CN105820219B - There is polypeptide and its application of binding affinity to HPV18 E7 albumen - Google Patents

Abstract

The invention discloses polypeptide and its application that a kind of couple of HPV18E7 has binding affinity, the E7 albumen for disclosing a kind of couple of HPV18 for the first time has the polypeptide of binding affinity；Diagnosis or therapeutical uses the present invention also provides the polypeptide as drug or molecular targeted reagent.

Description

There is polypeptide and its application of binding affinity to HPV18 E7 albumen

Technical field

The invention belongs to bioengineering field, the E7 egg of specifically a kind of pair of 18 type of HPV18E7(human papilloma virus It is white) with binding affinity polypeptide and its application.

Background technique

Cervical carcinoma is to threaten the principal disease of human health and life, occupies second in the cancer morbidity of women, only secondary It is more common in developing country in breast cancer, even occur the trend of rejuvenation in recent years.In China, annual cervical carcinoma New case is probably 13.15 ten thousand, accounts for global 1/7, the morbidity and mortality of cervical carcinoma rank the second in the world.Although nearly 60 Nian Lai, scholars both domestic and external have conducted extensive research, and the prevention vaccine of cervical carcinoma has listed, but due to it is many-sided because The common presence of element, the clinical treatment for cervical carcinoma, effect are still undesirable at present.

Human papilloma virus (Human Papillomavirus, HPV) infection is the Important cause of disease of cervical carcinoma.Epidemic disease It learns research shows that 90% or more cervical carcinoma is related to HPV infection, the especially HPV16 in high-risk HPV and 18 types, uterine neck squama In shape cell cancer, 16 type of the HPV infection positive accounts for 51% or more, and 18 type of the HPV infection positive accounts for 56% in adenocarcinoma of the uterine cervix.HPV base Because group is there are about 8 000 bp, by early transcription area (area Early transcribed region, E), late transcription area in structure (area Late transcribed region, L) and noncoding region (Non-coding region, URR) composition.The area E have E1, 6 opening code-reading frames such as E2, E4, E5, E6, E7 (Open reading frame, ORF), the albumen that they are encoded both participate in disease The process of poison duplication and transcription, wherein E6 and E7 is tumor antigenicity albumen, and E6 is in conjunction with P53, E7 and pRB protein binding, induction Infection cell converts, and cell causes to immortalize because duplication is out of control.

Affine body molecule, English name affibody are a kind of brand-new affinity ligands, and structure is by 58 amino acid Composition, relative molecular weight are small, and about 6.5 × 10³, it is functionally similar to antibody, it can be with corresponding antigen, receptor or ligand knot It closes, but has the unexistent property of some antibody, if relative molecular weight is small, only 6.5 × 10³, Folding rate is fast, selectivity With affinity height and stable in physicochemical property, chemical modification etc. can tolerate, therefore people are called " artificial antibody ". These peculiar properties that affibody molecule has, welcome it in each field of immunology and bio-science field Wide application prospect, from 20th century since the 80's were reported for the first time, researchers just to which create keen interest, because This its development is also extremely rapid.

Based on above description, the novel drugs or new of magnetic target therapy HPV related neoplasms disease are still urgently studied in this field Method, to improve current clinical status.

Summary of the invention

The purpose of the invention is to overcome shortcoming and defect of the existing technology, and providing a kind of HPV18E7 has The polypeptide of binding affinity and its application.

The first purpose of this invention, technical solution are that the E7 albumen of a kind of pair of 18 type of human papilloma virus has knot Close affinity polypeptide, it is characterised in that: the polypeptide be using Z sections of staphylococcus A albumen of amino acid sequence as skeleton, into The polypeptide obtained after 12-20 amino acid variation of row.

Further setting is the amino acid sequence relative to Z sections of staphylococcus A albumen, and the polypeptide is in 9-11,13- 14,17-18,24-25,27-28,32,35,43 upper generation amino acid mutations.

Further setting is the amino acid sequence relative to Z sections of staphylococcus A albumen, the polypeptide:

9th amino acids sport P, F or A；

10th amino acids sport F, L or V；

11st amino acids sport C, L or R；

13rd amino acids sport R, M or S；

14th amino acids sport Q, V or G；

17th amino acids sport S, F or A；

18th amino acids sport P, W or V；

24th amino acids sport H, E or P；

25th amino acids sport H or E；

27th amino acids sport E, A or D；

28th amino acids sport P, E or L；

32nd amino acids sport G, R or P；

35th amino acids sport P, H, Q or V；

43rd amino acids sport E, D or K.

It is 1 × 10 that further setting, which is the KD value of the E7 protein-interacting of the polypeptide and HPV 18,^-6M To 3 × 10^-7M。

Second object of the present invention provides a kind of targeting molecule for targeting 18 type of human papilloma virus, the target Tropism molecule includes previously described any polypeptide, and the conjugate of (or coupling) of being connected with the polypeptide, the coupling Object includes: cysteine residues, and Polypeptide tags inhibit the drug of 18 type of human papilloma virus or detectable marker (such as glimmering Signal, enzyme, biotin or radioactive isotope).

In this setting, the conjugate has combination affine with the E7 albumen to 18 type of human papilloma virus The polypeptide of power constitutes fused polypeptide.In this setting, the Polypeptide tags include but is not limited to: His label (such as 6 × His), Myc label, GST label, Flag label, the enzyme includes but is not limited to: alkaline phosphatase or horseradish peroxidase.

It includes: toxin that further setting, which is the drug of 18 type of inhibition human papilloma virus,；The toxin is tool There is the toxin for inhibiting 18 type virus infection of human papilloma virus or function of tumor inhibition, such as diphtheria toxin, ricin (WA), green pus The functional fragment of bacillus exotoxin or the toxin；And the tumour is the tumour of the human papilloma virus 18 type positive.

In this setting, the toxin is that the functional fragment of Pseudomonas aeruginosa Exotoxin A or the toxin is Pseudomonas aeruginosa The active fragment PE38KDEL of exotoxin A.Preferably, the Pseudomonas aeruginosa Exotoxin A or its functional fragment be connected to it is described The E7 albumen to HPV 18 have binding affinity polypeptide carboxyl terminal (end C).In this setting, The conjugate and the E7 albumen to 18 type of human papilloma virus have the polypeptide of binding affinity with flexible peptide Connection, the flexibility peptide includes but is not limited to: (Gly4Ser) 3.

