CN111748021A

CN111748021A - Polypeptide with binding affinity to chlamydia trachomatis MOMP and application thereof

Info

Publication number: CN111748021A
Application number: CN202010520616.7A
Authority: CN
Inventors: 朱珊丽; 张丽芳; 董海艳; 陈俊; 石威; 李文姝; 李明洋
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-06-09
Filing date: 2020-06-09
Publication date: 2020-10-09
Anticipated expiration: 2040-06-09
Also published as: CN111748021B

Abstract

The invention relates to a polypeptide specifically bound to major outer membrane protein of chlamydia trachomatis and application thereof, and discloses a polypeptide with binding affinity to major outer membrane protein of chlamydia trachomatis for the first time; the invention also provides the application of the polypeptide in diagnostic detection, and the polypeptide can be used as a targeting carrier in the diagnosis or treatment of drugs or molecular targeting agents.

Description

Polypeptide with binding affinity to chlamydia trachomatis MOMP and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a polypeptide with binding affinity to Major Outer Membrane Protein (MOMP) of chlamydia trachomatis and application thereof.

Background

Chlamydia trachomatis (Ct) is one of the main pathogens of sexually transmitted diseases, mainly causes male urethritis and female cervicitis, is often infected in a mixed manner with other pathogens, infects women through sexual transmission to cause poor pregnancy such as abortion, dead fetus and the like, and is closely related to the occurrence of cervical cancer. In recent years, STDs caused by Ct in China are in a continuously rising trend, and have great harm to human health. For Ct infectious diseases, an effective control method is not yet available. As a class of strictly intracellular parasitic, prokaryotic cell-type microorganisms with a unique developmental cycle, Ct exists extracellularly as an infectious pathogen, and exists intracellularly as a promoiety with reproductive properties. The division and the propagation of the primordium generate a large amount of protomers, and the mature protomers are released from the cells to infect new susceptible cells, and then are propagated and converted into primordium in cycles. Ct has now identified 19 serogroups, of which serotypes D, E, F, G and H are the major urogenital infections, and of which serotype E is the most common type reported at home and abroad. The Major Outer Membrane Protein (MOMP) is a high-content and wide-function transmembrane protein existing on the surfaces of Ct protomer and initiator, plays an important role in mediating the Ct to adhere to host cells, contains a plurality of antigen epitopes, and can be used as an important immunogen to stimulate the immune response of an organism. MOMP contains more cysteine, and can form a complex three-dimensional structure and polymer through intrachain and interchain disulfide bonds, so that the preparation of MOMP with complete structure and natural conformation is very difficult, and the development of a MOMP-based chlamydia vaccine is greatly limited. Researches show that MOMP VD2 and VD4 regions positioned outside a cell membrane contain a plurality of B cell epitopes and T cell epitopes, so the structural domain can be used as an important target for researching Ct vaccines, targeted therapy and diagnostic preparations.

At present, with the generation of the problem of persistent infection of pathogens and the development of breakthrough research brought by the molecular targeted therapy of tumors, scholars at home and abroad have attempted to switch the research on the treatment of infectious diseases to the molecular targeted therapy. Targeted therapies represented by monoclonal antibodies (mabs) have brought new promise for the treatment of infectious diseases and tumors, such as palivizumab for the prevention of respiratory syncytial virus infection in infants and young children, resisituzumab for the treatment of inhalation anthrax, trastuzumab (herceptin) for the treatment of metastatic breast cancer, etc., which have been approved by the FDA. However, the mAb-based targeted therapy still has its application limitations, such as weak tissue permeability, large toxic and side effects, high cost, etc., which seriously affect the broad application of the mAb in targeted therapy.

Based on the above description, there is still a need in the art to develop new methods for targeted treatment of Ct infection to improve the current clinical situation.

Disclosure of Invention

The invention aims to provide a polypeptide with binding affinity to Ct MOMP and application thereof.

In a first aspect of the present invention, there is provided a polypeptide having binding affinity for Ct MOMP, which is obtained by performing 12 to 20 (preferably 13 to 16, such as 14 or 15) amino acid variations on an amino acid sequence of Z segment (Z domain) of Staphylococcal Protein A (SPA) as a backbone.

In a preferred embodiment, the polypeptide having binding affinity for Ct MOMP is mutated at positions 9-11, 13-14, 17-18, 24-25, 27-28, 32, 35, 43 with respect to the amino acid sequence of Z fragment of staphylococcal protein A (SEQ ID NO: 1).

In another preferred embodiment, the polypeptide having binding affinity for Ct MOMP has, relative to the amino acid sequence of stretch Z of staphylococcal protein a:

the 9 th amino acid is mutated into H;

the 10 th amino acid is mutated into L;

the 11 th amino acid is mutated into A;

the 13 th amino acid is mutated into L;

the 14 th amino acid is mutated into M;

the 17 th amino acid is mutated into W;

the 18 th amino acid is mutated into T;

the 24 th amino acid is mutated into R;

the 25 th amino acid is mutated into H;

the 27 th amino acid is mutated into V;

the 28 th amino acid is mutated into H;

the 32 th amino acid is mutated into H;

the 35 th amino acid is mutated into R;

the 43 th amino acid was mutated to E.

In another preferred embodiment, the amino acid sequence of the polypeptide having binding affinity for Ct MOMP is as shown in seq id NO: 2, respectively.

In another preferred embodiment, the polypeptide having binding affinity for Ct MOMP has a KD value of 9.86 × 10 for interaction with Ct MOMP protein^-7M。

In another aspect of the present invention, there is provided a targeting molecule for targeting Ct MOMP, the targeting molecule comprising a polypeptide as described in any of the above, and a conjugate linked (or conjugated) to the polypeptide, the conjugate including (but not limited to): cysteine residues, polypeptide tags, drugs that inhibit Ct, or detectable labels (e.g., fluorescent labels, enzymes, biotin, or radioisotopes).

In a preferred embodiment, the conjugate is a peptide, and the conjugate and the polypeptide having binding affinity for Ct MOMP form a fusion polypeptide.

In another preferred embodiment, the polypeptide tags include, but are not limited to: his tag (e.g., 6 × His), Myc tag, GST tag, Flag tag.

In another preferred embodiment, the enzymes include, but are not limited to: alkaline phosphatase or horseradish peroxidase.

In another preferred embodiment, the conjugate is linked to the polypeptide having binding affinity for Ct MOMP in flexible peptides including (but not limited to): (Gly4Ser) 3.

In another aspect of the invention, there is provided an isolated polynucleotide encoding a polypeptide having binding affinity for Ct MOMP as described above.

In another aspect of the invention, there is provided a polynucleotide encoding said Ct MOMP targeting molecule, and wherein said conjugate is a peptide.

In another aspect of the invention, there is provided a recombinant vector comprising said polynucleotide.

