-9 and Caspase-3 are activated, leukocidin C is released, and apoptosis is induced. At high concentrations, PVL may non-specifically adsorb to the lipid bilayer, forming larger octamer channels, and when Luks-PV binds to PMNs, protein kinases A or C phosphorylate Luks-PV to activate Ca 2+ Channel, ca 2+ The internal flow generates interleukin and inflammatory mediators, so that the PMNs are dissolved, and oxidative metabolites and inflammatory mediators are released to initiate a series of inflammatory reactions, and severe cases cause tissue necrosis.
No antibody produced by aiming at staphylococcus aureus gamma-hemolysin HlgB antigen exists in the prior art.
Disclosure of Invention
The invention aims to: overcomes the defects of the prior art, provides a protein antibody or a functional fragment thereof which is specifically combined with staphylococcus aureus gamma-hemolysin HlgB and can be cross-combined with other toxin components of staphylococcus aureus, and provides the application thereof based on the antibody or other functional fragments.
In order to achieve the above object, the present invention provides the following technical solutions:
an antibody or fragment thereof capable of specifically binding to staphylococcus aureus gamma-hemolysin HlgB component and generating cross reaction with other hemolysin components of the hemolysin;
the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL);
wherein the heavy chain variable region (VH) comprises the following combination of CDRs: VH-CDR1, VH-CDR2 and VH-CDR3;
the light chain variable region (VL) comprises a CDR combination of: VL-CDR1, VL-CDR2 and VL-CDR3;
the heavy chain variable region is:
Figure BDA0001891127140000021
VH-CDR1 amino acids: the content of the GYTFTDYN is determined,
VH-CDR2 amino acids: the number of the INPNYDST is less than the number of the INPNYDST,
VH-CDR3 amino acids: ARDGSSAMDY of the formula,
the light chain variable region is:
Figure BDA0001891127140000031
VL-CDR1 amino acids: QSLLNSGNQKNY of the formula,
VL-CDR2 amino acids: the GAS is a GAS that is a mixture of gases,
VL-CDR3 amino acids: QNDHRYPLT.
Further, the heavy chain variable region (VH) comprises a sequence selected from: and SEQ ID NO:1, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence set forth in 1 has at least 75% identity. Preferably, the at least 75% identity is any percentage identity greater than or equal to 75%, such as at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity.
And/or, the light chain variable region (VL) comprises a sequence selected from: and SEQ ID NO:2, or a pharmaceutically acceptable salt thereof, and an amino acid sequence having at least 75% identity to the amino acid sequence set forth in figure 2. Preferably, the at least 75% identity is any percentage identity greater than or equal to 75%, such as at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity.
Further, comprising a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:
as shown in SEQ ID NO:1 or an amino acid sequence substantially identical to SEQ ID NO:1, a heavy chain variable region having an amino acid sequence with at least 75% identity to the amino acid sequence set forth in seq id no;
and to SEQ ID NO:2 having an amino acid sequence of at least 75% identity thereto.
Further, the antibody is any one of a monoclonal antibody, a single-chain antibody, a single-domain antibody, a fully or partially humanized antibody or a chimeric antibody; preferably, the antibody is IgA, igD, igE, igG or IgM, more preferably IgG1;
the fragment is selected from the scFv, fab, F (ab') 2 or Fv fragment of the antibody.
Further, the heavy chain constant region of the antibody is of an IgG1 subtype, and the light chain constant region is of a kappa type. Preferably, the antibody or fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO:1 and/or a heavy chain constant region as set forth in SEQ ID NO:2, or a light chain constant region.
In another aspect, the present invention also provides a conjugate comprising the aforementioned antibody or a fragment thereof.
In another aspect, the invention also provides a nucleic acid molecule encoding a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, a heavy chain or a light chain as in the aforementioned antibody or fragment thereof.
In another aspect, the present invention also provides a vector comprising a nucleic acid molecule as described above.
The vector is a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome or a phage vector.
In another aspect, the present invention also provides a pharmaceutical composition comprising an antibody or fragment thereof as described above, or a conjugate of the foregoing, or a nucleic acid molecule of the foregoing, or a vector of the foregoing;
and optionally pharmaceutically acceptable excipients.
In another aspect, the present invention also provides a use of the aforementioned antibody or fragment thereof, or the aforementioned conjugate, or the aforementioned nucleic acid molecule, or the aforementioned vector, or the aforementioned pharmaceutical composition for preparing a medicament for preventing or treating staphylococcus aureus infection.
The medicine can be combined with other optional anti-staphylococcus aureus medicines to treat pyogenic infection, pneumonia, pseudomembranous enteritis, pericarditis and other local infections caused by staphylococcus aureus, and septicemia, sepsis and other systemic infections.
Preferably, the staphylococcus aureus is a pathogenic staphylococcus aureus capable of causing an infection in a mammal, such as a human. The other anti-staphylococcus aureus drug can be vancomycin or linezolid injection.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages and positive effects as examples: the antibody can be specifically combined with a staphylococcus aureus gamma-hemolysin HlgB component, and can further generate cross activity with other virulence factors of the hemolysin, including HlgA, lukD and LukF. The antibody can neutralize staphylococcus aureus hemolysin virulence factors to the maximum extent, and further realizes the combined treatment of staphylococcus aureus infection by single or combined anti-staphylococcus aureus chemical drugs.
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FIG. 1 shows recombinant expression of His tag fused Candida albicans HlgB protein provided by the embodiment of the invention.
FIG. 2 shows recombinant expression of His-tag fused Candida albicans and HlgA protein provided by the embodiments of the present invention.
FIG. 3 shows recombinant expression of His tag fused Candida albicans and HlgC protein provided by the embodiment of the invention.
FIG. 4 shows the LukD protein recombinant expression fused with His tag provided in the embodiments of the present invention.
FIG. 5 shows the LukF protein recombinant expression fused with His tag provided in the embodiments of the present invention.
FIG. 6 shows the purification of recombinant proteins according to the present invention.
FIG. 7 is a graph showing the analysis of the results of the detection of the binding of an antibody to a Staphylococcus aureus virulence protein molecule by ELISA, according to an embodiment of the invention.
Detailed Description
The anti-staphylococcus aureus toxin antibody and the application thereof disclosed by the invention are further explained in detail with reference to the attached drawings and specific examples. It should be noted that technical features or combinations of technical features described in the following embodiments should not be considered in isolation, and they may be combined with each other to achieve better technical effects. In the drawings of the embodiments described below, the same reference numerals appearing in the respective drawings denote the same features or components, and may be applied to different embodiments. Thus, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.
It is noted that techniques, methods and apparatus that are known to one of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate. In all examples shown and discussed herein, any particular value should be construed as merely illustrative, and not limiting. Thus, other examples of the exemplary embodiments may have different values.
Example 1: his tag fused candida albicans HlgB protein recombinant expression
Taking protein amino acids of staphylococcus aureus HlgB and HlgA as target sequences, artificially synthesizing corresponding base sequences, and cloning the base sequences into a Pet-21a plasmid containing a His tag by utilizing enzyme cutting sites NdeI and XhoI. The recombinant plasmid was transformed into competent cells BL21 (DE 3) pLysS, and a single colony was picked the next day and inoculated into LB liquid medium containing 100. Mu.g/ml ampicillin, followed by shaking culture at 37 ℃ overnight. Inoculating overnight culture solution at a ratio of 1: 100 into LB liquid culture medium containing 100. Mu.g/ml ampicillin, and performing shaking culture at 200rpm at 37 deg.C until OD 600 About 0.6-0.8, IPTG was added to the bacterial suspension to a final concentration of 0.5mM, and induction was carried out at 37 ℃ for 4.5 hours. Taking the induced bacteria liquid, centrifuging at 8,000rpm for 3min, collecting thallus, and storing at-80 deg.C.
Full-length amino acid sequence of HlgB protein:
Figure BDA0001891127140000061
expressing the full-length base sequence of the HlgB protein:
Figure BDA0001891127140000062
example 2: recombinant expression of HlgA protein fused with His tag
The method takes staphylococcus aureus HlgA protein amino acid as a target sequence, artificially synthesizes a corresponding base sequence, and clones the protein into Pet-21a plasmid containing His label by utilizing enzyme cutting sites NdeI and XhoI. The recombinant plasmid was transformed into competent cells BL21 (DE 3) pLysS, and a single colony was picked the next day and inoculated into LB liquid medium containing 100. Mu.g/ml ampicillin, followed by shaking culture at 37 ℃ overnight. Inoculating overnight culture solution at a ratio of 1: 100 into LB liquid medium containing 100 μ g/ml ampicillin, and performing shaking culture at 200rpm at 37 deg.C to OD 600 About 0.6-0.8, IPTG was added to the bacterial solution to a final concentration of 0.5mM, and induction was carried out at 37 ℃ for 4.5 hours. Taking the induced bacterial liquid, centrifuging at 8,000rpm for 3min, collecting thallus, and preserving at-80 ℃.