Third object of the present invention, provides a kind of isolated polynucleotides, and coding is any described to human milk above The E7 albumen of head 18 type of tumor virus has the polypeptide of binding affinity.

Fourth object of the present invention provides a kind of polynucleotides, encodes previously described targeting human papilloma virus The targeting molecule of 18 types, and the wherein described conjugate is peptide.

5th purpose of the invention, provides a kind of recombinant vector, which includes previously described polynucleotides.

6th purpose of the invention, provides a kind of host cell, which includes previously described recombinant vector, It includes have or genome in be integrated with previously described polynucleotides.

7th purpose of the invention provides a kind of 18 type of previously described any pair of human papilloma virus for preparing The method that E7 albumen has the polypeptide of binding affinity, which is characterized in that the described method includes:

(1) previously described cell is cultivated, to express any E7 to 18 type of human papilloma virus described previously Albumen has the polypeptide of binding affinity；

(2) polypeptide of (1) acquisition is isolated and purified.

8th purpose of the invention provides a kind of E7 albumen of 18 type of previously described any pair of human papilloma virus The purposes of the targeting molecule of polypeptide or 18 type of targeting human papilloma virus with binding affinity, is used to prepare The drug for treating 18 type of human papilloma virus infectious disease or 18 type of human papilloma virus expression positive tumor；Or it is used to prepare Detect the detection reagent of 18 type virus infection of human papilloma virus；Or it is used to prepare 18 type of diagnosis of human papilloma viral infection disease The diagnostic reagent of disease or 18 type of human papilloma virus expression positive tumor.

9th purpose of the invention, provides a kind of pharmaceutical composition comprising previously described to human papilloma virus Targeting HPV 18 described in polypeptide or claim 5 or 6 of the E7 albumen of 18 types with binding affinity Targeting molecule；And pharmaceutically acceptable carrier.

Tenth purpose of the invention, provide it is a kind of for diagnose or treat 18 type of human papilloma virus infectious disease or 18 type of human papilloma virus expresses the medicine box of positive tumor, includes: previously described any pair of human papilloma in the medicine box The E7 albumen of viral 18 types has the polypeptide of binding affinity or the target of previously described targeting 18 type of human papilloma virus Tropism molecule or previously described pharmaceutical composition.

The present invention is described further with specific embodiment with reference to the accompanying drawings of the specification.

Detailed description of the invention

Fig. 1 recombinant plasmid pET21a (+)/HPV16E7 digestion qualification figure；

In Fig. 1: M1:1kb DNA marker;1:pET21a (+) plasmid 2:pET21a (+)/HPV18E7; 3: pET21a(+)/HPV18E7//NdeI+XhoI；4:HPV18E7 DNA fragmentation；M2: DL2000 DNA marker；

The SDS-PAGE electrophoresis and Western Blot of Fig. 2 HPV18E7 albumen are analyzed；

In Fig. 2, M:marker;1-3. recombinant plasmid pET21a (+)/HPV18E7 respectively through IPTG induction 9h, 6h, 3h protein expression；4. recombinant plasmid pET21a (+)/HPV18E7 is expressed without IPTG inducible protein；5. E.coli. BL21 (DE3) bacterial strain.A:SDS-PAGE；B:Western Blotting analysis (primary antibody be mouse resist 6 × His monoclonal antibody (1: 5000))；

Fig. 3 Elisa detects the datagram of the compatibility of three-level library monoclonal；

Fig. 4 selects the 3 clone's amino acid sequences studied；

Fig. 5 A pET21a (+)/Z _{HPV18 E7}Construction of recombinant plasmid schematic diagram;

Fig. 5 B recombinant plasmid pET21a (+)/Z_{HPV18 E7}Digestion qualification figure；

In Fig. 5 B, M1:DL2000 DNA marker;1:Z HPV18 E7 DNA fragmentation 2:pET21a (+)/Z_{HPV18 E7}/NdeI+EcoRI; 3: pET21a(+)/Z _{HPV18 E7}Recombinant plasmid；4:pET21a (+) plasmid；M2:. 1kb DNA marker；

Fig. 6 A recombinant protein pET21a (+)/Z_{HPV18 E7}Expression；

In Fig. 6 A, M:marker;E.coli. BL21 1. (DE3) bacterial strain;2. recombinant plasmid pET21a (+)/Z_{HPV18 E7}Before induction；3. pET21a(+)/Z_{HPV18 E7:228}Recombinant protein；4. pET21a(+)/Z_{HPV18 E7:242}Recombinant protein；5 . pET21a(+)/Z _{HPV18 E7:310}Recombinant protein；6. SPAZ albumen；

Fig. 6 B recombinant protein pET21a (+)/Z_{HPV18 E7}Purifying figure；

In Fig. 6 B, M:marker;；1. pET21a (+)/Z after purification_{HPV18 E7:228}Albumen；2. pET21a after purification (+)/Z _{HPV18 E7:242}Albumen；3. pET21a (+)/Z after purification_{HPV18 E7:310}Albumen；4. Zwt albumen after purification；

Fig. 7 Z_{HPV18 E7}It is identified with the surface plasma resonance of HPV18 E7 affinity；

Fig. 7 A: various concentration Z_{HPV18 E7:228}The sensing of acquisition;Fig. 7 B: various concentration Z_{HPV18 E7:242}The biography of acquisition Sense;Fig. 7 C: various concentration Z_{HPV18 E7:310} The sensing of acquisition;Fig. 7 D:Z_{HPV18 E7:228,}Z _{HPV18 E7:242、}Z_{HPV18 E7:310}And its compared with surface plasma resonance of the SPA-z reference protein in conjunction with HPV18 E7；

Fig. 8 Z_{HPV18 E7}(confocal laser is identified with the cellular immunofluorescence method of HPV18 E7 native protein affinity Microscope × 400)；

Fig. 8 A:Z_{HPV18 E7:228}The indirect cellular immunofluorescence of albumen detects；Fig. 8 B:Z_{HPV18 E7:242}Albumen it is indirect Cellular immunofluorescence detection；Fig. 8 C:Z_{HPV18 E7:310}The indirect cellular immunofluorescence of albumen detects；

Each sets of numbers of Fig. 9 HeLa cell is inoculated with transplanted tumor in nude mice growth curve；

Figure 10 HeLa cell Xenografts in nude mice tissue HE stained slice observes (× 400)；

A:4x10⁷/ ml HeLa groups of cells; B:1x10⁷/ ml HeLa groups of cells; C:1x10⁶/ ml HeLa cell Group；

Figure 11 normal nude mice injects dy-Z_{HPV18 E7:228}Biological near infrared signal distribution map afterwards

A: normal nude mice injects dy-Z_{HPV18 E7:228}The biological near infrared signal image of each period afterwards；B: normal naked Mouse injects dy-Z_{HPV18 E7:228}The ratio figure of the kidney of each period and normal tissue near infrared signal intensity afterwards.