In another aspect of the invention, there is provided a host cell comprising said recombinant vector, or comprising or having integrated into its genome said polynucleotide.

In another aspect of the invention, there is provided a method of preparing a polypeptide having binding affinity for Ct MOMP as described in any one of the preceding, the method comprising: (1) culturing said cell, thereby expressing said polypeptide having binding affinity for Ct MOMP; (2) and (3) separating and purifying the polypeptide obtained in the step (1).

In another aspect of the invention, there is provided a use of the polypeptide having binding affinity for Ct MOMP or the Ct MOMP-targeting molecule for preparing a medicament for treating Ct MOMP protein expression positive cells; or

The kit is used for preparing a detection reagent for detecting Ct MOMP; or

Used for preparing a diagnostic reagent for diagnosing Ct infection.

In a preferred embodiment, in the targeting molecule targeting Ct MOMP, the conjugate is an anti-Ct drug (e.g., toxin), the polypeptide having binding affinity for Ct MOMP, or the targeting molecule of Ct MOMP is used for treating Ct genital tract infection.

In another preferred embodiment, in the targeting molecule targeting Ct MOMP, the conjugate is a detectable label (such as a fluorescent label or an enzyme), the polypeptide having binding affinity for Ct MOMP, or the targeting molecule targeting Ct MOMP is used for Ct MOMP expression positive cells.

In another preferred example, the Ct MOMP expression positive cells include: urogenital tract infected epithelial cells, and the like.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising: any one of the polypeptides having binding affinity for Ct MOMP described above or any one of the targeting molecules targeting Ct MOMP described above; and a pharmaceutically acceptable carrier.

In another aspect of the present invention, there is provided a kit for diagnosing or treating Ct MOMP expression positive cells, the kit comprising: any of the aforementioned polypeptides having binding affinity for Ct MOMP, or any of the aforementioned targeting molecules targeting Ct MOMP, or the pharmaceutical composition.

In a preferred embodiment, said polypeptide having binding affinity for Ct MOMP or said Ct MOMP targeting molecule is in an effective amount.

In another aspect of the invention, there is provided a method for treating Ct infection, comprising administering to a subject in need thereof the polypeptide having binding affinity for Ct MOMP, or the targeting molecule targeting Ct MOMP.

In another aspect of the invention, there is provided a method for diagnosing Ct MOMP expression positive cells, comprising administering to a subject in need of treatment a targeted molecule of said targeted Ct MOMP; in the targeting molecule, the conjugate is a detectable label (e.g., a fluorescent label or an enzyme).

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

The invention is further described with reference to the drawings and the detailed description.

Drawings

FIG. 1, Z_{Ct MOMP}And comparison of Zwt sequences. The polypeptide Z of the invention_{Ct MOMP}The modified amino acid position in (A) is indicated in bold font (SEQ ID NO: 2).

FIG. 2, Z produced in example 1_{Ct MOMP}A polypeptide recombinant plasmid construction diagram and a composition schematic diagram of recombinant protein.

(A)Z_{Ct MOMP}Constructing a diagram of the recombinant plasmid; (B) constructing a Zwt recombinant plasmid map; (C) and (D) are each Z_{Ct MOMP}And Zwt schematic composition of prokaryotic expression of full-length recombinant protein, Z_{Ct MOMP}Represents a polypeptide having an amino acid sequence selected from SEQ ID NOs: 2, Zwt represents an amino acid sequence selected from SEQ ID NO: 1, 6xHis for a six histidine tag, HM for ndei (catatg) translated amino acids, LE for xhoi (ctcgag) translated amino acids.

FIG. 3, pET21a (+)/Z_{Ct MOMP}Electrophoretogram of the recombinant plasmid (1).

A is pET21a (+)/Z_{Ct MOMP}. M1: 1kb DNAmarker; 1: pET21a (+) vector plasmid; 2: pET21a (+)/Z_{Ct MOMP}An Affibody recombinant plasmid; 3: pET21a (+)/Z_{Ct MOMP}Affibody/NdeI+XhoI；4：Z_{Ct MOMP}Affibody DNA fragments; m2: DL2000 DNAmarker. B is pET21a (+)/Zwt. M1: 1kb DNA marker; 1: pET21a (+) vector plasmid; 2: pET21a (+)/Zwt recombinant plasmid; 3: pET21a (+)/Zwt/NdeI + XhoI; 4: a Zwt DNA fragment; m2: DL2000 DNA marker.

FIG. 4, Z_{Ct MOMP}The recombinant protein is subjected to prokaryotic expression and purification of SDS-PAGE (A) and Westernblot (B) for analysis.

M: a protein Marker; 1-2 are each purified Z_{Ct MOMP}And Zwt. In the Westernblot experiment, the primary antibody is Histag monoclonal antibody.

FIG. 5, MOMP recombinant protein prokaryotic expression identification and rabbit serum antibody preparation analysis.

(A) SDS-PAGE analysis of MOMP protein, M: a protein Marker; 1: coli.bl21(DE3) strain; pET21a (+)/MOMP transformed E.coli.BL21(DE3) strain; pET21a (+)/MOMP transformed E.coli.BL21(DE3) strain prior to IPTG induction; pET21a (+)/MOMP transformed E.coli.BL21(DE3) strain after IPTG induction; 5: purifying MOMP protein; (B) western blot analysis of MOMP recombinant purified protein, wherein the primary antibody is a his-tag monoclonal antibody, 1: purified MOMP recombinant protein; 2: a carrier protein; (C) the titer of the mouse serum antibody after the MOMP recombinant protein immunization is shown.

FIG. 6, culture and identification of Ct.

(A) After HeLa229 cells were infected with Ct, cytoplasmic inclusion bodies (containing large amounts of protomers and primordia) were visualized by iodine staining and Giemsa staining, respectively. (B) And detecting the Ct inclusion body by an indirect immunofluorescence method. The rabbit anti-MOMP polyclonal antibody is a primary antibody, FITC-goat anti-rabbit IgG is a secondary antibody, and DAPI stains cell nucleus.

FIG. 7, Z_{Ct MOMP}affinity of affibody and MOMP recombinant proteins was determined by SPR on a ProteOn XPR36 instrument.

A to B are each Z_{Ct MOMP}And the affinity analysis of the Zwt protein and the target protein MOMP protein.

FIG. 8, Z_{Ct MOMP}Western blot identification of binding of affibodies to MOMP proteins.

A to B are each Z_{Ct MOMP}Western blot detection of binding of affibody and Zwt proteins to MOMP protein in HeLa229 cells infected with Ct. The internal reference is GAPDH.

FIG. 9, Z_{Ct MOMP}Immunoprecipitation identification of binding of affibodies to MOMP proteins.

A to B are each Z_{Ct MOMP}Western blot detection of binding of affibody and Zwt proteins to MOMP protein in HeLa229 cells infected with Ct. The internal references are the heavy and light chains of immunoglobulins.