Full-length amino acid sequence of staphylococcus aureus HlgA protein:
Figure BDA0001891127140000071
expressing the full-length base sequence of the staphylococcus aureus HlgA protein:
Figure BDA0001891127140000072
example 3: recombinant expression of HlgC protein fused with His tag
The method takes staphylococcus aureus HlgC protein amino acid as a target sequence, artificially synthesizes a corresponding base sequence, and clones the protein into Pet-21a plasmid containing His label by utilizing enzyme cutting sites NdeI and XhoI. The recombinant plasmid was transformed into competent cells BL21 (DE 3) pLysS, and a single colony was picked the next day and inoculated into LB liquid medium containing 100. Mu.g/ml ampicillin, followed by shaking culture at 37 ℃ overnight. Inoculating overnight culture solution at a ratio of 1: 100 into LB liquid culture medium containing 100. Mu.g/ml ampicillin, and performing shaking culture at 200rpm at 37 deg.C until OD 600 About 0.6-0.8, adding IPTG to the bacterial liquid to final concentrationThe degree was 0.5mM and induction was carried out at 37 ℃ for 4.5h. Taking the induced bacteria liquid, centrifuging at 8,000rpm for 3min, collecting thallus, and storing at-80 deg.C.
Full-length amino acid sequence of staphylococcus aureus protein:
Figure BDA0001891127140000081
expressing the full-length base sequence of the staphylococcus aureus HlgC protein:
Figure BDA0001891127140000082
example 4: recombinant expression of LukD protein fused with His tag
Taking a staphylococcus aureus LukD protein amino acid as a target sequence, artificially synthesizing a corresponding base sequence, and cloning the corresponding base sequence into a Pet-21a plasmid containing a His label by utilizing enzyme cutting sites NdeI and XhoI. The recombinant plasmid was transformed into competent cells BL21 (DE 3) pLysS, and on the following day, a single colony was picked up and inoculated into LB liquid medium containing 100. Mu.g/ml ampicillin, followed by shaking culture at 37 ℃ overnight. Inoculating overnight culture solution into LB liquid culture medium containing 100 μ g/ml ampicillin at a ratio of 1: 100, and performing shaking culture at 200rpm at 37 deg.C to obtain OD 600 About 0.6-0.8, IPTG was added to the bacterial suspension to a final concentration of 0.5mM, and induction was carried out at 37 ℃ for 4.5 hours. Taking the induced bacteria liquid, centrifuging at 8,000rpm for 3min, collecting thallus, and storing at-80 deg.C.
Staphylococcus aureus protein LukD full-length amino acid sequence:
Figure BDA0001891127140000091
expressing the full-length base sequence of the LukD protein of the staphylococcus aureus:
Figure BDA0001891127140000092
example 5: recombinant expression of LukF protein fused with His tag
The method takes staphylococcus aureus LukF protein amino acid as a target sequence, artificially synthesizes a corresponding base sequence, and clones the corresponding base sequence into a Pet-21a plasmid containing a His label by utilizing enzyme cutting sites NdeI and XhoI. The recombinant plasmid was transformed into competent cells BL21 (DE 3) pLysS, and a single colony was picked the next day and inoculated into LB liquid medium containing 100. Mu.g/ml ampicillin, followed by shaking culture at 37 ℃ overnight. Inoculating overnight culture solution at a ratio of 1: 100 into LB liquid culture medium containing 100. Mu.g/ml ampicillin, and performing shaking culture at 200rpm at 37 deg.C until OD 600 About 0.6-0.8, IPTG was added to the bacterial solution to a final concentration of 0.5mM, and induction was carried out at 37 ℃ for 4.5 hours. Taking the induced bacteria liquid, centrifuging at 8,000rpm for 3min, collecting thallus, and storing at-80 deg.C.
Full-length amino acid sequence of staphylococcus aureus protein LukF:
Figure BDA0001891127140000101
expressing the full-length base sequence of the staphylococcus aureus LukF protein:
Figure BDA0001891127140000102
example 6: purification of recombinant staphylococcus aureus HlgB, hlgA, hlgC, lukD and LukF proteins
Crushing the escherichia coli induced to express the recombinant staphylococcus aureus HlgB protein by using an ultrasonic crusher, and working at 180W for 3s with the interval of 3s for 7-9min; centrifuging at 13,000rpm for 30min, collecting supernatant, and filtering with 0.22 μmL filter for sterilization; mixing the Ni column and the supernatant on a rotary mixer for 1h at room temperature, and loading the Ni column into a filler column; eluting with BD solution (containing imidazole at concentration of 30 mM) with 5 times of bed volume and Ni column nonspecific binding protein until the protein color developing solution does not change color, and eluting with BB solution (containing imidazole at concentration of 300 mM) with 5 times of bed volume to obtain target protein; the eluent containing the target protein is concentrated by a 10KD concentration tube to replace the solvent into PBS buffer.
The electrophoretogram is shown in FIG. 6.
Example 7: balb/c mice immunized by recombinant staphylococcus aureus HlgB protein
Referring to Antibodies a Laboratory Manual, second Edition (Edward A. Greenfield 2012), 8-week-old Balb/c mice were immunized in a total of 42 days at 14-day intervals. The immunogen candida albicans Eno1 protein is emulsified in complete or incomplete freund's adjuvant and injected in a unilateral manner into the subcutaneous tissue and peritoneal cavity of the dorsum cervicovarian, caudate root, groin 3 of the mouse. On the 35 th day of immunization, tail vein blood was collected, and spleen cells of immunized mice were fused with myeloma cells after antibody titer detection by ELISA.
Example 8: screening and identification of anti-HlgB protein monoclonal antibody hybridoma cell strain and determination of antibody sequence
Taking recombinant staphylococcus aureus HlgB protein immune Balb/c mouse spleen cells and myeloma cells P3X63Ag8.653 to fuse by using a PEG or electrofusion method, inoculating the fused hybridoma cells into a 96-well plate, adding a culture medium containing HAT culture medium and HT after 24 hours, and screening the hybridoma cells; after culturing in a 96-well plate for 10-14 days, cell supernatants were taken for ELISA experiments to screen hybridoma clones capable of secreting anti-Eno 1 antibody.
Adding the hybridoma parent clone secreting the anti-HlgB antibody into a 96-well plate paved with feeder cells by a limiting dilution method, observing and marking monoclonal cells under a microscope after 2-3 days, and screening monoclonal hybridoma cells capable of secreting the anti-HlgB monoclonal antibody through an ELISA (enzyme-linked immunosorbent assay) experiment after 7 days.
After the monoclonal hybridoma secreting the anti-HlgB monoclonal antibody is subjected to expanded culture, total RNA of the cell is extracted according to the steps of an RNAfast200 kit (Shanghai Feijie Biotechnology Co., ltd.); reverse transcribing hybridoma cell total RNA to cDNA using 5 XPrimeScript RT Master Mix (Takara); amplification of antibody light chain variable region IgVL (kappa) and heavy chain variable region V using degenerate primers (Anke Krebber. 1997) and Extaq PCR reagents (Takara) H Sequencing; purifying the PCR amplification product by using a PCR clean-up Gel extraction kit (Macherey-Nagel Co.); test with pClone007 Simple Vector KitKit (Otsugaku Biotechnology Co., ltd.) instructions will amplify PCR products connected to T vector and transform Escherichia coli competent cells, strain amplification, plasmid extraction and DNA sequencing to obtain monoclonal antibody variable region sequence.
Example 9: ELISA method for detecting binding of antibody and HlgB protein
Fungal HlgB protein was diluted to 1. Mu.g/ml with PBS buffer and 100. Mu.l per well was coated in 96-well plates (Microwell 96F 167008, thermo) and incubated overnight at 4 ℃; the next day the 96-well plates were removed and washed with PBST (0.5% PBS) and the residual water was spun off thoroughly after 1min of each soak. 200. Mu.l of BSA-containing PBST (5% assay) was added to each well and blocked at 37 ℃ for 1 hour; the plates were then washed with PBST and the wells were spin dried. 100. Mu.l of each sample to be tested was added to the 96-well plate and incubated overnight at 4 ℃. After the 96-well plate was removed and washed with PBST, 100. Mu.l of a secondary antibody was added to each well, and incubated at 37 ℃ for 1 hour. Washing with PBST for 5 times, adding 100 μ l of Substrate Solution (Invitrogen) per well, and incubating at 37 deg.C for 10min; after the reaction was terminated by adding 50. Mu.l of 2N sulfuric acid to each well, absorbance was measured at a wavelength of 450nm using a microplate reader (Multiskcin FC, thermo).