Figure 12 HeLa mice with tumor injects the Z of dylight label_{HPV18 E7:228}, Z_{HPV18 E7:310}, Z_{HPV18 E7:242}, Z_WTAfter give birth to Object fluorescence distribution figure；

Figure 13 HeLa mice with tumor injects the Z of dylight label_HPV18E7The bio distribution of tumour and kidney after affibody Curve.A, B, C, D are respectively HeLa mice with tumor injection Dy755-Z_{HPV18 E7:228}；Dy755-Z_{HPV18 E7:310}；Dy755-Z_{HPV18 E7:224}；The fluorescence intensity ratio of each period tumour, left kidney and right kidney and normal tissue after Dy755-Zwt.E is each time Section HeLa mice with tumor injects Dy755-Z_{HPV18 E7:228}；Dy755-Z_{HPV18 E7:310}；Dy755-Z_{HPV18 E7:224}；Dy755-Zwt The fluorescence intensity ratio of each period tumour and normal tissue afterwards.

Figure 14 pET21a (+)/Z_{HPV18 E7}The construction of recombinant plasmid schematic diagram of/PE38KDEL；

Figure 15 recombinant plasmid pET21a (+)/Z_{HPV18 E7/}PE38KDEL digestion qualification figure；

In Figure 15, M1:DL10000Bp Marker; 1: pET21a(+) / Z_{HPV18 E7:228}/ PE38KDEL plasmid 2: pET21a(+)/₂₂₈/ PE38KDEL plasmid; 3: pET21a(+)/ Z_{HPV18 E7:228}/PE38KDEL/NdeI+EcoRI；4: Z_{HPV18 E7:228}DNA fragmentation；M2:DL2000 DNA marker；

Figure 16 Z_{HPV18 E7} Affitoxin protein SDS-PAGE electrophoresis and WB identification.

In Figure 16 A, M:marker;E.coli. BL21 1. (DE3) bacterial strain;2. recombinant plasmid pET21a (+)/Z_{HPV18 E7:228}Before/PE38KDEL induction；3. recombinant plasmid pET21a (+)/Z_{HPV18 E7:228}After/PE38KDEL induction；4. pET21a(+)/Z _{HPV18 E7:310}After/PE38KDEL induction；Figure 16 B recombinant protein Z_HPV18E7 The purifying M of affitoxin: marker；1. 228 albumen of affitoxin after purification；2. 310 albumen of affitoxin after purification；Figure 16 C, primary antibody his- Tag mouse monoclonal antibody: 1. 228 albumen of affitoxin after purification；2. 310 albumen of affitoxin after purification；

Figure 17 affitoxin 228, (A) and affitoxin 310 (B) analyze the IC50 of HeLa cell；

Figure 18 various concentration affitoxin 228, (A) and (B) albumen of affitoxin 310 are to HeLa cell Growth inhibition effect；

Figure 19 Z_{HPV18 E7}Tumor-inhibiting action effect of the affitoxin to HeLa cell tumor bearing nude mice；

In Figure 19, three groups of nude mices are long to 100 ~ 200 mm to tumour after being inoculated with HeLa cell³When size, tail vein note 5mg/kg affitoxin228 albumen or 310 albumen of affitoxin and 0.2 ml PBS are penetrated, it is primary every injection in 3 days, totally 6 It is secondary, in figure shown in arrow；

Tumor-inhibiting action of Figure 20 ZHPV18 E7 affitoxin to HeLa cell tumor bearing nude mice；

In Figure 20, A is by the tumor tissues that execution each group nude mice removes after 45 days；B removes tumor tissues by each group Average weight.

Specific embodiment

The present invention is specifically described below by embodiment, is served only for that invention is further explained, no It can be interpreted as limiting the scope of the present invention, the technician in the field can be according to the content of foregoing invention to the present invention Make some nonessential modifications and adaptations.

As used herein, " polypeptide to HPV18 E7 with binding affinity " refers to staphylococcal protein A Z sections of amino acid sequence carries out the polypeptide obtained after 12-20 amino acid variation as skeleton, and the polypeptide can be special Property combination HPV18 E7, have few or there is no a non-specific binding.

As used herein, " polypeptide of the invention ", " to HPV18 E7 have binding affinity polypeptide ", " HPV18E7 combination polypeptide ", " Z_HPV18E7Affibody polypeptide ", " Z_HPV18E7 affibody”、 “Z_{HPV18E7”、“}affibody Albumen ", " affibody recombinant protein ", " Z_{HPV18 E7}Recombinant protein " may be used interchangeably;SPAZ can be interchanged with Zwt to be made With.

As used herein, " targeting molecule ", which refers to, has binding affinity to HPV18 E7 for of the invention Polypeptide connect with other functional conjugates after obtain, molecule that HPV18E7 can be targeted.The conjugate can be with It is cysteine residues, Polypeptide tags inhibit the drug of HPV 18, enzyme or detectable marker etc..

As used herein, " fused polypeptide " is the subordinate concept of " targeting molecule ", refers to the present invention To HPV18 E7 have binding affinity polypeptide and other functional peptides (such as toxin protein or functional protein segment) Molecule obtained after connection, that HPV18 E7 can be targeted.