Detailed Description

The invention is described in detail below with reference to examples, which are intended to be illustrative only and not to be construed as limiting the scope of the invention, and many insubstantial modifications and variations of the invention can be made by an engineer skilled in the art based on the teachings of the invention.

As used herein, the term "polypeptide having binding affinity for Ct MOMP" refers to a polypeptide obtained by 12-20 amino acid variations using the amino acid sequence of Z fragment of staphylococcal protein A as a backbone, and which is capable of specifically binding Ct MOMP with little or no non-specific binding.

As used herein, the terms "polypeptide of the invention", "polypeptide having binding affinity for Ct MOMP", "Ct MOMP-binding polypeptide", "Z_{Ct MOMP}affibody polypeptides "," Z_{Ct MOMP}affibody”、“Z_{Ct MOMP}: 461, "affibody protein", "affibody recombinant protein", "Z_{Ct MOMP}Recombinant protein "may be used interchangeably; ct MOMP and MOMP can be used interchangeably; SPAZ and Zwt may be used interchangeably.

As used herein, the "targeting molecule" refers to a molecule that can target Ct MOMP obtained by linking the polypeptide having binding affinity for Ct MOMP of the present invention to other functional conjugates. The conjugate can be cysteine residue, polypeptide label, drug for inhibiting Ct, enzyme or detectable marker, etc.

As used herein, the "fusion polypeptide" is a subordinate concept of the "targeting molecule" and refers to a molecule that can target Ct MOMP and is obtained by linking the polypeptide having binding affinity for Ct MOMP of the present invention to other functional peptides (e.g., toxin protein or functional protein fragment).

The inventors selected Ct MOMP as the target antigen. The inventor takes a Z structural domain (Zwt, SEQ ID NO: 1) of staphylococcal protein A as a scaffold, carries out random mutation on a surface amino acid residue simulation antibody binding site, constructs a mutant library by a phage display technology, carries out affinity screening on the library by taking Ct MOMP as a target antigen, and finally obtains a polypeptide with high affinity for the Ct MOMP through a large amount of screening work.

The polypeptide of the invention is obtained by taking the amino acid sequence of the Z structural domain of the staphylococcal protein A as a framework and carrying out 14-20 (preferably 14) amino acid variations. As a preferred mode of the present invention, the polypeptide of the present invention has amino acid mutations at positions 9-11, 13-14, 17-18, 24-25, 27-28, 32, 35, 43 relative to the amino acid sequence of Z-fragment of staphylococcal protein A (SEQ ID NO: 1). More preferably, the polypeptide of the invention has the amino acid sequence of SEQ ID NO: 2, as shown in figure 1.

The invention also encompasses polypeptides formed by adding additional amino acid residues at either or both ends of the amino acid sequence of the Ct MOMP binding polypeptide. These additional amino acid residues may function when the polypeptide binds to Ct MOMPBut may also be used for other purposes as well, such as involving one or more of production, purification, stabilization, coupling or detection of the polypeptide. These additional amino acid residues may include one or more amino acid residues added for chemical coupling purposes. Such as the first or last addition of a cysteine residue at the N-or C-terminus of the polypeptide chain. Such additional amino acid residues may also include a "tag" for polypeptide purification or detection, such as a hexa-histidine peptide (His) that interacts with a tag antibody₆) A tag, either a "myc" tag or a "flag" tag. In addition, other alternatives known to those skilled in the art are also encompassed by the present invention.

The "additional amino acid residues" may also constitute one or more polypeptide domains with the desired function, such as the same binding function as the first, Ct MOMP binding domain, or other binding function, or an enzymatic function, or a fluorescent function, or a combination thereof.

The invention also includes polypeptides modified to increase their stability under alkaline conditions based on the Ct MOMP binding polypeptides. This stability includes site-directed substitution of any asparagine residue present in the unmodified sequence with an amino acid residue that is less sensitive to basic conditions. This property of reduced sensitivity to alkali, which is advantageous for using the polypeptides of the invention as affinity ligands in affinity chromatography, enables a prolonged lifetime of the affinity chromatography matrix, since the affinity chromatography column is subjected to frequent strong alkali treatments for elution between different reactions.

The invention also includes polypeptides obtained by other modifications based on the Ct MOMP binding polypeptides of the invention. These modified (usually without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

The Ct MOMP binding polypeptide can be connected with a conjugate to form a functional targeting molecule, and the connection can be connected or adsorbed through chemical bonds (including peptide bonds); the chemical bond is a covalent bond or a non-covalent bond. Preferably, the linkage is by peptide bond, thereby forming a fusion polypeptide. The Ct MOMP binding polypeptide and the conjugate may be linked directly or via a polypeptide linker (linker peptide). The linker comprises, for example, 1-30 amino acids; preferably 1-20 amino acids. The arrangement of the linker peptide does not substantially affect the activity of each polypeptide in the fusion protein. Preferably, the linkage may be performed using a flexible peptide (Gly4Ser) 3. Other linker peptides well known to those skilled in the art may also be used in the present invention.

In "heterologous" fusion polypeptides, where the Ct MOMP binding polypeptide constitutes a first domain or first moiety, and the second and other moieties have other functions than binding to the Ct MOMP, these contemplated results are also within the scope of the invention. The second and further portions of the fusion polypeptide may comprise a binding domain having affinity for other target molecules than Ct MOMP. Such binding domains may also be associated with the SPA domain, but have substitution mutations at 1 to about 20 positions. The result is a fusion polypeptide having at least one Ct MOMP binding domain and at least one domain with affinity for the other target molecule. This extends the utility of the polypeptides of the invention, e.g., as therapeutic agents or as capture, detection or isolation reagents.

Other options for the second and further portions of the fusion polypeptides of the invention include one or more portions for therapeutic use. In therapeutic applications, other molecules may be covalently or non-covalently coupled to the polypeptides of the invention by other means, such as by linking engineered P.aeruginosa exotoxin PE38KDEL or granzyme (GrB), etc., via a flexible peptide to the C-terminus of the Ct MOMP binding polypeptide, to form a fusion protein. Non-limiting examplesIncluded are enzymes that direct effector enzymes (e.g., carboxypeptidase) for "ADEPT" (antibody-mediated enzyme prodrug therapy); proteins including proteins to recruit effector cells and other components of the immune system; including cytokines such as IL-2, IFN γ, IL-12, TNFa, IP 10; including procoagulant factors such as tissue factor, von Willebrand factor; including toxins such as ricin A, calcheamicin, maytansinoids; including toxic small molecules such as auristatin analogs, doxorubicin, and the like. At the same time, for more convenient incorporation of radionuclides (e.g. for facilitating incorporation⁶⁸Ga、⁷⁶Br、¹¹¹In、⁹⁹Tc、¹²⁴I、¹²⁵I) For diagnosis or radionuclides (e.g. of the type⁹⁰y、¹³¹I、²¹¹At) for therapeutic use, the additional amino acids listed above (in particular hexa-histidine tag and cysteine) may be considered, with the aim of coupling the chelator of radioisotopes to the polypeptide sequence.