Example 10: hemolysis experiment for detecting antibody inhibition bi-component toxin HlgAB protein by ELISA method
The HlgB and HlgA proteins were mixed and adjusted to a concentration of 2. Mu.g/ml. The proteins were assembled to form the HlgAB protein by incubation at 37 ℃ for 10min, and the HlgB antibody was added and incubated with the HlgAB protein for 10min. 5% rabbit blood was added in 75. Mu.l and PBS was added to make the final volume 150. Mu.l. The mixture was whipped and incubated at 37 ℃ for 60min, and the sample was centrifuged at 3000rpm,3min. The absorbance value of OD405 was measured by a microplate reader (Multiskcin FC, thermo).
The results of the test are shown in FIG. 7.
Example 11: animal model replication of gamma hemolysin sepsis in mice and detection of treatment effect of monoclonal antibody
The HlgB and HlgA proteins were mixed and adjusted to a concentration of 50. Mu.g/ml, and incubated at 37 ℃ for 10min. The proteins are assembled to form the HlgAB protein. The tail vein was injected with 50. Mu.g of anti-HlgB antibody, and 30min later, the tail vein was injected with 20. Mu.g of HlgAB protein. Mice were observed for survival time.
Other embodiments
In the foregoing description, the disclosure of the present invention is not intended to limit itself to these aspects. Rather, the various components may be selectively and operatively combined in any number within the intended scope of the present disclosure. In addition, terms like "comprising" and "having" should be interpreted as inclusive or open-ended, rather than exclusive or closed-ended, by default, unless explicitly defined to the contrary. All technical, scientific, or other terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs unless defined otherwise. Common terms found in dictionaries should not be interpreted as being too idealized or too impractical in the context of related art documents unless the present disclosure expressly limits them to that. Any changes and modifications of the present invention based on the above disclosure will be within the scope of the appended claims.
Sequence listing
<110> Shanghai Kopara-technology of medicine Co., ltd
<120> anti-staphylococcus aureus toxin antibody and use thereof
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Cys Thr Cys Cys Gly Ala Ala Ala Ala Ala Cys Cys Ala Gly Ala Ala
305 310 315 320
Cys Gly Ala Ala Gly Ala Ala Thr Thr Cys Cys Ala Gly Gly Thr Thr
325 330 335
Cys Ala Gly Ala Ala Cys Ala Cys Cys Cys Thr Gly Gly Gly Thr Thr
340 345 350
Ala Cys Ala Cys Cys Thr Thr Cys Gly Gly Thr Gly Gly Thr Gly Ala
355 360 365
Cys Ala Thr Cys Thr Cys Thr Ala Thr Cys Thr Cys Thr Ala Ala Cys
370 375 380
Gly Gly Thr Cys Thr Gly Thr Cys Thr Gly Gly Thr Gly Gly Thr Cys
385 390 395 400
Thr Gly Ala Ala Cys Gly Gly Thr Ala Ala Cys Ala Cys Cys Gly Cys
405 410 415
Thr Thr Thr Cys Thr Cys Thr Gly Ala Ala Ala Cys Cys Ala Thr Cys
420 425 430
Ala Ala Cys Thr Ala Cys Ala Ala Ala Cys Ala Gly Gly Ala Ala Thr
435 440 445
Cys Thr Thr Ala Cys Cys Gly Thr Ala Cys Cys Ala Cys Cys Cys Thr
450 455 460
Gly Thr Cys Thr Cys Gly Thr Ala Ala Cys Ala Cys Cys Ala Ala Cys
465 470 475 480
Thr Ala Cys Ala Ala Ala Ala Ala Cys Gly Thr Thr Gly Gly Thr Thr
485 490 495
Gly Gly Gly Gly Thr Gly Thr Thr Gly Ala Ala Gly Cys Thr Cys Ala
500 505 510
Cys Ala Ala Ala Ala Thr Cys Ala Thr Gly Ala Ala Cys Ala Ala Cys
515 520 525
Gly Gly Thr Thr Gly Gly Gly Gly Thr Cys Cys Gly Thr Ala Cys Gly
530 535 540
Gly Thr Cys Gly Thr Gly Ala Cys Thr Cys Thr Thr Thr Cys Cys Ala
545 550 555 560
Cys Cys Cys Gly Ala Cys Cys Thr Ala Cys Gly Gly Thr Ala Ala Cys
565 570 575
Gly Ala Ala Cys Thr Gly Thr Thr Cys Cys Thr Gly Gly Cys Thr Gly
580 585 590
Gly Thr Cys Gly Thr Cys Ala Gly Thr Cys Thr Thr Cys Thr Gly Cys
595 600 605
Thr Thr Ala Cys Gly Cys Thr Gly Gly Thr Cys Ala Gly Ala Ala Cys
610 615 620
Thr Thr Cys Ala Thr Cys Gly Cys Thr Cys Ala Gly Cys Ala Cys Cys
625 630 635 640
Ala Gly Ala Thr Gly Cys Cys Gly Cys Thr Gly Cys Thr Gly Thr Cys
645 650 655
Thr Cys Gly Thr Thr Cys Thr Ala Ala Cys Thr Thr Cys Ala Ala Cys
660 665 670
Cys Cys Gly Gly Ala Ala Thr Thr Cys Cys Thr Gly Thr Cys Thr Gly
675 680 685
Thr Thr Cys Thr Gly Thr Cys Thr Cys Ala Cys Cys Gly Thr Cys Ala
690 695 700
Gly Gly Ala Cys Gly Gly Thr Gly Cys Thr Ala Ala Ala Ala Ala Ala
705 710 715 720
Thr Cys Thr Ala Ala Ala Ala Thr Cys Ala Cys Cys Gly Thr Thr Ala
725 730 735
Cys Cys Thr Ala Cys Cys Ala Gly Cys Gly Thr Gly Ala Ala Ala Thr
740 745 750
Gly Gly Ala Cys Cys Thr Gly Thr Ala Cys Cys Ala Gly Ala Thr Cys
755 760 765
Cys Gly Thr Thr Gly Gly Ala Ala Cys Gly Gly Thr Thr Thr Cys Thr
770 775 780
Ala Cys Thr Gly Gly Gly Cys Thr Gly Gly Thr Gly Cys Thr Ala Ala
785 790 795 800
Cys Thr Ala Cys Ala Ala Ala Ala Ala Cys Thr Thr Cys Ala Ala Ala
805 810 815
Ala Cys Cys Cys Gly Thr Ala Cys Cys Thr Thr Cys Ala Ala Ala Thr
820 825 830
Cys Thr Ala Cys Cys Thr Ala Cys Gly Ala Ala Ala Thr Cys Gly Ala
835 840 845
Cys Thr Gly Gly Gly Ala Ala Ala Ala Cys Cys Ala Cys Ala Ala Ala
850 855 860
Gly Thr Thr Ala Ala Ala Cys Thr Gly Cys Thr Gly Gly Ala Cys Ala
865 870 875 880
Cys Cys Ala Ala Ala Gly Ala Ala Ala Cys Cys Gly Ala Ala Ala Ala
885 890 895
Cys Ala Ala Cys Ala Ala Ala Cys Thr Cys Gly Ala Gly
900 905
<210> 5
<211> 283