The present inventor selects HPV18 E7 as target antigen.The present inventor is with the Z structural domain of staphylococcus A albumen (Zwt, SEQ ID NO:1) is used as bracket, its surface amino groups acid residue analog antibody binding site is carried out random mutation, is led to Display technique of bacteriophage building mutated library is crossed, affine screening is carried out to the library using HPV18 E7 as target antigen, through excessive The screening operation of amount is finally obtained the polypeptide for having high affinity for HPV18 E7.

Polypeptide of the invention is to carry out 14-20 using the amino acid sequence of staphylococcal protein A Z structural domain as skeleton The polypeptide obtained after (preferably 14) amino acid variation.As preferred embodiment of the invention, polypeptide of the invention relative to The amino acid sequence (SEQ ID NO:1) that Z sections of staphylococcus A albumen, in 9-11,13-14,17-18,24-25,27- 28,32,35,43 upper generation amino acid mutations.It is highly preferred that polypeptide of the invention has any institute of SEQ ID NO:1-4 The amino acid sequence shown, as shown in table 1.

Present invention also contemplates that volume is added in the amino acid sequence either end of the HPV18 E7 combination polypeptide or both ends Outer amino acid residue and the polypeptide formed.These additional amino acid residues can be acted as in polypeptide combination HPV18 E7 With, but other purposes can also be equally used for, such as it is related to the production, purifying, stabilization, one kind or several of coupling or detection of the polypeptide Kind.These additional amino acid residues may include the amino acid residue that one or more are added for chemical coupling purpose.Such as It is added in first of polypeptide chain or last position, i.e., adds cysteine residues etc. in the end N or C.It is this additional Amino acid residue also may include one " label " for peptide purification or detection, such as six with labelled antibody interaction Histidine peptide (His₆) label, or " myc " label or " flag " label.In addition, other substitutions well known to those skilled in the art Mode is also included in the present invention.

" the additional amino acid residue " also may make up one or more polypeptide domains with expectation function, such as with First, the identical binding function of HPV18E7 binding structural domain or other binding functions or a kind of enzymatic functions, or It is a kind of fluorescent functional or a combination thereof.

The present invention is also contained on the basis of the HPV18 E7 combination polypeptide, is modified and then increases it in alkaline item The polypeptide of stability under part.This stability includes being substituted in amino acid residue less sensitive for alkaline condition fixed point Any asparagus fern phthalein amine residue occurred in the sequence being not decorated.Since affinity column will be subjected to frequency between different reactions Numerous highly basic is handled to be eluted, and this characteristic that sensibility is reduced to alkali is conducive to use polypeptide of the invention as parent With the affinity ligand in chromatography, it is able to extend the service life of affinity chromatography matrices.

The present invention is also contained on the basis of HPV18 E7 combination polypeptide of the invention, obtain after other modifications more Peptide.These modification (not changing primary structure usually) forms include: the chemical derivative form such as acetyl of internal or external polypeptide Change or carboxylated.Modification further includes glycosylation, is carried out in the synthesis and processing of polypeptide or in further processing step such as those Glycosylation modified and generation polypeptide.This modification can carry out glycosylated enzyme (such as mammal by the way that polypeptide to be exposed to Glycosylase or deglycosylation enzyme) and complete.Modified forms further include with phosphorylated amino acid residue (such as phosphoric acid junket ammonia Acid, phosphoserine, phosphothreonine) sequence.It further include being modified to improve its anti-proteolytic properties or optimization The polypeptide of solubility property.

HPV18 E7 combination polypeptide of the invention can be connect with conjugate, so that functional targeting molecule is constituted, This connection can be connected or be adsorbed by chemical bond (including peptide bond)；The chemical bond is covalent bond or non-covalent bond.As A kind of preferred embodiment, is keyed by peptide, to form fused polypeptide.The HPV18 E7 combination polypeptide and conjugate it Between can be connected directly, or by polypeptide linker (link peptide) connect.The connexon is for example including 1-30 ammonia Base acid；Preferably 1-20 amino acid.The setting of link peptide has no substantial effect on the activity of each polypeptide in fusion protein.Compared with Goodly, it can use flexible peptide (Gly4Ser) 3 to be attached.Other link peptides well known to those skilled in the art can also be applied In the present invention.

In " heterologous " fused polypeptide, the HPV18 E7 combination polypeptide constitutes first structural domain or first portion Point, second and other parts have in addition to combine HPV18 E7 other than other functions, these expected results are also in the present invention In the range of.Second and other parts of the fused polypeptide may be comprising having other target molecules other than HPV18 E7 The binding structural domain of affinity.This binding structural domain may also be related to SPA structural domain, but 1 to about 20 positions It is mutated with replacing.The result is that fused polypeptide have at least one HPV18 E7 binding structural domain and at least one with it is described other Target molecule has the structural domain of affinity.This extends the application of polypeptide of the invention, such as treatment preparation or as capture, Detection or separation agent.

Other selections of fused polypeptide second and other parts of the present invention include one or more portions for therapeutic application Point.In therapeutic application, other molecules can also be covalently or non-covalently coupled on polypeptide of the invention by other methods. As the preferred embodiment of the present invention, the pseudomonas aeruginosa exotoxin PE38KDEL of transformation is connected to by flexible peptide The end C- of HPV18 E7 combination polypeptide constitutes fusion protein.Non-limitative example includes using polypeptide guiding effect enzyme of the present invention (such as carboxypeptidase) and carry out " ADEPT " (antibody-mediated enzyme prodrug treatment, antibody-directed enzyme Prodrug therapy) enzyme；Protein including effector cell and other components to raise immune system；Including thin Intracellular cytokine, such as IL-2, IFN γ, IL-12, TNF α, IP10；Including clot-promoting factor, such as tissue factor, von Willebrand The factor；Including toxin, such as ricin A, calcheamicin, maytansinoid；Including toxic small molecule, such as Auristatin analog, adriamycin etc..Meanwhile in order to be more convenient incorporation radionuclide (such as⁶⁸Ga、⁷⁶Br、¹¹¹In、⁹⁹Tc 、¹²⁴I、¹²⁵I) (such as diagnosis or radionuclide⁹⁰Y、¹³¹I、²¹¹At) for treating, it may be considered that it is above-mentioned enumerate it is additional Amino acid (especially six histidines label and cysteine), the purpose is to radioisotopic intercalating agent is coupled to polypeptide Sequence.