The invention also covers the purpose of detecting Ct MOMP positive cells based on the specificity of the polypeptide of the invention by connecting a detectable marker (such as a fluorescent marker, biotin or a radioactive isotope) to the Ct MOMP binding polypeptide.

"Ct MOMP binding affinity" means that binding affinity can be determined, for example, by using a surface plasmon resonance (surf plasmon resonance) technique such as

A polypeptide property detected by the device. Ct MOMP binding affinity can be detected by an experiment in which Ct MOMP protein is immobilized on the sensor chip of the device and then a sample containing the polypeptide to be detected is passed through the chip. Alternatively, the polypeptide to be detected may be immobilized on a sensor chip of the device, and then the sample containing the Ct MOMP may be passed through the chip. One skilled in the art can use the obtained sensor image to establish at least one qualitative measure of Ct MOMP binding affinity of the polypeptide. If quantitative measurement methods are required, for example to establish a certain KD-value between the interactions, a table can also be usedSurface plasmon resonance method. For example, the binding value may utilize

Ct MOMP protein was immobilized on the sensor chip of the device, and a sample of the polypeptide whose affinity is to be detected was prepared by serial dilution and injected in random order^-6M。。

The invention also provides an isolated nucleic acid encoding a Ct MOMP binding polypeptide or targeting molecule or fusion polypeptide of the invention, as well as the complementary strand thereof. The nucleic acid can be artificially synthesized in a complete sequence, and can also be obtained by a PCR amplification method respectively.

The invention also provides vectors comprising the nucleic acid molecules encoding the same. The vector may further comprise an expression control sequence operably linked to the sequence of the nucleic acid molecule to facilitate expression of the fusion protein. As used herein, "operably linked" or "operably linked" refers to a condition in which certain portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the coding sequence.

In the present invention, any suitable vector may be used, such as some vectors for cloning and expression of bacterial, fungal, yeast and mammalian cells, e.g., Pouwels et al, cloning vectors: as described in laboratory manuals.

In addition, recombinant cells containing the nucleic acid sequences are also encompassed by the present invention. The term "host cell" includes prokaryotic and eukaryotic cells. Commonly used prokaryotic host cells include E.coli, Bacillus subtilis, and the like; coli cells (e.coli), such as e.coli HMS174(DE3), or BL21(DE3), may be mentioned, for example. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells.

Methods of producing Ct MOMP binding polypeptides or targeting molecules or fusion polypeptides of the invention are also encompassed by the invention. The method includes culturing a recombinant cell containing a nucleic acid encoding a corresponding polypeptide to obtain a product polypeptide. The polypeptide prepared as described above may be purified to substantially homogeneous properties, for example, as a single band on SDS-PAGE.

Based on the information to be expressed and the current state of the art for recombinant expression of proteins, the skilled artisan, in conjunction with the present disclosure, can readily prepare the polypeptides of the invention. For example, a plasmid expressing an unmodified Z domain may be used as starting material. The desired substitution mutations can be introduced into this plasmid using known techniques to obtain the expression vectors of the invention.

When chemical polypeptide synthesis methods are used to prepare the polypeptides or targeting molecules or fusion proteins of the invention, any naturally occurring amino acid residues in the above polypeptides may be substituted with any corresponding, non-naturally occurring amino acid residue or derivative thereof, provided that the function of the product polypeptide is not substantially impaired.

The invention also relates to applications of the Ct MOMP binding polypeptide or the targeting molecule or the fusion polypeptide in different aspects, including treatment, diagnosis and/or detection.

The Ct MOMP binding polypeptide can be used as a substitute of a Ct MOMP antibody in different applications.

As a non-limiting example, it may be used to treat diseases characterized by Ct MOMP expression, such as urogenital chlamydia trachomatis infection, and the like. By binding to intracellular Ct MOMP, for in vivo and in vitro diagnosis of related diseases. The polypeptide of the invention can be used as a detection reagent, a capture reagent or a separation reagent, and can also be directly used as a therapeutic preparation or a means for targeting other therapeutic preparations to Ct MOMP protein. Methods of using the polypeptides of the invention in vitro can be performed in different ways, such as microtiter plates, protein arrays, biosensor surfaces, and tissue sections, among others. In order to adapt the polypeptides of the invention for specific uses, modifications and/or additions may be made to the polypeptides of the invention without departing from the scope of the invention.

These modifications and additions are described in detail below, which may include additional amino acids contained in the same polypeptide chain, or labels and/or therapeutic agents that chemically modify or otherwise bind to the polypeptides of the invention. In addition, fragments of the polypeptide that retain the ability to bind Ct MOMP are also encompassed by the invention.

The Ct MOMP binding properties of the polypeptides of the invention and the stability of the binding molecules for producing targeting molecules (including fusion proteins) and/or labels with the polypeptides mean that the polypeptides can also be used to target other active substances to sites of chlamydial infection, including cells expressing Ct MOMP. Thus, another aspect of the invention provides the use of a CtMOMP binding polypeptide as described herein conjugated to a substance having anti-cancer activity to transport the substance to cells expressing Ct MOMP, resulting in damage or apoptosis of the target cell.

Such an anti-cancer active substance may be a protein fused or coupled to a Ct MOMP binding polypeptide by a chemical bond, such as a protein selected from effector enzymes for "ADEPT" (antibody-directed enzyme promoter therapy) applications, proteins for recruiting effector cells and other components of the immune system, cytokines such as IL-2, IFN γ, IL-12, TNF α, IP10, etc., procoagulant factors such as tissue factor, von Willebrand factor, etc., toxins such as ricin A, Pseudomonas exotoxin, calcheamicin, maytansinoids, etc⁹⁰y、¹³¹I、²¹¹At, etc.), such an isotope may bind directly to the Ct MOMP binding polypeptide or may bind to the Ct MOMP binding polypeptide through a chelating agent, such as the well-known chelating agents DOTA or DTPA.

In a related aspect, the invention also provides a method of targeting a substance having anti-Ct activity to Ct MOMP expressing cells in vivo comprising administering to a patient a conjugate of the active substance described herein and a Ct MOMP binding polypeptide. Such conjugates have been described appropriately hereinbefore.

The invention also includes the use of the polypeptide that binds to Ct MOMP to detect Ct MOMP in a sample.

For example, such assays can be used to diagnose disease conditions characterized by expression of Ct MOMP. Detection of the presence of Ct MOMP can be performed in vivo or in vitro. The preferred choice for in vivo diagnosis is the use of Westernblot, ELISA, etc. The sample to be tested may for example be a biological fluid sample or a tissue sample. The current general method is to use antibodies aiming at CtMOMP, and the method can be applicable to the polypeptide which is combined with Ct MOMP and is detected by methods such as Westernblot, ELISA and the like and is used for identifying the expression of CtMOMP in fresh and frozen genital tract secretion samples.