<212> PRT
<213> Artificial Synthesis (HlgA protein)
<400> 5
Met Glu Asn Lys Ile Glu Asp Ile Gly Gln Gly Ala Glu Ile Ile Lys
1 5 10 15
Arg Thr Gln Asp Ile Thr Ser Lys Arg Leu Ala Ile Thr Gln Asn Ile
20 25 30
Gln Phe Asp Phe Val Lys Asp Lys Lys Tyr Asn Lys Asp Ala Leu Val
35 40 45
Val Lys Met Gln Gly Phe Ile Ser Ser Arg Thr Thr Tyr Ser Asp Leu
50 55 60
Lys Lys Tyr Pro Tyr Ile Lys Arg Met Ile Trp Pro Phe Gln Tyr Asn
65 70 75 80
Ile Ser Leu Lys Thr Lys Asp Ser Asn Val Asp Leu Ile Asn Tyr Leu
85 90 95
Pro Lys Asn Lys Ile Asp Ser Ala Asp Val Ser Gln Lys Leu Gly Tyr
100 105 110
Asn Ile Gly Gly Asn Phe Gln Ser Ala Pro Ser Ile Gly Gly Ser Gly
115 120 125
Ser Phe Asn Tyr Ser Lys Thr Ile Ser Tyr Asn Gln Lys Asn Tyr Val
130 135 140
Thr Glu Val Glu Ser Gln Asn Ser Lys Gly Val Lys Trp Gly Val Lys
145 150 155 160
Ala Asn Ser Phe Val Thr Pro Asn Gly Gln Val Ser Ala Tyr Asp Gln
165 170 175
Tyr Leu Phe Ala Gln Asp Pro Thr Gly Pro Ala Ala Arg Asp Tyr Phe
180 185 190
Val Pro Asp Asn Gln Leu Pro Pro Leu Ile Gln Ser Gly Phe Asn Pro
195 200 205
Ser Phe Ile Thr Thr Leu Ser His Glu Arg Gly Lys Gly Asp Lys Ser
210 215 220
Glu Phe Glu Ile Thr Tyr Gly Arg Asn Met Asp Ala Thr Tyr Ala Tyr
225 230 235 240
Val Thr Arg His Arg Leu Ala Val Asp Arg Lys His Asp Ala Phe Lys
245 250 255
Asn Arg Asn Val Thr Val Lys Tyr Glu Val Asn Trp Lys Thr His Glu
260 265 270
Val Lys Ile Lys Ser Ile Thr Pro Lys Leu Glu
275 280
<210> 6
<211> 852
<212> PRT
<213> Artificial Synthesis (HlgA protein fused with His tag)
<400> 6
Cys Ala Thr Ala Thr Gly Gly Ala Ala Ala Ala Cys Ala Ala Ala Ala
1 5 10 15
Thr Cys Gly Ala Ala Gly Ala Cys Ala Thr Cys Gly Gly Thr Cys Ala
20 25 30
Gly Gly Gly Thr Gly Cys Thr Gly Ala Ala Ala Thr Cys Ala Thr Cys
35 40 45
Ala Ala Ala Cys Gly Thr Ala Cys Cys Cys Ala Gly Gly Ala Cys Ala
50 55 60
Thr Cys Ala Cys Cys Thr Cys Thr Ala Ala Ala Cys Gly Thr Cys Thr
65 70 75 80
Gly Gly Cys Thr Ala Thr Cys Ala Cys Cys Cys Ala Gly Ala Ala Cys
85 90 95
Ala Thr Cys Cys Ala Gly Thr Thr Cys Gly Ala Cys Thr Thr Cys Gly
100 105 110
Thr Thr Ala Ala Ala Gly Ala Cys Ala Ala Ala Ala Ala Ala Thr Ala
115 120 125
Cys Ala Ala Cys Ala Ala Ala Gly Ala Cys Gly Cys Thr Cys Thr Gly
130 135 140
Gly Thr Thr Gly Thr Thr Ala Ala Ala Ala Thr Gly Cys Ala Gly Gly
145 150 155 160
Gly Thr Thr Thr Cys Ala Thr Cys Thr Cys Thr Thr Cys Thr Cys Gly
165 170 175
Thr Ala Cys Cys Ala Cys Cys Thr Ala Cys Thr Cys Thr Gly Ala Cys
180 185 190
Cys Thr Gly Ala Ala Ala Ala Ala Ala Thr Ala Cys Cys Cys Gly Thr
195 200 205
Ala Cys Ala Thr Cys Ala Ala Ala Cys Gly Thr Ala Thr Gly Ala Thr
210 215 220
Cys Thr Gly Gly Cys Cys Gly Thr Thr Cys Cys Ala Gly Thr Ala Cys
225 230 235 240
Ala Ala Cys Ala Thr Cys Thr Cys Thr Cys Thr Gly Ala Ala Ala Ala
245 250 255
Cys Cys Ala Ala Ala Gly Ala Cys Thr Cys Thr Ala Ala Cys Gly Thr
260 265 270
Thr Gly Ala Cys Cys Thr Gly Ala Thr Cys Ala Ala Cys Thr Ala Cys
275 280 285
Cys Thr Gly Cys Cys Gly Ala Ala Ala Ala Ala Cys Ala Ala Ala Ala
290 295 300
Thr Cys Gly Ala Cys Thr Cys Thr Gly Cys Thr Gly Ala Cys Gly Thr
305 310 315 320
Thr Thr Cys Thr Cys Ala Gly Ala Ala Ala Cys Thr Gly Gly Gly Thr
325 330 335
Thr Ala Cys Ala Ala Cys Ala Thr Cys Gly Gly Thr Gly Gly Thr Ala
340 345 350
Ala Cys Thr Thr Cys Cys Ala Gly Thr Cys Thr Gly Cys Thr Cys Cys
355 360 365
Gly Thr Cys Thr Ala Thr Cys Gly Gly Thr Gly Gly Thr Thr Cys Thr
370 375 380
Gly Gly Thr Thr Cys Thr Thr Thr Cys Ala Ala Cys Thr Ala Cys Thr
385 390 395 400
Cys Thr Ala Ala Ala Ala Cys Cys Ala Thr Cys Thr Cys Thr Thr Ala
405 410 415
Cys Ala Ala Cys Cys Ala Gly Ala Ala Ala Ala Ala Cys Thr Ala Cys
420 425 430
Gly Thr Thr Ala Cys Cys Gly Ala Ala Gly Thr Thr Gly Ala Ala Thr
435 440 445
Cys Thr Cys Ala Gly Ala Ala Cys Thr Cys Thr Ala Ala Ala Gly Gly
450 455 460
Thr Gly Thr Thr Ala Ala Ala Thr Gly Gly Gly Gly Thr Gly Thr Thr
465 470 475 480
Ala Ala Ala Gly Cys Thr Ala Ala Cys Thr Cys Thr Thr Thr Cys Gly
485 490 495
Thr Thr Ala Cys Cys Cys Cys Gly Ala Ala Cys Gly Gly Thr Cys Ala
500 505 510
Gly Gly Thr Thr Thr Cys Thr Gly Cys Thr Thr Ala Cys Gly Ala Cys
515 520 525
Cys Ala Gly Thr Ala Cys Cys Thr Gly Thr Thr Cys Gly Cys Thr Cys
530 535 540
Ala Gly Gly Ala Cys Cys Cys Gly Ala Cys Cys Gly Gly Thr Cys Cys
545 550 555 560
Gly Gly Cys Thr Gly Cys Thr Cys Gly Thr Gly Ala Cys Thr Ala Cys
565 570 575
Thr Thr Cys Gly Thr Thr Cys Cys Gly Gly Ala Cys Ala Ala Cys Cys
580 585 590
Ala Gly Cys Thr Gly Cys Cys Gly Cys Cys Gly Cys Thr Gly Ala Thr
595 600 605
Cys Cys Ala Gly Thr Cys Thr Gly Gly Thr Thr Thr Cys Ala Ala Cys
610 615 620
Cys Cys Gly Thr Cys Thr Thr Thr Cys Ala Thr Cys Ala Cys Cys Ala
625 630 635 640
Cys Cys Cys Thr Gly Thr Cys Thr Cys Ala Cys Gly Ala Ala Cys Gly
645 650 655
Thr Gly Gly Thr Ala Ala Ala Gly Gly Thr Gly Ala Cys Ala Ala Ala
660 665 670
Thr Cys Thr Gly Ala Ala Thr Thr Cys Gly Ala Ala Ala Thr Cys Ala
675 680 685
Cys Cys Thr Ala Cys Gly Gly Thr Cys Gly Thr Ala Ala Cys Ala Thr
690 695 700
Gly Gly Ala Cys Gly Cys Thr Ala Cys Cys Thr Ala Cys Gly Cys Thr
705 710 715 720
Thr Ala Cys Gly Thr Thr Ala Cys Cys Cys Gly Thr Cys Ala Cys Cys
725 730 735
Gly Thr Cys Thr Gly Gly Cys Thr Gly Thr Thr Gly Ala Cys Cys Gly
740 745 750
Thr Ala Ala Ala Cys Ala Cys Gly Ala Cys Gly Cys Thr Thr Thr Cys
755 760 765
Ala Ala Ala Ala Ala Cys Cys Gly Thr Ala Ala Cys Gly Thr Thr Ala