Present invention also contemplates that connecting a detectable marker (such as fluorescence on the HPV18 E7 combination polypeptide Label, biotin or radioactive isotope), so as to the specificity based on polypeptide of the invention, realize detection HPV18 infection or The purpose of HPV18 infection related disease.

" HPV18 E7 binding affinity " refers to can be for example by utilizing surface plasma resonance (surfaceplasmon resonance) technology such as Biocore^®A kind of polypeptide nature that device is detected.HPV18 E7 Binding affinity can be detected by an experiment, wherein HPV18 E7 is fixed on the induction chip of the device, so The sample containing candidate polypeptide is passed through into the chip afterwards.Alternatively, polypeptide to be detected can also be fixed on to the induction of the device On chip, the sample containing HPV18 E7 is then passed through into the chip.Those skilled in the art can use induction obtained Image establishes at least one observational measurement method of the HPV18 E7 binding affinity of polypeptide.If necessary to method for quantitative measuring, Such as some KD value in order to establish between interaction, surface plasma resonance method also can be used.For example, associated value can To utilize Biocore^®2000 devices (Biocore AB) are measured.HPV18 E7 is fixed on the induction chip of the device On, and affinity polypeptide sample to be detected is prepared by serial dilution and is injected with random sequence.It then can be from result Middle calculating KD value.In an embodiment of the present invention, the KD value of the polypeptide reaches 1 × 10^-6M to 3 × 10^-7M。

The present invention also provides points for encoding HPV18 E7 combination polypeptide or targeting molecule or fused polypeptide of the invention From nucleic acid, be also possible to its complementary strand.The nucleic acid can be artificial synthesized with complete sequence, it is also possible to the method difference of PCR amplification It obtains.

The present invention also provides the carriers comprising encoding the nucleic acid molecules.The carrier also may include and the nucleic acid The connected expression regulation sequence of the series of operations of molecule, in order to the expression of the fusion protein.As used herein, " operation Property be connected " or " being operably coupled to " refer to such a situation, i.e. certain parts of linear DNA sequence can influence same line The activity of property DNA sequence other parts.For example, it can exactly be operated if promoter control is with the transcription of coded sequence Ground is connected in coded sequence.

In the present invention, any suitable carrier can use, such as some for bacterium, fungi, yeast and lactation The clone of zooblast and the carrier of expression, such as Pouwels, cloning vector: described in laboratory manual.

In addition, the recombinant cell containing the nucleic acid sequence is also included in the present invention.Term " host cell " includes original Nucleus and eukaryocyte.Common prokaryotic host cell includes Escherichia coli, hay bacillus etc.；It is thin to may be, for example, Escherichia coli Born of the same parents (E. coli), such as Escherichia coli HMS174 (DE3) or BL21 (DE3).Common eukaryotic host cell include yeast cells, Insect cell and mammalian cell.

The method for producing HPV18 E7 combination polypeptide or targeting molecule or fused polypeptide of the invention has also been included in this In invention.The method includes cultivating the recombinant cell of the code nucleic acid containing corresponding polypeptide, product polypeptide is obtained.It can will be above-mentioned The peptide purification prepared is substantially uniform property, such as is in single band on SDS-PAGE electrophoresis.

The technical level of information and current recombinant expression protein based on polypeptide to be expressed, in conjunction in of the invention disclose Hold, the easily prepared polypeptide of the invention of those skilled in the art.For example, the plasmid for the Z structural domain that expression is not modified can be used Make starting material.Using known technology, required substitution mutation, which can be introduced into, obtains expression of the invention in this plasmid Carrier.

It is above-mentioned more when preparing polypeptide or targeting molecule or fusion protein of the invention using chemical polypeptide synthesis method Any naturally-produced amino acid residue in peptide can by amino acid residue that any corresponding, non-natural generates or its spread out Replaced biology, as long as the function of product polypeptide is not compromised substantially.

The invention further relates to the HPV18 E7 combination polypeptide or targeting molecules or fused polypeptide in different aspect Using, including it is applied to treatment, diagnosis and/or detection.

HPV18 E7 combination polypeptide of the invention can be used as HPV18 E7 antibody in one of different application substitute.

As unrestricted example, it can be used for treating feature characterized by HPV18 E7 expression or HPV18 are infected Disease, such as tumour (such as cervical carcinoma, head and neck neoplasm) etc..By combining HPV18 E7 intracellular to inhibit cell signal to pass It leads, the internal and external diagnosis for related disease.Polypeptide of the invention can be used as a kind of detection reagent, a kind of capture reagent or Person's separation agent, but also can be directly used as it is a kind of treat preparation or by other treatment preparations target HPV18 E7 albumen Means.It can be carried out in different ways using the method for polypeptide of the invention in vitro, such as microtiter plate, protein arrays, biology Sensor surface and histotomy etc..In order to make polypeptide of the present invention be suitable for special purposes, without departing from model of the invention In the case where enclosing, polypeptide of the invention can be modified and/or be added.

These modifications and addition has been discussed in more detail below, may be included in the additional ammonia for including in same polypeptide chain Base acid, or label and/or treatment preparation, chemical modification or in other ways combine polypeptide of the invention.In addition, of the invention Also cover to remain the segment of the polypeptide in conjunction with HPV18 E7 ability.

HPV18 E7 combination polypeptide of the invention can be used as treatment preparation, and therapeutic effect is based at least one following Mechanism: (i) reinforces Chemotherapy, applies polypeptide of the invention and acts synergistically at present with the chemotherapeutic treatment in future.It blocks intracellular Destruction of the HPV18 E7 albumen to tumor suppressor gene pRb；(ii) inhibit the proliferation of tumour cell.It may be in the cell There are following mechanism: when entering cell interior and HPV18 E7 protein binding in conjunction with polypeptide, having blocked HPV18 E7 and pRb Combination, to block growth and proliferation of cell signal.

The HPV18 E7 binding characteristic of polypeptide of the present invention and with the polypeptide produce targeting molecule (including fusion protein) And/or the stability of the binding molecule of label means that the polypeptide can be used for other active material target tumors position, These tumours include the cell for expressing HPV18 E7.Therefore, there is provided HPV18 as described herein for another aspect of the present invention E7 combination polypeptide and a kind of application of the substance coupling with anticancer activity, are transported to expression HPV18 E7 for the substance Cell, generate damage or the apoptosis of target cell.