The polypeptides of the invention can also be used as part of a fusion protein, wherein the other domain is a reporter enzyme or a fluorescent enzyme. Alternatively, it may be labeled with one or more fluorescent agents and/or radioisotopes, optionally labeled with a chelator. Suitable radioisotopes include⁶⁸Ga、⁷⁶Br、¹¹¹In、⁹⁹Tc、¹²⁴I and¹²⁵i, and the like.

The invention also comprises the application of the Ct MOMP binding polypeptide in the detection of Ct MOMP in biological liquid samples. This method comprises the steps of: (1) providing a sample of biological fluid from a patient to be tested, (2) adding a Ct MOMP binding polypeptide as described herein to the sample under conditions that allow the polypeptide to bind to any Ct MOMP present in the sample, (3) removing unbound polypeptide, and (4) detecting bound polypeptide. The amount of bound polypeptide detected correlates with the amount of CtMOMP present in the sample. In step (2), the Ct MOMP binding polypeptide may be added to the sample in any suitable form, including, for example, when the Ct MOMP binding polypeptide is immobilized on a solid support, by which the sample is contacted, or the Ct MOMP binding polypeptide is present in solution.

Other applications of the Ct MOMP binding polypeptide include: the method for detecting Ct MOMP in the sample comprises the following steps: (1) providing a tissue sample, such as a frozen section, suspected of containing Ct MOMP, (2) adding a Ct MOMP binding polypeptide of the invention to the sample under suitable conditions conducive to binding of the polypeptide to any Ct MOMP present in the sample, (3) removing unbound polypeptide, and (4) detecting bound polypeptide. The amount of bound polypeptide detected correlates with the amount of Ct MOMP present in the sample.

The invention also provides a kit for diagnosing Ct MOMP expression in a tissue sample, which comprises the Ct MOMP binding polypeptide fused with a reporter enzyme (such as alkaline phosphatase or horseradish peroxidase), a reagent for detecting the activity of the enzyme, and positive and negative control tissue sections.

The invention also provides a kit for diagnosing Ct MOMP expression in a tissue sample, which comprises the Ct MOMP binding polypeptide fused with a marker (such as a flag marker or a myc marker) detected by an antibody, a primary antibody specific to the marker, a secondary antibody specific to the primary antibody and coupled with a reporter enzyme, a reagent for detecting enzyme activity, and positive and negative control tissue sections.

One area of diagnostic application is the detection of Ct-infected cells or aggregates thereof in vivo. The invention provides a kit for performing such a diagnosis, comprising a Ct MOMP binding polypeptide of the invention labelled with a chelator, a diagnostic radioisotope (a non-limiting example being⁶⁸Ga、⁷⁶Br、¹¹¹In、⁹⁹Tc、¹²⁴I and¹²⁵i, etc.), and reagents for assaying incorporation efficiency.

As noted above, the invention encompasses the use of Ct MOMP binding polypeptides of the invention to target active substances to cells expressing Ct MOMP, such as urogenital mucosal cells. The invention also provides a kit for this purpose comprising a Ct MOMP binding polypeptide of the invention labeled with a chelator, a therapeutic radioisotope (a non-limiting example being⁹⁰Y、¹³¹I、2¹¹At), and reagents for assaying incorporation efficiency.

The present invention also provides a pharmaceutical composition comprising: the effective amount of the polypeptide with binding affinity to Ct MOMP protein or the targeting molecule of the targeting Ct MOMP protein, and a pharmaceutically acceptable carrier.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity), i.e., with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Sufficient details regarding pharmaceutically acceptable carriers can be found in Remington's pharmaceutical Sciences (mackpub.co., n.j.1991). Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, saline, glycerin and sorbitol. In addition, auxiliary substances, such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and stabilizers, such as albumin and the like, may also be present in these carriers.

The compositions may be formulated into a variety of dosage forms suitable for mammalian administration including, but not limited to: injection, capsule, tablet, emulsion, and suppository.

In use, a safe and effective amount of a polypeptide or targeting molecule of the invention having binding affinity for Ct MOMP protein is administered to a mammal (e.g., a human), wherein the safe and effective amount is typically at least about 1 microgram per kilogram of body weight, and in most cases no more than about 10 milligrams per kilogram of body weight, preferably the dose is from about 1 microgram per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The invention will be further illustrated with reference to the following specific examples.

Example 1 library construction and screening Studies of Ct MOMP binding Polypeptides

Random combinatorial libraries of phage-displayed Ct MOMP-binding polypeptides, i.e., libraries of many different SPA domain-associated polypeptides, are constructed, from which Ct MOMP-binding polypeptides are screened and their affinity identified.

1. Construction and identification of random combinatorial phage display libraries of Ct MOMP binding polypeptides

According to the amino acid sequence and structure of wild type SPA-Z (Nilsson B et al, Protein Eng. 1987; 1 (2): 107-. 113), random primers are designed for the corresponding coding sequences of the three helical structural regions, and the SPA coding sequence capable of causing random amino acid mutation is obtained by PCR amplification and named SPA-N. According to the conventional molecular cloning method, the SPA-N coding sequence is cloned to a pCANTAB5E vector through SfiI and NotI sites to construct a pCANTAB5E/SPA-N recombinant plasmid, the recombinant plasmid is transformed into a competent E.coli TG1 cell, a 2YT-A plate is coated, and the recombinant plasmid is cultured at 37 ℃ overnight. Namely the primary library, marked as affibody primary library for standby. Randomly picking 20 single clone colonies growing on the plate, identifying the extracted plasmid as positive clone by SfiI and Not I double enzyme digestion, sequencing and analyzing the randomness.

As a result, 18 clones out of 20 clones to be sequenced were sequenced according to the sequencing results, and since the randomness was completely different, the recombination rate was 18/20-90%, and the diversity was 18/18-100%, the above-mentioned transformed and cultured bacterial solution was diluted at a ratio of 2 × YT medium (1: 10 )²...), coating SOB-AG plate, calculating the number of single colony on the plate, calculating the storage capacity, accumulating the storage capacity by increasing the connection transformation times, and making the number of clone reach 2.4 × 10 after multiple connection transformation⁶Z protein variants (affibody molecules) having random amino acid residues at

positions

9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32, 35 and 43.