770 775 780
Cys Cys Gly Thr Thr Ala Ala Ala Thr Ala Cys Gly Ala Ala Gly Thr
785 790 795 800
Thr Ala Ala Cys Thr Gly Gly Ala Ala Ala Ala Cys Cys Cys Ala Cys
805 810 815
Gly Ala Ala Gly Thr Thr Ala Ala Ala Ala Thr Cys Ala Ala Ala Thr
820 825 830
Cys Thr Ala Thr Cys Ala Cys Cys Cys Cys Gly Ala Ala Ala Cys Thr
835 840 845
Cys Gly Ala Gly
850
<210> 7
<211> 287
<212> PRT
<213> Artificial Synthesis (HlgC protein)
<400> 7
Met Ala Asn Asp Thr Glu Asp Ile Gly Lys Gly Ser Asp Ile Glu Ile
1 5 10 15
Ile Lys Arg Thr Glu Asp Lys Thr Ser Asn Lys Trp Gly Val Thr Gln
20 25 30
Asn Ile Gln Phe Asp Phe Val Lys Asp Lys Lys Tyr Asn Lys Asp Ala
35 40 45
Leu Ile Leu Lys Met Gln Gly Phe Ile Ser Ser Arg Thr Thr Tyr Tyr
50 55 60
Asn Tyr Lys Lys Thr Asn His Val Lys Ala Met Arg Trp Pro Phe Gln
65 70 75 80
Tyr Asn Ile Gly Leu Lys Thr Asn Asp Lys Tyr Val Ser Leu Ile Asn
85 90 95
Tyr Leu Pro Lys Asn Lys Ile Glu Ser Thr Asn Val Ser Gln Thr Leu
100 105 110
Gly Tyr Asn Ile Gly Gly Asn Phe Gln Ser Ala Pro Ser Leu Gly Gly
115 120 125
Asn Gly Ser Phe Asn Tyr Ser Lys Ser Ile Ser Tyr Thr Gln Gln Asn
130 135 140
Tyr Val Ser Glu Val Glu Gln Gln Asn Ser Lys Ser Val Leu Trp Gly
145 150 155 160
Val Lys Ala Asn Ser Phe Ala Thr Glu Ser Gly Gln Lys Ser Ala Phe
165 170 175
Asp Ser Asp Leu Phe Val Gly Tyr Lys Pro His Ser Lys Asp Pro Arg
180 185 190
Asp Tyr Phe Val Pro Asp Ser Glu Leu Pro Pro Leu Val Gln Ser Gly
195 200 205
Phe Asn Pro Ser Phe Ile Ala Thr Val Ser His Glu Lys Gly Ser Ser
210 215 220
Asp Thr Ser Glu Phe Glu Ile Thr Tyr Gly Arg Asn Met Asp Val Thr
225 230 235 240
His Ala Ile Lys Arg Ser Thr His Tyr Gly Asn Ser Tyr Leu Asp Gly
245 250 255
His Arg Val His Asn Ala Phe Val Asn Arg Asn Tyr Thr Val Lys Tyr
260 265 270
Glu Val Asn Trp Lys Thr His Glu Ile Lys Val Lys Gly Gln Asn
275 280 285
<210> 8
<211> 870
<212> PRT
<213> Artificial Synthesis (HlgC protein fused with His tag)
<400> 8
Cys Ala Thr Ala Thr Gly Gly Cys Ala Ala Ala Thr Gly Ala Thr Ala
1 5 10 15
Cys Cys Gly Ala Ala Gly Ala Thr Ala Thr Ala Gly Gly Thr Ala Ala
20 25 30
Ala Gly Gly Thr Ala Gly Thr Gly Ala Thr Ala Thr Ala Gly Ala Ala
35 40 45
Ala Thr Cys Ala Thr Cys Ala Ala Ala Cys Gly Thr Ala Cys Ala Gly
50 55 60
Ala Ala Gly Ala Thr Ala Ala Gly Ala Cys Cys Ala Gly Thr Ala Ala
65 70 75 80
Thr Ala Ala Ala Thr Gly Gly Gly Gly Ala Gly Thr Gly Ala Cys Ala
85 90 95
Cys Ala Gly Ala Ala Thr Ala Thr Thr Cys Ala Ala Thr Thr Thr Gly
100 105 110
Ala Thr Thr Thr Thr Gly Thr Gly Ala Ala Gly Gly Ala Cys Ala Ala
115 120 125
Gly Ala Ala Gly Thr Ala Thr Ala Ala Cys Ala Ala Ala Gly Ala Cys
130 135 140
Gly Cys Ala Cys Thr Gly Ala Thr Ala Thr Thr Ala Ala Ala Gly Ala
145 150 155 160
Thr Gly Cys Ala Gly Gly Gly Cys Thr Thr Thr Ala Thr Cys Ala Gly
165 170 175
Cys Ala Gly Cys Ala Gly Ala Ala Cys Cys Ala Cys Cys Thr Ala Thr
180 185 190
Thr Ala Thr Ala Ala Cys Thr Ala Cys Ala Ala Gly Ala Ala Ala Ala
195 200 205
Cys Ala Ala Ala Cys Cys Ala Cys Gly Thr Gly Ala Ala Ala Gly Cys
210 215 220
Ala Ala Thr Gly Ala Gly Ala Thr Gly Gly Cys Cys Gly Thr Thr Cys
225 230 235 240
Cys Ala Gly Thr Ala Thr Ala Ala Thr Ala Thr Thr Gly Gly Cys Cys
245 250 255
Thr Gly Ala Ala Ala Ala Cys Ala Ala Ala Thr Gly Ala Cys Ala Ala
260 265 270
Gly Thr Ala Thr Gly Thr Thr Ala Gly Cys Thr Thr Ala Ala Thr Cys
275 280 285
Ala Ala Cys Thr Ala Cys Cys Thr Gly Cys Cys Gly Ala Ala Ala Ala
290 295 300
Ala Cys Ala Ala Ala Ala Thr Thr Gly Ala Ala Ala Gly Cys Ala Cys
305 310 315 320
Cys Ala Ala Cys Gly Thr Cys Ala Gly Cys Cys Ala Ala Ala Cys Cys
325 330 335
Cys Thr Gly Gly Gly Thr Thr Ala Cys Ala Ala Cys Ala Thr Thr Gly
340 345 350
Gly Thr Gly Gly Gly Ala Ala Thr Thr Thr Thr Cys Ala Gly Thr Cys
355 360 365
Gly Gly Cys Ala Cys Cys Thr Ala Gly Cys Cys Thr Gly Gly Gly Thr
370 375 380
Gly Gly Thr Ala Ala Thr Gly Gly Thr Thr Cys Ala Thr Thr Thr Ala
385 390 395 400
Ala Thr Thr Ala Cys Ala Gly Cys Ala Ala Ala Thr Cys Ala Ala Thr
405 410 415
Cys Ala Gly Cys Thr Ala Cys Ala Cys Cys Cys Ala Gly Cys Ala Ala
420 425 430
Ala Ala Thr Thr Ala Thr Gly Thr Gly Ala Gly Thr Gly Ala Ala Gly
435 440 445
Thr Cys Gly Ala Ala Cys Ala Ala Cys Ala Gly Ala Ala Thr Ala Gly
450 455 460
Cys Ala Ala Ala Ala Gly Thr Gly Thr Thr Cys Thr Gly Thr Gly Gly
465 470 475 480
Gly Gly Thr Gly Thr Thr Ala Ala Ala Gly Cys Thr Ala Ala Thr Ala
485 490 495
Gly Cys Thr Thr Thr Gly Cys Ala Ala Cys Gly Gly Ala Gly Thr Cys
500 505 510
Cys Gly Gly Cys Cys Ala Gly Ala Ala Ala Ala Gly Cys Gly Cys Ala
515 520 525
Thr Thr Thr Gly Ala Cys Ala Gly Cys Gly Ala Thr Cys Thr Gly Thr
530 535 540
Thr Thr Gly Thr Thr Gly Gly Cys Thr Ala Thr Ala Ala Ala Cys Cys
545 550 555 560
Gly Cys Ala Thr Ala Gly Thr Ala Ala Ala Gly Ala Thr Cys Cys Gly
565 570 575
Cys Gly Gly Gly Ala Thr Thr Ala Thr Thr Thr Thr Gly Thr Thr Cys
580 585 590
Cys Thr Gly Ala Thr Ala Gly Thr Gly Ala Ala Thr Thr Ala Cys Cys
595 600 605
Gly Cys Cys Gly Thr Thr Ala Gly Thr Thr Cys Ala Gly Ala Gly Cys
610 615 620
Gly Gly Ala Thr Thr Thr Ala Ala Thr Cys Cys Cys Thr Cys Ala Thr
625 630 635 640
Thr Thr Ala Thr Thr Gly Cys Cys Ala Cys Gly Gly Thr Thr Thr Cys
645 650 655
Ala Cys Ala Thr Gly Ala Gly Ala Ala Ala Gly Gly Thr Ala Gly Cys
660 665 670
Ala Gly Cys Gly Ala Thr Ala Cys Cys Ala Gly Cys Gly Ala Ala Thr
675 680 685
Thr Thr Gly Ala Ala Ala Thr Ala Ala Cys Cys Thr Ala Thr Gly Gly