The substance of this anticancer activity may be by merging or being coupled on HPV18 E7 combination polypeptide by chemical bond Protein, such as selected from for " ADEPT " (antibody-directed enzyme prodrug therapy) application effect Answer enzyme；For raising the effector cell of immune system and the albumen of other components；Cell factor, as IL-2, IFN γ, IL-12, TNF α a, IP 10 etc.；Clot-promoting factor, such as tissue factor, the von Willebrand factor；Toxin, such as ricin A, Pseudomonas exotoxin, calcheamicin, maytansinoid etc..Alternatively, the active material is also likely to be cell Drug toxicity, (such as such as auristatin analog or adriamycin or radioactive isotope⁹⁰Y、¹³¹I、 ²¹¹At etc.), it is this same Position element polypeptide can be bound directly in conjunction with HPV18 E7, or pass through a kind of intercalating agent, intercalating agent DOTA or DTPA as the well-known And in conjunction with HPV18 E7 in conjunction with polypeptide.

In related fields, the substance with anticancer activity is oriented to expression in vivo the present invention also provides a kind of The method of HPV18E7 cell, including it is administered to patient's active material as described herein and the polypeptide in conjunction with HPV18 E7 Conjugate.This conjugate is suitably described above.

The invention also includes the HPV18 E7 albumen used in the polypeptide test sample in conjunction with HPV18 E7.

For example, it is the disease event for expressing HPV18 E7 that this detection, which can be used to diagnostic characteristic,.Detect HPV18 E7 Presence can carry out in vivo or in vitro.Preferably selecting for in-vivo diagnostic is taken the photograph using positron emission X line tomography Shadow art, PET.Detected sample may, for example, be biological fluid sample or tissue sample.Present common method is to use needle To the antibody with HPV18 E7, this method can be adapted for the polypeptide of the invention in conjunction with HPV18 E7, and this method is Histochemical method's detection and the presence of HPV18 E7, for identify fresh, freezing or formalin fix, paraffin embedding In tissue sample with the expression of HPV18 E7 albumen.In order to detect HPV18 E7,

Polypeptide of the invention can also serve as a part of fusion protein, and wherein other structures domain is reporter enzyme or luciferase. Alternatively, it is also possible to optionally pass through intercalating agent mark by one or more fluorescent reagents and/or labelled with radioisotope Note.Suitable radioactive isotope includes⁶⁸Ga、⁷⁶ Br、¹¹¹In、⁹⁹Tc、¹²⁴I and¹²⁵I etc..

The invention also includes the HPV18 E7 combination polypeptide is applied in detection biological fluid sample HPV18E7.For this method the following steps are included: (1) provides the biological fluid sample for being detected patient, (2) will be described herein HPV18 E7 combination polypeptide be added under conditions of can make in conjunction with any HPV18 E7 present in the polypeptide and sample In sample, (3) remove uncombined polypeptide, and the polypeptide that (4) detection combines.The amount and sample of the polypeptide for the combination being detected Present in HPV18 E7 amount it is related.In step (2), HPV18 E7 combination polypeptide can be added in any suitable form Into sample, include the case where for example such, when HPV18 E7 combination polypeptide is fixed on a kind of solid support, passes through It is contacted the sample with or HPV18 E7 combination polypeptide exists in solution.

The other application of the HPV18 E7 combination polypeptide further include: the method for HPV18 E7 in test sample, packet It includes following steps: a kind of tissue sample of the suspection containing HPV18 E7 (1) is provided, such as frozen section or with formalin packet HPV18 E7 combination polypeptide of the invention is added into the sample in the histotomy buried, (2) under optimum conditions, the item Part is to combine beneficial to any HPV18 E7 present in the polypeptide and sample, and (3) remove the polypeptide being not associated with, and (4) are detected In conjunction with polypeptide.The amount of the polypeptide of the combination detected is related to the amount of HPV18 E7 present in sample.

The present invention also provides the kits that one diagnoses HPV18 E7 expression in tissue sample, including (such as with reporter enzyme Alkaline phosphatase or horseradish peroxidase) fusion, the reagent of of the invention HPV18 E7 combination polypeptide, detection enzymatic activity, With positive and negative control tissue slice.

The present invention also provides the kits that one diagnoses HPV18 E7 expression in tissue sample, including are examined by antibody Survey in conjunction with the HPV18 E7 of the invention that label (such as flag label or myc mark) merges polypeptide, one be specific to mark The primary antibody of note is specific to primary antibody and the reagent with the secondary antibody of reporter enzyme coupling, detection enzymatic activity, and positive and negative control tissue Slice.

One field of diagnostic application is exactly to detect cancer cell or its aggregation in vivo.The present invention provides a progress The kit of this diagnosis, the kit include being marked with a chelating object, of the invention HPV18 E7 combination polypeptide, one (unrestricted example is with radioactive isotope for kind of diagnosis⁶⁸Ga、⁷⁶Br、¹¹¹In、⁹⁹Tc、¹²⁴I and¹²⁵I etc.), and be used for Analyze the reagent of doping efficiency.

As described above, present invention encompasses HPV18 E7 combination polypeptides of the invention by active material targeted expression HPV18 The application of for example certain form of cancer cell of the cell of E7.The present invention also provides a kit for this purpose, the examinations Agent box includes (non-with HPV18 E7 combination polypeptide of the invention, a therapeutic radioisotopes for a chelating substance markers Restrictive example is⁹⁰Y、¹³¹I、²¹¹), and the reagent for analyzing doping efficiency At.

The present invention also provides a kind of pharmaceutical composition, it includes: it is a effective amount of of the present invention to human papilloma virus The E7 albumen of 18 types has the polypeptide of binding affinity or targets the targeting molecule of HPV 18, and pharmaceutically Acceptable carrier.