2. Screening and titer determination of Ct MOMP binding polypeptides

Sealing the purified MOMP coated 96-well enzyme label plate, adding phage library (primary library) for incubation, adding E.coli TG137 deg.C, slightly shaking for incubation, taking 100. mu.l, diluting with 2. multidrug with 2. multidot YT culture medium, taking 100. mu.l of diluent, coating SOB-AG plate, standing overnight at 30 deg.C, counting the number of colonies infected by combined phage, calculating the titer of Ct MOMP combined phage, and finding the plate with visible colonies, the titer is 1 × 10⁵(ii) a At this time, the first round of elutriation is completed, and 10 is added to the other part of the bacterial liquid¹⁰Culturing helper phage M13KO7 and kanamycin overnight, centrifuging, collecting supernatant, filtering with 0.22 μ M filter membrane to obtain CtRepeating the 3-round enrichment screening to obtain the phage library with Ct MOMP affinity screening, which is the second-grade phage library with titer of 1 × 10⁶(ii) a Repeating the above 4 rounds of enrichment screening on the basis of the secondary library to obtain a tertiary library. Meanwhile, a blank control without phage is set for synchronous screening.

3. Preparation of Ct MOMP (tumor necrosis factor-associated protein) binding polypeptide monoclonal phage and ELISA (enzyme-linked immunosorbent assay) identification

ELISA was used to screen for phages expressing Ct MOMP binding affibody molecules. Coating a 96-well enzyme label plate with Ct MOMP at 2 mu g/well, and standing overnight at 4 ℃; washing with PBS, and sealing with 2% skimmed milk powder for 2 h; washing, mixing phage obtained after four rounds of screening with equal volume of 3% skimmed milk powder, 200 μ l/hole, 37 deg.C, 2 h. Washing, adding HRP/anti-M13 enzyme-labeled secondary antibody (rabbit anti-M13, Abcam # ab6188) diluted by 1: 10000, 200 mul/hole, 37 ℃, 1 h; washing, adding OPD color development liquid 200 μ l/hole, 37 deg.C, 15 min; 2M H₂8O₄Stop the reaction at 50. mu.l/well; microplate reader (ELx 800)^TMBIO-TEK, Winooski, USA) read the OD490 values.

Selecting affibody molecules binding to antigen in four rounds of panning cycles, further performing phage ELISA assays to analyze their Ct MOMP binding activity after these four rounds of selection cycles, identifying phage encoding Ct MOMP binding polypeptides using ELISA values of a490 above 0.5 as selection criteria, and selecting 45 clones above this ELISA signal value for DNA sequence analysis.

4. Sequence detection and screening of Ct MOMP affibody molecules

A total of 45 single clones were sent to Shanghai, China for sequencing with the sequencing primer CATATGGTTGACAACAAATTCAACAAAGAA (SEQ ID NO: 5). Sequencing results the standard sequences Zwt and SPA-N were further analyzed for randomness and diversity of their three helical regions by DNA STAR software analysis. As a result, 20 clones with completely correct sequences were obtained, and after combining the repeated sequences, 20 clones with completely correct sequences were obtained.

According to the analysis of the DNA sequencing result, 1 single gram which has the strongest binding activity with Ct MOMP is selected from the 20 clones which are correctly sequencedDNA sequence (Z) of a Rongphage (i.e., a monoclonal phage displaying Ct MOMP affibody molecules)_{Ct MOMP}) The study was performed as a target with the amino acid sequence shown as SEQ ID NO: 2, and the coding sequence is shown as SEQ ID NO: 3. used for the next molecular cloning and expression of Ct MOMP binding affinity body and function detection.

Example 2 construction of Ct MOMP-binding polypeptide recombinant plasmid and expression and purification of prokaryotic protein

1 clone with a higher ELISA reading (Z in FIG. 1) was selected as before_{Ct MOMP}) And Zwt as a negative control for Ct MOMP binding polypeptides. In order to carry out functional detection on the screened affibody molecules, recombinant plasmid construction, prokaryotic protein expression and identification are carried out on the affibody molecules, and purified proteins are prepared.

Construction and characterization of recombinant plasmid pET21a (+)/affibody

PCR primers were designed with reference to the affibody gene sequence (GenBank: GY324633.1), the upstream primer

5’GGGAATTCCATATGGTTGACAACAAATTCAACAAAGAA 3 '(SEQ ID NO: 6, in italics and underlined Ned I cleavage site), downstream primer 5' CCGGAATTCCGTTTCGGAGCCTGAGCGT 3' (SEQ ID NO: 7, italic and underlined indicates an XhoI cleavage site); monoclonal affibody Z from the correct sequencing triple pool of the screen_{Ct MOMP}As a template, the affibody target gene (SEQ ID NO: 3) was amplified by PCR, while the full sequence (SEQ ID NO: 4) of the affibody Zwt was synthesized after prokaryotic codon optimization as a negative control. The PCR-amplified target gene was cloned into pET21a (+) vector by NdeI and XhoI to construct pET21a (+)/Z_{Ct MOMP}The recombinant plasmid of (4), and sequencing identification (FIG. 2, FIG. 3).

2.Z_{Ct MOMP}Prokaryotic protein preparation

The recombinant plasmid was transformed into E.coli (E.coli) BL21(DE3), cultured at 37 ℃ for 16 hours, and induced to culture for 6 hours by adding 0.8mM isopropylthio- β -D-thiogalactopyranoside (IPTG) (Merck, Germany) IPTG to express His-tagged Z_{Ct MOMP}And a Zwt protein. The recombinant protein expressed after induction was subjected to affinity chromatography using Ni-NTA Agarose (QIAGEN, USA) as affinity layerAnalytically purified and characterized by SDS-PAGE analysis. As a result, pET21a (+)/Z was successfully constructed using molecular biology techniques_{Ct MOMP}Recombinant plasmid and prokaryotic expression system is adopted to prepare purified Z_{ct MOMP}And Zwt recombinant fusion protein, which is analyzed by SDS-PAGE electrophoresis and detected by Western blot with His tag antibody as primary antibody (figure 4), and the molecular mass of the band stained with Coomassie brilliant blue is about 7.8kDa, and the expected Z is_{Ct MOMP}The molecular mass of the protein is consistent, and the Western blot result verifies that the protein at the position is the target protein again. The invention selects pET21a (+) vector, the starting enzyme site of its multiple cloning site is NdeI (CATATG) in design, its codon ATG is the amino acid (M) starting codon for target protein translation, thus the protein expressed by prokaryotic expression system is the full-length target protein Z_{Ct MOMP}And the carrier protein fragment is not contained, so that the interference of the carrier protein on the experimental result is avoided.

Example 3, Z_{Ct MOMP}Binding to Ct MOMP protein

To identify Z_{Ct MOMP}Specificity of binding to Ct MOMP, Z analytically screened by Surface Plasmon Resonance (SPR)_{Ct MOMP}And its control Zwt affibody binds to the target protein MOMP with affinity and specificity.