690 695 700
Thr Cys Gly Thr Ala Ala Cys Ala Thr Gly Gly Ala Thr Gly Thr Gly
705 710 715 720
Ala Cys Cys Cys Ala Cys Gly Cys Cys Ala Thr Thr Ala Ala Ala Cys
725 730 735
Gly Thr Ala Gly Cys Ala Cys Ala Cys Ala Thr Thr Ala Thr Gly Gly
740 745 750
Thr Ala Ala Thr Ala Gly Cys Thr Ala Cys Thr Thr Ala Gly Ala Cys
755 760 765
Gly Gly Ala Cys Ala Thr Cys Gly Thr Gly Thr Gly Cys Ala Thr Ala
770 775 780
Ala Thr Gly Cys Ala Thr Thr Thr Gly Thr Thr Ala Ala Thr Ala Gly
785 790 795 800
Ala Ala Ala Thr Thr Ala Cys Ala Cys Cys Gly Thr Gly Ala Ala Ala
805 810 815
Thr Ala Cys Gly Ala Ala Gly Thr Thr Ala Ala Thr Thr Gly Gly Ala
820 825 830
Ala Ala Ala Cys Cys Cys Ala Thr Gly Ala Ala Ala Thr Thr Ala Ala
835 840 845
Gly Gly Thr Gly Ala Ala Ala Gly Gly Ala Cys Ala Gly Ala Ala Cys
850 855 860
Cys Thr Cys Gly Ala Gly
865 870
<210> 9
<211> 302
<212> PRT
<213> Artificial Synthesis (LukD protein)
<400> 9
Met Ala Gln His Ile Thr Pro Val Ser Glu Lys Lys Val Asp Asp Lys
1 5 10 15
Ile Thr Leu Tyr Lys Thr Thr Ala Thr Ser Asp Asn Asp Lys Leu Asn
20 25 30
Ile Ser Gln Ile Leu Thr Phe Asn Phe Ile Lys Asp Lys Ser Tyr Asp
35 40 45
Lys Asp Thr Leu Val Leu Lys Ala Ala Gly Asn Ile Asn Ser Gly Tyr
50 55 60
Lys Lys Pro Asn Pro Lys Asp Tyr Asn Tyr Ser Gln Phe Tyr Trp Gly
65 70 75 80
Gly Lys Tyr Asn Val Ser Val Ser Ser Glu Ser Asn Asp Ala Val Asn
85 90 95
Val Val Asp Tyr Ala Pro Lys Asn Gln Asn Glu Glu Phe Gln Val Gln
100 105 110
Gln Thr Leu Gly Tyr Ser Tyr Gly Gly Asp Ile Asn Ile Ser Asn Gly
115 120 125
Leu Ser Gly Gly Leu Asn Gly Ser Lys Ser Phe Ser Glu Thr Ile Asn
130 135 140
Tyr Lys Gln Glu Ser Tyr Arg Thr Thr Ile Asp Arg Lys Thr Asn His
145 150 155 160
Lys Ser Ile Gly Trp Gly Val Glu Ala His Lys Ile Met Asn Asn Gly
165 170 175
Trp Gly Pro Tyr Gly Arg Asp Ser Tyr Asp Pro Thr Tyr Gly Asn Glu
180 185 190
Leu Phe Leu Gly Gly Arg Gln Ser Ser Ser Asn Ala Gly Gln Asn Phe
195 200 205
Leu Pro Thr His Gln Met Pro Leu Leu Ala Arg Gly Asn Phe Asn Pro
210 215 220
Glu Phe Ile Ser Val Leu Ser His Lys Gln Asn Asp Thr Lys Lys Ser
225 230 235 240
Lys Ile Lys Val Thr Tyr Gln Arg Glu Met Asp Arg Tyr Thr Asn Gln
245 250 255
Trp Asn Arg Leu His Trp Val Gly Asn Asn Tyr Lys Asn Gln Asn Thr
260 265 270
Val Thr Phe Thr Ser Thr Tyr Glu Val Asp Trp Gln Asn His Thr Val
275 280 285
Lys Leu Ile Gly Thr Asp Ser Lys Glu Thr Asn Pro Gly Val
290 295 300
<210> 10
<211> 915
<212> PRT
<213> Artificial Synthesis (fusion His tag LukD protein)
<400> 10
Cys Ala Thr Ala Thr Gly Gly Cys Thr Cys Ala Gly Cys Ala Cys Ala
1 5 10 15
Thr Cys Ala Cys Cys Cys Cys Gly Gly Thr Thr Thr Cys Thr Gly Ala
20 25 30
Ala Ala Ala Ala Ala Ala Ala Gly Thr Thr Gly Ala Cys Gly Ala Cys
35 40 45
Ala Ala Ala Ala Thr Cys Ala Cys Cys Cys Thr Gly Thr Ala Cys Ala
50 55 60
Ala Ala Ala Cys Cys Ala Cys Cys Gly Cys Thr Ala Cys Cys Thr Cys
65 70 75 80
Thr Gly Ala Cys Ala Ala Cys Gly Ala Cys Ala Ala Ala Cys Thr Gly
85 90 95
Ala Ala Cys Ala Thr Cys Thr Cys Thr Cys Ala Gly Ala Thr Cys Cys
100 105 110
Thr Gly Ala Cys Cys Thr Thr Cys Ala Ala Cys Thr Thr Cys Ala Thr
115 120 125
Cys Ala Ala Ala Gly Ala Cys Ala Ala Ala Thr Cys Thr Thr Ala Cys
130 135 140
Gly Ala Cys Ala Ala Ala Gly Ala Cys Ala Cys Cys Cys Thr Gly Gly
145 150 155 160
Thr Thr Cys Thr Gly Ala Ala Ala Gly Cys Thr Gly Cys Thr Gly Gly
165 170 175
Thr Ala Ala Cys Ala Thr Cys Ala Ala Cys Thr Cys Thr Gly Gly Thr
180 185 190
Thr Ala Cys Ala Ala Ala Ala Ala Ala Cys Cys Gly Ala Ala Cys Cys
195 200 205
Cys Gly Ala Ala Ala Gly Ala Cys Thr Ala Cys Ala Ala Cys Thr Ala
210 215 220
Cys Thr Cys Thr Cys Ala Gly Thr Thr Cys Thr Ala Cys Thr Gly Gly
225 230 235 240
Gly Gly Thr Gly Gly Thr Ala Ala Ala Thr Ala Cys Ala Ala Cys Gly
245 250 255
Thr Thr Thr Cys Thr Gly Thr Thr Thr Cys Thr Thr Cys Thr Gly Ala
260 265 270
Ala Thr Cys Thr Ala Ala Cys Gly Ala Cys Gly Cys Thr Gly Thr Thr
275 280 285
Ala Ala Cys Gly Thr Thr Gly Thr Thr Gly Ala Cys Thr Ala Cys Gly
290 295 300
Cys Thr Cys Cys Gly Ala Ala Ala Ala Ala Cys Cys Ala Gly Ala Ala
305 310 315 320
Cys Gly Ala Ala Gly Ala Ala Thr Thr Cys Cys Ala Gly Gly Thr Thr
325 330 335
Cys Ala Gly Cys Ala Gly Ala Cys Cys Cys Thr Gly Gly Gly Thr Thr
340 345 350
Ala Cys Thr Cys Thr Thr Ala Cys Gly Gly Thr Gly Gly Thr Gly Ala
355 360 365
Cys Ala Thr Cys Ala Ala Cys Ala Thr Cys Thr Cys Thr Ala Ala Cys
370 375 380
Gly Gly Thr Cys Thr Gly Thr Cys Thr Gly Gly Thr Gly Gly Thr Cys
385 390 395 400
Thr Gly Ala Ala Cys Gly Gly Thr Thr Cys Thr Ala Ala Ala Thr Cys
405 410 415
Thr Thr Thr Cys Thr Cys Thr Gly Ala Ala Ala Cys Cys Ala Thr Cys
420 425 430
Ala Ala Cys Thr Ala Cys Ala Ala Ala Cys Ala Gly Gly Ala Ala Thr
435 440 445
Cys Thr Thr Ala Cys Cys Gly Thr Ala Cys Cys Ala Cys Cys Ala Thr
450 455 460
Cys Gly Ala Cys Cys Gly Thr Ala Ala Ala Ala Cys Cys Ala Ala Cys
465 470 475 480
Cys Ala Cys Ala Ala Ala Thr Cys Thr Ala Thr Cys Gly Gly Thr Thr
485 490 495
Gly Gly Gly Gly Thr Gly Thr Thr Gly Ala Ala Gly Cys Thr Cys Ala
500 505 510
Cys Ala Ala Ala Ala Thr Cys Ala Thr Gly Ala Ala Cys Ala Ala Cys
515 520 525
Gly Gly Thr Thr Gly Gly Gly Gly Thr Cys Cys Gly Thr Ala Cys Gly
530 535 540
Gly Thr Cys Gly Thr Gly Ala Cys Thr Cys Thr Thr Ala Cys Gly Ala
545 550 555 560
Cys Cys Cys Gly Ala Cys Cys Thr Ala Cys Gly Gly Thr Ala Ala Cys
565 570 575
Gly Ala Ala Cys Thr Gly Thr Thr Cys Cys Thr Gly Gly Gly Thr Gly