As used herein, the ingredient of " pharmaceutically acceptable " is suitable for people and/or mammal and without excessively bad Side reaction (such as toxicity), i.e., with the substance of reasonable benefit/risk ratio.Term " pharmaceutically acceptable carrier ", which refers to, to be used for The carrier of Therapeutic Administration, including various excipient and diluent.The term refers to medicament carriers some in this way: themselves is not It is necessary active constituent, and does not have excessive toxicity after applying.Suitable carrier is well known to those of ordinary skill in the art 's.Can be found in Remington ' s Pharmaceutical Sciences (Mack Pub. Co., N.J.1991) about Pharmaceutically acceptable carrier absolutely proves.Pharmaceutically acceptable carrier can contain liquid, Ru Shui, salt in the composition Water, glycerol and sorbierite.In addition, there is likely to be complementary substances in these carriers, such as lubricant, glidant, wetting agent Or emulsifier, pH buffer substance and stabilizer, such as albumin.

The composition can be made to the various dosage forms for being suitable for mammal administration, the dosage form includes but unlimited In: injection, capsule, tablet, emulsion, suppository.

It when in use, is that there is the E7 albumen of the present invention to HPV 18 of safe and effective amount The polypeptide or targeting molecule of binding affinity are applied to mammal (such as people), wherein the safe and effective amount typically at least about 1 Microgram/kg body weight, and in most cases no more than about 10 mg/kg weight, preferably the dosage is about 1 micro- G kg about 1 mg/kg weight of weight-.Certainly, specific dosage be also contemplated that administration route, patient health situation etc. because Element, within the scope of these are all skilled practitioners technical ability.

Present invention will be further explained below with reference to specific examples.

(1) preparation of HPV18 E7 albumen: pET32a (+)/HPV18 E7 recombinant plasmid transformed saved with laboratory E.coli BL21 (DE3), after IPTG inducing expression, SDS-PAGE electrophoresis, an obvious protein band appears in average molecular matter The position for measuring (Mr) about 20kDa is consistent (Fig. 5 A) with expected albumen Mr size.Through being analysed with affinity column after purification, through SDS- There is 1 more single protein band (Fig. 2A) at the position of Mr 20kDa in PAGE analysis.Resist 6 × His with mouse MAb is that primary antibody carries out Western blot analysis, it is seen that occurs signal reaction band (Fig. 2 B) at Mr 20kDa, illustrates this Band is HPV18 E7 albumen, and band is more single after purification.

(2) library phage random affibody that laboratory saves is washed in a pan using the HPV18 E7 albumen of preparation It washes: using HPV18 E7 albumen as target protein, carrying out four-wheel elutriation with the library phage random affibody；By the HPV18 of purifying E7 albumen is coated with 96 hole elisa Plates, is closed plus phage library (primary library) is incubated for, adds E. coli TG1 37 DEG C, jog incubates；100 μ l are taken, do gradient doubling dilution with 2*YT culture medium, take 100 μ l of dilution coating SOB-AG flat Plate, 30 DEG C overnight, counts and combines phage-infect clump count, calculates HPV18E7 combination phage titre；As a result plate is visible Bacterium colony, titre are 1 × 10²；It completes the first round at this time to eluriate, another part bacterium solution adds 10¹⁰Helper phage M13KO7 is (purchased from north Jing Baokewei Shi An biotech firm) and kanamycins overnight incubation, it takes supernatant through 0.22 μm of membrane filtration after centrifugation, obtains Phage library after screening that HPV18 E7 molecule is affine is the library level-one affibody.Above-mentioned 3 wheel enrichment isolation is repeated, respectively Phage library after obtaining the affine screening of HPV18 E7 molecule, is second level, and titre is 1 × 10⁶；On the basis of second level library again Above-mentioned 2 wheel enrichment isolation is repeated, is three-level library, titre is 1 × 10⁸, level Four library is obtained with method, titre is 2 × 10⁸.Simultaneously It is arranged and the blank control of bacteriophage is not added makees Synchronous Screening.

(3) the affine body affibody of high-affinity of the screening in conjunction with HPV18 E7 protein-specific, is designated as Z_HPV18E7:

Affibody monoclonal after random picking four-wheel elutriation chooses affibody points with the methods of ELISA, sequencing Son, i.e. Z_HPV18E7；235 plants of monoclonal further progress Elisa in the HPV18E7 affine body level Four library after the elutriation of picking fourth round Verifying is read according to OD450 value, is chosen OD450 value and is read higher 80 strain clone, i.e., reading is higher than 0.6 (OD450 > 0.6), It is sent to sequencing (such as Fig. 3).It identifies that 10 cloned sequences are completely correct by sequencing, and there is no terminator after translating.Therefrom select 3 higher monoclonals of affinity are taken, they are 228,242,310 clones, carry out subsequent research, these three monoclonals difference Labeled as Z_{HPV18 E7:228}、Z_{HPV18 E7:242}、Z _{HPV18 E7:310}(amino acid sequence such as table 1, also reference can be made to attached drawing 4, nucleotides sequence Column are referring to attached drawing 4).

(4) Z is prepared_HPV18E7Albumen, and identify the compatibility of itself and HPV18 E7 albumen:

The affibody positive colony Z that screening is obtained_HPV18E7It is cloned into carrier pET21a (+), is built into recombinant plasmid pET21a(+)/Z_HPV18E7, expression, purifying Z_HPV18E7Albumen；Cervical Cancer HeLa Cells strain is cultivated, cellular immunofluorescence and table are utilized Surface plasma resonance (SPR) technical appraisement Z_HPV18E7The compatibility and specificity of albumen and HPV18 E7 albumen；Specifically:

① pET21a(+)/Z _{HPV18 E7}Construction of recombinant plasmid

To screen obtained Z_{HPV18 E7:228}、Z _{HPV18 E7:242}、Z _{HPV18 E7:310}Clone is template, through PCR amplification, is utilizedNdeⅠWithEcoRⅠRestriction enzyme site is cloned into pET21a (+) vector construction recombinant plasmid (Fig. 5 A) as insertion vector site；Structure PET21a (+)/Z built_{HPV18 E7}Recombinant plasmid warpNdeⅠWithEcoRⅠAfter digestion, digestion products in agarose gel electrophoresis about One single band (Fig. 5 B) of each appearance and pET21a (+)/Z at 5400 bp and 174 bp_{HPV18 E7}Theoretical size be consistent. Through being sequenced, with NTI Software match recombinant plasmid sequence and theoretical sequence, the two is coincide, and illustrates construction of recombinant plasmid success (figure 6).