Preparation and identification of Ct MOMP

pET21a (+)/Ct MOMP constructed and stored in a laboratory is transformed into escherichia coli BL21(DE3), recombinant protein is expressed after IPTG induction, protein is prepared by purification of Ni-NTA affinity chromatography, and serum antibody is prepared by conventional immunization of white fungus rabbit in Japan. As a result, SDS-PAGE showed a distinct protein band to appear at a position of about 40kDa relative to the molecular mass (Mr), consistent with the expected size of the protein Mr (FIG. 5A); western blot analysis with mouse anti-6 XHis mAb as the primary antibody showed a single signal response band at Mr 40kDa (FIG. 5B), indicating that Ct MOMP protein can be specifically recognized and bound by His-tag antibody. ELISA detection shows that the rabbit has high titer antibody reaction after being immunized by Ct MOMP protein, which indicates that the high titer Ct MOMP specific rabbit serum antibody is successfully prepared (figure 5C).

Culture and identification of Ct

Cell culture: HeLa229 cells were treated as per 10⁵Inoculating the cells into 6-hole cell culture plates, removing cell culture solution after the cells grow into a compact monolayer, adding 2ml of PBS solution containing DEAE-D (30 mu g/ml) into each hole, acting at room temperature for 30min, taking out the E-type Ct standard strain frozen at-80 ℃, repeatedly freezing and thawing twice, adding 2 sterile glass beads, vortex oscillating for 1min, placing in a low-speed centrifuge at 32 ℃, 3000r/min, centrifuging for 5min, reserving supernatant, removing DEAE-D solution from the cell holes, adding 2ml of RPMI1640 culture medium and 45 mu 1E-type Ct standard strain (7.1 × 10)⁴IFU/bore). Centrifuging at 1500r/min for 1h, sucking off liquid in the holes, adding 2ml of chlamydia infection liquid (RPMI1640 culture medium 180ml, fetal calf serum 20ml, cycloheximide 1mg/L, gentamicin 50mg/L, vancomycin 25mg/L) into each hole, and continuing culturing. After 44-48 h, identifying an inclusion body formed by Ct by adopting iodine dyeing and Giemsa dyeing; meanwhile, the rabbit MOMP polyclonal antibody prepared by the method is used as a primary antibody, and a FITC-goat anti-rabbit antibody is used as a secondary antibody for indirect immunofluorescence detection.

The results show that, after infection of HeLa229 cells with Ct, (A) iodine staining and Giemsa staining both observed cytoplasmic inclusion bodies (containing large amounts of protomers and primordia). Iodine staining resulted in the observation of tan inclusions in the cytoplasm, and Giemsa staining resulted in the observation of lightly stained inclusions in the cytoplasm, often squeezing the nucleus to one side (fig. 6A). (B) The indirect immunofluorescence method is used for detecting the Ct inclusion body, a blue-green fluorescence block with larger volume is the Ct inclusion body (figure 6B), and the inclusion body does not appear in an uninfected cell group and a PBS control group. The above results show that the Ct culture is successful, and the prepared rabbit anti-MOMP antibody can effectively identify MOMP protein.

3.Z_{Ct MOMP}Sensor analysis of polypeptides

Ct MOMP protein and Z were performed in a ProteOn XPR36 systematic apparatus (Bio-Rad Co., Ltd.)_{Ct MOMP}Affinity analysis of the interaction between the polypeptides, i.e., analysis of the His-tagged Z by Surface Plasmon Resonance (SPR)_{Ct MOMP}And its interaction between the control Zwt affibody molecule and the Ct MOMP protein. According to the operating manual, the Ct MOMP protein is fixed on different flow cells by coupling to a GLH chip, and then the protein is sievedAffinity determination between selected polypeptides. The 7 th flow cell surface was activated and deactivated to serve as a blank at the time of injection. Affibody molecules were diluted in 5 different gradient concentrations, 4000nM, 2000nM, 1000nM, 500nM, 250nM, 125nM, respectively, and bound to Ct MOMP, respectively. All analyses were performed at 25 ℃ with an injection sample volume of 200. mu.l and random sequential injections at a flow rate of 30. mu.l/min, followed by a wash with 100mM HCl (BIO-RAD cat # 176:. sup. 2250100mM HCl) for 6min (dissociation) using a ProteOn Manager^TM1: the 1 langmuir binding model analyzes the binding curve (sensorgram).

The result follows Z_{Ct MOMP}The molecular concentration is increased, the capability of the molecular concentration to interact with the target protein Ct MOMP is enhanced, and the affinity equilibrium dissociation constant KD value, Z_{Ct MOMP}And their control Zwt molecules are 9.86 × 10 respectively^-7mol/L and 8.83 × 10^-3mol/L (FIG. 7). Z_{Ct MOMP}The KD values of the molecules differ by a factor of 10000. Z obtained by screening_{Ct MOMP}Can be combined with Ct MOMP recombinant protein with high affinity, and meanwhile, wild Zwt molecules and Ct MOMP protein have almost no binding force. Indicating the selected Z_{Ct MOMP}The molecule has higher specific affinity with Ct MOMP protein, and simultaneously shows that Z of prokaryotic induction expression_{Ct MOMP}The molecule and Ct MOMP protein have biological activity.

Thus, Z of the invention_{Ct MOMP}The molecule and MOMP have the ability to bind and recognize each other. Verification of Z from the protein level_{Ct MOMP}Affinity to MOMP.

Example 4, Z_{Ct MOMP}Binding of polypeptide to intracellular expressed Ct MOMP protein

Z screened for further validation_{Ct MOMP}Affinity with Ct MOMP, using HeLa229 cell infected by E-type Ct as research object, taking total cell protein after cracking, further verifying Z by Western blot and immunoprecipitation method respectively_{Ct MOMP}Binding between the molecule and the Ct MOMP protein molecule.

Cell culture: HeLa229 cells were treated as per 10⁵The amount of each well is inoculated into a 6-well cell culture plate, and the cells are discarded after the cells grow into a compact monolayerAdding 2ml of PBS solution containing DEAE-D (30 mu g/ml) into each well, acting at room temperature for 30min, taking out the E type Ct standard strain frozen at-80 ℃, repeatedly freezing and thawing twice, adding 2 sterile glass beads, performing vortex oscillation for 1min, placing in a low-speed centrifuge at 32 ℃, 3000r/min, centrifuging for 5min, taking out supernatant, discarding DEAE-D solution in the cell wells, adding 2ml of RPMI1640 culture medium and 45 mu l E type Ct standard strain (7.1 × 10) into each well⁴IFU/bore). Centrifuging at 1500r/min for 1h, sucking off liquid in the holes, adding 2ml of chlamydia infection liquid (RPMI1640 culture medium 180ml, fetal calf serum 20ml, cycloheximide 1mg/L, gentamicin 50mg/L, vancomycin 25mg/L) into each hole, and continuing culturing. And after 44-48 h, adding cell lysate, centrifuging for 15 minutes at 10000r/min, and taking supernatant protein.