580 585 590
Gly Thr Cys Gly Thr Cys Ala Gly Thr Cys Thr Thr Cys Thr Thr Cys
595 600 605
Thr Ala Ala Cys Gly Cys Thr Gly Gly Thr Cys Ala Gly Ala Ala Cys
610 615 620
Thr Thr Cys Cys Thr Gly Cys Cys Gly Ala Cys Cys Cys Ala Cys Cys
625 630 635 640
Ala Gly Ala Thr Gly Cys Cys Gly Cys Thr Gly Cys Thr Gly Gly Cys
645 650 655
Thr Cys Gly Thr Gly Gly Thr Ala Ala Cys Thr Thr Cys Ala Ala Cys
660 665 670
Cys Cys Gly Gly Ala Ala Thr Thr Cys Ala Thr Cys Thr Cys Thr Gly
675 680 685
Thr Thr Cys Thr Gly Thr Cys Thr Cys Ala Cys Ala Ala Ala Cys Ala
690 695 700
Gly Ala Ala Cys Gly Ala Cys Ala Cys Cys Ala Ala Ala Ala Ala Ala
705 710 715 720
Thr Cys Thr Ala Ala Ala Ala Thr Cys Ala Ala Ala Gly Thr Thr Ala
725 730 735
Cys Cys Thr Ala Cys Cys Ala Gly Cys Gly Thr Gly Ala Ala Ala Thr
740 745 750
Gly Gly Ala Cys Cys Gly Thr Thr Ala Cys Ala Cys Cys Ala Ala Cys
755 760 765
Cys Ala Gly Thr Gly Gly Ala Ala Cys Cys Gly Thr Cys Thr Gly Cys
770 775 780
Ala Cys Thr Gly Gly Gly Thr Thr Gly Gly Thr Ala Ala Cys Ala Ala
785 790 795 800
Cys Thr Ala Cys Ala Ala Ala Ala Ala Cys Cys Ala Gly Ala Ala Cys
805 810 815
Ala Cys Cys Gly Thr Thr Ala Cys Cys Thr Thr Cys Ala Cys Cys Thr
820 825 830
Cys Thr Ala Cys Cys Thr Ala Cys Gly Ala Ala Gly Thr Thr Gly Ala
835 840 845
Cys Thr Gly Gly Cys Ala Gly Ala Ala Cys Cys Ala Cys Ala Cys Cys
850 855 860
Gly Thr Thr Ala Ala Ala Cys Thr Gly Ala Thr Cys Gly Gly Thr Ala
865 870 875 880
Cys Cys Gly Ala Cys Thr Cys Thr Ala Ala Ala Gly Ala Ala Ala Cys
885 890 895
Cys Ala Ala Cys Cys Cys Gly Gly Gly Thr Gly Thr Thr Cys Thr Cys
900 905 910
Gly Ala Gly
915
<210> 11
<211> 302
<212> PRT
<213> Artificial Synthesis (LukF protein)
<400> 11
Met Ala Gln His Ile Thr Pro Val Ser Glu Lys Lys Val Asp Asp Lys
1 5 10 15
Ile Thr Leu Tyr Lys Thr Thr Ala Thr Ser Asp Ser Asp Lys Leu Lys
20 25 30
Ile Ser Gln Ile Leu Thr Phe Asn Phe Ile Lys Asp Lys Ser Tyr Asp
35 40 45
Lys Asp Thr Leu Ile Leu Lys Ala Ala Gly Asn Ile Tyr Ser Gly Tyr
50 55 60
Thr Lys Pro Asn Pro Lys Asp Thr Ile Ser Ser Gln Phe Tyr Trp Gly
65 70 75 80
Ser Lys Tyr Asn Ile Ser Ile Asn Ser Asp Ser Asn Asp Ser Val Asn
85 90 95
Val Val Asp Tyr Ala Pro Lys Asn Gln Asn Glu Glu Phe Gln Val Gln
100 105 110
Gln Thr Val Gly Tyr Ser Tyr Gly Gly Asp Ile Asn Ile Ser Asn Gly
115 120 125
Leu Ser Gly Gly Gly Asn Gly Ser Lys Ser Phe Ser Glu Thr Ile Asn
130 135 140
Tyr Lys Gln Glu Ser Tyr Arg Thr Ser Leu Asp Lys Arg Thr Asn Phe
145 150 155 160
Lys Lys Ile Gly Trp Asp Val Glu Ala His Lys Ile Met Asn Asn Gly
165 170 175
Trp Gly Pro Tyr Gly Arg Asp Ser Tyr His Ser Thr Tyr Gly Asn Glu
180 185 190
Met Phe Leu Gly Ser Arg Gln Ser Asn Leu Asn Ala Gly Gln Asn Phe
195 200 205
Leu Glu Tyr His Lys Met Pro Val Leu Ser Arg Gly Asn Phe Asn Pro
210 215 220
Glu Phe Ile Gly Val Leu Ser Arg Lys Gln Asn Ala Ala Lys Lys Ser
225 230 235 240
Lys Ile Thr Val Thr Tyr Gln Arg Glu Met Asp Arg Tyr Thr Asn Phe
245 250 255
Trp Asn Gln Leu His Trp Ile Gly Asn Asn Tyr Lys Asp Glu Asn Arg
260 265 270
Ala Thr His Thr Ser Ile Tyr Glu Val Asp Trp Glu Asn His Thr Val
275 280 285
Lys Leu Ile Asp Thr Gln Ser Lys Glu Lys Asn Pro Met Ser
290 295 300
<210> 12
<211> 915
<212> PRT
<213> Artificial Synthesis (fusion His tag LukF protein)
<400> 12
Cys Ala Thr Ala Thr Gly Gly Cys Thr Cys Ala Gly Cys Ala Cys Ala
1 5 10 15
Thr Cys Ala Cys Cys Cys Cys Gly Gly Thr Thr Thr Cys Thr Gly Ala
20 25 30
Ala Ala Ala Ala Ala Ala Ala Gly Thr Thr Gly Ala Cys Gly Ala Cys
35 40 45
Ala Ala Ala Ala Thr Cys Ala Cys Cys Cys Thr Gly Thr Ala Cys Ala
50 55 60
Ala Ala Ala Cys Cys Ala Cys Cys Gly Cys Thr Ala Cys Cys Thr Cys
65 70 75 80
Thr Gly Ala Cys Thr Cys Thr Gly Ala Cys Ala Ala Ala Cys Thr Gly
85 90 95
Ala Ala Ala Ala Thr Cys Thr Cys Thr Cys Ala Gly Ala Thr Cys Cys
100 105 110
Thr Gly Ala Cys Cys Thr Thr Cys Ala Ala Cys Thr Thr Cys Ala Thr
115 120 125
Cys Ala Ala Ala Gly Ala Cys Ala Ala Ala Thr Cys Thr Thr Ala Cys
130 135 140
Gly Ala Cys Ala Ala Ala Gly Ala Cys Ala Cys Cys Cys Thr Gly Ala
145 150 155 160
Thr Cys Cys Thr Gly Ala Ala Ala Gly Cys Thr Gly Cys Thr Gly Gly
165 170 175
Thr Ala Ala Cys Ala Thr Cys Thr Ala Cys Thr Cys Thr Gly Gly Thr
180 185 190
Thr Ala Cys Ala Cys Cys Ala Ala Ala Cys Cys Gly Ala Ala Cys Cys
195 200 205
Cys Gly Ala Ala Ala Gly Ala Cys Ala Cys Cys Ala Thr Cys Thr Cys
210 215 220
Thr Thr Cys Thr Cys Ala Gly Thr Thr Cys Thr Ala Cys Thr Gly Gly
225 230 235 240
Gly Gly Thr Thr Cys Thr Ala Ala Ala Thr Ala Cys Ala Ala Cys Ala
245 250 255
Thr Cys Thr Cys Thr Ala Thr Cys Ala Ala Cys Thr Cys Thr Gly Ala
260 265 270
Cys Thr Cys Thr Ala Ala Cys Gly Ala Cys Thr Cys Thr Gly Thr Thr
275 280 285
Ala Ala Cys Gly Thr Thr Gly Thr Thr Gly Ala Cys Thr Ala Cys Gly
290 295 300
Cys Thr Cys Cys Gly Ala Ala Ala Ala Ala Cys Cys Ala Gly Ala Ala
305 310 315 320
Cys Gly Ala Ala Gly Ala Ala Thr Thr Cys Cys Ala Gly Gly Thr Thr
325 330 335
Cys Ala Gly Cys Ala Gly Ala Cys Cys Gly Thr Thr Gly Gly Thr Thr
340 345 350
Ala Cys Thr Cys Thr Thr Ala Cys Gly Gly Thr Gly Gly Thr Gly Ala
355 360 365
Cys Ala Thr Cys Ala Ala Cys Ala Thr Cys Thr Cys Thr Ala Ala Cys
370 375 380
Gly Gly Thr Cys Thr Gly Thr Cys Thr Gly Gly Thr Gly Gly Thr Gly
385 390 395 400
Gly Thr Ala Ala Cys Gly Gly Thr Thr Cys Thr Ala Ala Ala Thr Cys
405 410 415
Thr Thr Thr Cys Thr Cys Thr Gly Ala Ala Ala Cys Cys Ala Thr Cys
420 425 430
Ala Ala Cys Thr Ala Cys Ala Ala Ala Cys Ala Gly Gly Ala Ala Thr
435 440 445
Cys Thr Thr Ala Cys Cys Gly Thr Ala Cys Cys Thr Cys Thr Cys Thr
450 455 460
Gly Gly Ala Cys Ala Ala Ala Cys Gly Thr Ala Cys Cys Ala Ala Cys
465 470 475 480
Thr Thr Cys Ala Ala Ala Ala Ala Ala Ala Thr Cys Gly Gly Thr Thr
485 490 495
Gly Gly Gly Ala Cys Gly Thr Thr Gly Ala Ala Gly Cys Thr Cys Ala
500 505 510
Cys Ala Ala Ala Ala Thr Cys Ala Thr Gly Ala Ala Cys Ala Ala Cys
515 520 525
Gly Gly Thr Thr Gly Gly Gly Gly Thr Cys Cys Gly Thr Ala Cys Gly
530 535 540
Gly Thr Cys Gly Thr Gly Ala Cys Thr Cys Thr Thr Ala Cys Cys Ala
545 550 555 560
Cys Thr Cys Thr Ala Cys Cys Thr Ala Cys Gly Gly Thr Ala Ala Cys
565 570 575
Gly Ala Ala Ala Thr Gly Thr Thr Cys Cys Thr Gly Gly Gly Thr Thr
580 585 590
Cys Thr Cys Gly Thr Cys Ala Gly Thr Cys Thr Ala Ala Cys Cys Thr
595 600 605
Gly Ala Ala Cys Gly Cys Thr Gly Gly Thr Cys Ala Gly Ala Ala Cys
610 615 620
Thr Thr Cys Cys Thr Gly Gly Ala Ala Thr Ala Cys Cys Ala Cys Ala
625 630 635 640
Ala Ala Ala Thr Gly Cys Cys Gly Gly Thr Thr Cys Thr Gly Thr Cys
645 650 655
Thr Cys Gly Thr Gly Gly Thr Ala Ala Cys Thr Thr Cys Ala Ala Cys
660 665 670
Cys Cys Gly Gly Ala Ala Thr Thr Cys Ala Thr Cys Gly Gly Thr Gly
675 680 685
Thr Thr Cys Thr Gly Thr Cys Thr Cys Gly Thr Ala Ala Ala Cys Ala
690 695 700
Gly Ala Ala Cys Gly Cys Thr Gly Cys Thr Ala Ala Ala Ala Ala Ala
705 710 715 720
Thr Cys Thr Ala Ala Ala Ala Thr Cys Ala Cys Cys Gly Thr Thr Ala
725 730 735
Cys Cys Thr Ala Cys Cys Ala Gly Cys Gly Thr Gly Ala Ala Ala Thr
740 745 750
Gly Gly Ala Cys Cys Gly Thr Thr Ala Cys Ala Cys Cys Ala Ala Cys
755 760 765
Thr Thr Cys Thr Gly Gly Ala Ala Cys Cys Ala Gly Cys Thr Gly Cys
770 775 780
Ala Cys Thr Gly Gly Ala Thr Cys Gly Gly Thr Ala Ala Cys Ala Ala
785 790 795 800
Cys Thr Ala Cys Ala Ala Ala Gly Ala Cys Gly Ala Ala Ala Ala Cys
805 810 815
Cys Gly Thr Gly Cys Thr Ala Cys Cys Cys Ala Cys Ala Cys Cys Thr
820 825 830
Cys Thr Ala Thr Cys Thr Ala Cys Gly Ala Ala Gly Thr Thr Gly Ala
835 840 845
Cys Thr Gly Gly Gly Ala Ala Ala Ala Cys Cys Ala Cys Ala Cys Cys
850 855 860
Gly Thr Thr Ala Ala Ala Cys Thr Gly Ala Thr Cys Gly Ala Cys Ala
865 870 875 880
Cys Cys Cys Ala Gly Thr Cys Thr Ala Ala Ala Gly Ala Ala Ala Ala
885 890 895
Ala Ala Ala Cys Cys Cys Gly Ala Thr Gly Thr Cys Thr Cys Thr Cys
900 905 910
Gly Ala Gly
915

Claims (8)

1. An antibody specifically binding to staphylococcus aureus toxin, which can specifically bind to staphylococcus aureus gamma-hemolysin HlgB component and can generate cross reaction with other hemolysin components of the hemolysin;
the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL);
wherein the heavy chain variable region (VH) comprises the following CDR combinations: VH-CDR1, VH-CDR2 and VH-CDR3;
the light chain variable region (VL) comprises the following CDR combinations: VL-CDR1, VL-CDR2 and VL-CDR3;
the heavy chain variable region is:
EVQLQQSGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSPEWIGDINPNYDSTRYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARDGSSAMDYWGQGTSVTVSS（SEQ ID NO: 1），
VH-CDR1 amino acids: the content of the GYTFTDYN is GYTFTDYN,
VH-CDR2 amino acids: the number of the INPNYDST is less than the number of the INPNYDST,
VH-CDR3 amino acids: ARDGSSAMDY of the formula,
the light chain variable region is:
DIVMTQSPSSLSVSAGEKVTMTCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHRYPLTFGAGTKLELK（SEQ ID NO: 2），
VL-CDR1 amino acids: QSLLNSGNQKNY of the formula,
VL-CDR2 amino acids: the GAS is a GAS that is a mixture of gases,
VL-CDR3 amino acids: QNDHRYPLT.
2. The antibody of claim 1, wherein: the antibody is any one of a monoclonal antibody, a single-chain antibody, a single-domain antibody, a fully or partially humanized antibody or a chimeric antibody.
3. The antibody of claim 1, wherein: the heavy chain constant region of the antibody is of an IgG1 subtype, and the light chain constant region of the antibody is of a kappa type.
4. A conjugate comprising the antibody of any one of claims 1 to 3.
5. A nucleic acid molecule encoding the antibody of any one of claims 1 to 3.
6. A vector comprising the nucleic acid molecule of claim 5;
the vector is a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome or a phage vector.
7. A pharmaceutical composition comprising the antibody of any one of claims 1 to 3, or comprising the conjugate of claim 4, or comprising the nucleic acid molecule of claim 5, or comprising the vector of claim 6;
and optionally pharmaceutically acceptable excipients.
8. Use of the antibody of any one of claims 1 to 3, or the conjugate of claim 4, or the nucleic acid molecule of claim 5, or the vector of claim 6, or the pharmaceutical composition of claim 7, in the manufacture of a medicament for the prevention or treatment of a staphylococcus aureus infection.
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CN110437334B (en) * 2019-07-26 2020-12-01 西南医科大学 Fully human alpha-hemolysin recombinant antibody against staphylococcus aureus
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CN110498854B (en) * 2019-09-28 2021-01-29 中国人民解放军陆军军医大学 Antibody for resisting staphylococcus aureus enterotoxin B and application thereof
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