Western blot detection: boiling part of the supernatant, performing SDS-PAGE electrophoresis, transferring to membrane, and adding Z_{Ct MOMP}The Western blot detection is carried out by taking a molecule as a primary antibody, taking a mouse anti-His tag as a secondary antibody and taking HRP-goat anti-mouse as a tertiary antibody.

Immunoprecipitation (IP): a portion of the supernatant was incubated with rabbit anti-MOMP polyclonal antibody and slowly shaken overnight at 4 ℃. Add 20. mu.l of well resuspended ProteinA + GAgarose and shake slowly at 4 ℃ for 3 hours. Centrifuge at 2500r/min for 5 minutes and carefully aspirate the supernatant. Washing the precipitate with PBS for 5 times, removing supernatant, adding 40 μ l 1XSDS-PAGE electrophoresis loading buffer solution, resuspending the precipitate, boiling, performing SDS-PAGE electrophoresis, transferring membrane, and performing Z-electrophoresis_{Ct MOMP}The Western blot detection is carried out by taking a molecule as a primary antibody, taking a rabbit anti-His tag as a secondary antibody and taking an HRP-goat anti-rabbit antibody as a tertiary antibody.

Western blot results show that Z_{Ct MOMP}The molecule can specifically recognize and combine with a trimer MOMP with the molecular weight of about 100kDa in Ct-infected HeLa229 cells, while the band can not be seen in the uninfected HeLa229 cells at the same position, and the target protein can not be recognized by a negative control Zwt (figure 8); IP results show that the protein specifically recognized and bound by rabbit MOMP polyclonal antibody can be really recognized and bound by Z_{Ct MOMP}The molecules bound and had molecular weights around 100kDa (FIG. 9), confirming the trimeric form of MOMP.

The above results further validate Z from the cellular level_{Ct MOMP}The molecule has strong affinity with Ct MOMPForce and specificity of binding.

Sequence listing

<110> Wenzhou university of medical science

<120> a polypeptide having binding affinity for MOMP of Chlamydia trachomatis and use thereof

<160>7

<170>PatentIn version 3.5

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<213>Staphylococcus aureus

<221>MISC_FEATURE

<222>(1)..(58)

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Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

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Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

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Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210>2

<211>58

<212>PRT

<213> Artificial sequence

<221>MISC_FEATURE

<222>(1)..(58)

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Val Asp Asn Lys Phe Asn Lys Glu His Leu Ala Ala Leu Met Glu Ile

1 5 10 15

Trp Thr Leu Pro Asn Leu Asn Arg His Gln Val His Ala Phe Ile His

20 25 30

Ser Leu Arg Asp Asp Pro Ser Gln Ser Ala Glu Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

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gaccctgccg aacctgaacc gacaccaggt acacgctttc atccactctc 100

tgcggaacaa cccgtctcag tctgctgagc tcctggctga agctaaaaaa 150

ctgaacgacg ctcaggctcc gaaa 174

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tgaaagacga cccgtctcag tctgctaacc tgctggctga agctaaaaaa 150

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catatggttgacaacaaattcaacaaagaa 30

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<222>(1)..(38)

<400>6

gggaattccatatggttgacaacaaattcaacaaagaa 38

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Claims

1. A polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis, wherein: the polypeptide is represented by SEQ ID NO: 1 as skeleton, and carrying out 12-20 amino acid variations to obtain the polypeptide.

2. A polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis according to claim 1, wherein the polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis is represented by the amino acid sequence as set forth in SEQ ID NO: 1, amino acid mutations occur at positions 9-11, 13-14, 17-18, 24-25, 27-28, 32 and 35 of the amino acid sequence of the Z segment of staphylococcal protein A.

3. A polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis according to claim 2, wherein the polypeptide has binding affinity for the major outer membrane protein of chlamydia trachomatis relative to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of fragment Z of staphylococcal protein a, said polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis:

the 9 th amino acid is mutated into H;

the 10 th amino acid is mutated into L;

the 11 th amino acid is mutated into A;

the 13 th amino acid is mutated into L;

the 14 th amino acid is mutated into M;

the 17 th amino acid is mutated into W;

the 18 th amino acid is mutated into T;

the 24 th amino acid is mutated into R;

the 25 th amino acid is mutated into H;

the 27 th amino acid is mutated into V;

the 28 th amino acid is mutated into H;

the 32 th amino acid is mutated into H;

the 35 th amino acid is mutated into R.

4. A polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis according to claim 3, wherein the amino acid sequence of said polypeptide is selected from the group consisting of: SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

5. The polypeptide having binding affinity for the major outer membrane protein of Chlamydia trachomatis according to claim 4, wherein the polypeptide interacts with the major outer membrane protein of Chlamydia trachomatis with a KD value of 9.86 × 10^-7M。

6. A targeting molecule for targeting the major outer membrane protein of chlamydia trachomatis, said targeting molecule comprising the polypeptide of any one of claims 1 to 5, and a conjugate linked to said polypeptide, said conjugate comprising: a cysteine residue, a polypeptide tag, a detectable label, or an agent that inhibits chlamydia trachomatis.

7. An isolated polynucleotide encoding the polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis according to claim 4, having the sequence of SEQ ID NO: 3, respectively.

8. A recombinant vector comprising the polynucleotide of claim 7.

9. A host cell comprising the recombinant vector of claim 8, or comprising a genome into which the polynucleotide of claim 7 has been integrated.

10. The use of the targeted chlamydia trachomatis targeting molecule of claim 6, wherein the conjugate is a medicament of the major outer membrane protein of chlamydia trachomatis for the preparation of a medicament for the treatment of a chlamydia trachomatis infectious disease;

or the conjugate is a polypeptide label or a detectable marker, and is used for preparing a detection reagent for detecting the chlamydia trachomatis infection or preparing a diagnostic reagent for diagnosing the chlamydia trachomatis infection disease.

11. A pharmaceutical composition, comprising: a polypeptide having binding affinity for the major outer membrane protein of chlamydia trachomatis according to any one of claims 1 to 5 or a targeting molecule targeting the major outer membrane protein of chlamydia trachomatis according to any one of claim 4; and a pharmaceutically acceptable carrier.

12. A kit for diagnosing a disease caused by chlamydia trachomatis infection, said kit comprising: the targeting molecule of claim 6 for targeting the major outer membrane protein of Chlamydia trachomatis, wherein the targeting molecule is a polypeptide tag or a detectable label, and a detection reagent for detecting the polypeptide tag or the detectable label.

13. A kit for treating a disease caused by chlamydia trachomatis infection, said kit comprising: a polypeptide having binding affinity for the major outer membrane protein of Chlamydia trachomatis according to any one of claims 1 to 5, or a targeting molecule targeting the major outer membrane protein of Chlamydia trachomatis according to any one of claims 6, or a pharmaceutical combination according to any one of claims 11.