② pET21a(+)/Z _{HPV18 E7}Recombinant protein expression, identification and purifying

pET21a(+)/Z _{HPV18 E7}Recombinant plasmid existsE.coli In BL21 (DE3) after IPTG inducing expression, through SDS- PAGE electroresis appraisal, relative molecular mass (M _r) about 7kDa position, Z_{HPV18 E7:228}、Z _{HPV18 E7:242}、Z _{HPV18 E7:310} All there is apparent protein band, the wild type affibody albumen originally constructed with this laboratory, SPAZ (Zwt) albumen, phase It coincide (Fig. 7 A).Due to recombinant plasmid pET21a (+)/Z_{HPV18 E7}C-terminal contain the His label in pET21a (+), so with Affinity column is analysed after purification, is analyzed through SDS-PAGE, Z_{HPV18 E7:228}、Z _{HPV18 E7:242}、Z _{HPV18 E7:310}Albumen is about Occur 1 more single protein band at the position of 7kDa, it is consistent with reference protein SPAZ albumen (Fig. 7 B), illustrate Z_{HPV18 E7:228}、Z _{HPV18 E7:242}、Z _{HPV18 E7:310}Albumen successful expression is simultaneously prepared for purifying protein.

In the present embodiment, the present invention selects pET21a (+) carrier, utilizes the starting enzyme position of its multiple cloning sites in design Point is NdeI (CATATG), and codon ATG is amino acid (M) initiation codon of destination protein translation, is utilized in this way The albumen of prokaryotic expression is the destination protein Affibody of overall length and without carrier protein segment, avoids carrier Interference of the albumen to experimental result, lays a good foundation for follow-up test.The affine body molecule Z that will be filtered out_{HPV18 E7}Structure It build pET21a(+ in) on carrier, corresponding albumen is expressed in protokaryon, it can be by recombinant protein using the His label of carrier itself The recombinant protein that higher degree is obtained by Ni affine series of strata Column methods purifying, through SDS-PAGE electroresis appraisal, Z_{HPV18 E7:228}、Z _{HPV18 E7:242}、Z _{HPV18 E7:310}Albumen size is expressed in the position of about 7.8 kDa.Due to affibody points Son amount is small, can penetrate 0.2 μm of pvdf membrane, therefore can not carry out Western Blot to it and further analyze.The present embodiment is adopted With the methods of following surface plasma resonance (surface plasmon resonance, SPR) and immunofluorescence to it in egg The affinity characteristic of white level and cellular level and target protein is further verified.

3. the analysis of surface plasma resonance (surface plasmon resonance, SPR) technology and identification

By target protein HPV 18E7 and affine body Z used_{HPV18 E7:228}、Z _{HPV18 E7:242}、Z _{HPV18 E7:310}Albumen with And it is spare after wild type SPAZ (Zwt) protein expression, purifying, concentration and the renaturation as control, regulatory protein concentration is extremely 500ug/ml.According to operation manual, HPV18E7 recombinant protein is fixed by being coupled to GLH chip on different flow cells, Carry out the affinity determination between screening polypeptide.6th flowing pool surface be activated and inactivate using as inject when blank Control.By Z_{HPV18 E7:228}、Z _{HPV18 E7:242}、Z _{HPV18 E7:310}, SPAZ albumen be diluted to various concentration, pass sequentially through chip list Face.All analyses are carried out at 25 DEG C, and flow velocity is 30 μ l/min, and the capacity of specimen injection is 200 μ l, and with 30 μ l of flow velocity/ Min random sequence injection, then with HCl(BIO-RAD article No.: 100 mM HCl of #176-2250) washing 6min (dissociation), benefit With ProteOn Manager^TMThe 1:1 Langmuir binding model of software (BIO-RAD) analyzes binding curve (influence chart).

As can be seen from Figure 9, Z_{HPV18 E7:228}Concentration range is from 0 to 20nM, minimum 0.625nM, Z_{HPV18 E7:242}Concentration range From 0 to 40nM, minimum 1.25nM, Z_{HPV18 E7:310}Concentration range is from 0 to 20nM, minimum 0.625nM.As can be known from these results, These three are affine, and body molecule has combination with target protein, however, Z_{HPV18 E7:228}、Z _{HPV18 E7:310}Minimum concentration it is lower, say The combination of bright they and target protein is stronger.Different affine body proteins are subjected to dynamic analysis with Dissociation equilibrium constant (KD), are sent out When present same protein concentration levels, Z_{HPV18 E7:228}Dissociation equilibrium constant highest, Z_{HPV18 E7:310}Dissociation equilibrium constant Take second place, Z_{HPV18 E7:242}Dissociation equilibrium constant it is minimum, and the numerical value of reference protein SPAZ be lower than 0, illustrate SPA Z and target protein HPV 18E7 is not bound with.

It follows that the Z filtered out_{HPV18 E7:228}、Z _{HPV18 E7:242}、Z _{HPV18 E7:310}Albumen can specific recognition HPV 18E7 albumen demonstrates the HPV 18E7 of the three recombination Affibody albumen and cell expression that filter out from protein level With very strong affinity.

4. cellular immunofluorescence analysis and identification

With the Z of purifying_{HPV18 E7:228}、Z _{HPV18 E7:242}、Z _{HPV18 E7:310}Albumen is incubated for the HPV18E7 protein expression positive Cervical Cancer HeLa Cells, after 6 hours, respectively with His monoclonal antibody, SPA-Z rabbit anteserum to analyze Z_{HPV18 E7}Albumen and cell HPV18E7 protein expression feminine gender but HPV16E7 is arranged in the identification and binding ability of the HPV18E7 albumen of strain expression at the same time The Siha cell strain of the protein expression positive compares.It is observed under confocal fluorescent microscopic, the nucleus of HeLa cell The dotted or lumps fluorescin of interior visible multiple greens, and Siha cell has no apparent fluorescence, and three Z_{HPV18 E7}Albumen the equal visible green of nucleus of HeLa cell dotted or lumps fluorescence, as seen in Figure 8.

HeLa cell is HPV 18DNA positive cell in the present embodiment, and Siha be then HPV 18DNA it is negative but The cervical cancer cell of the HPV16DNA positive, in the cellular immunofluorescence experiment of this research, three Z filtering out_{HPV18 E7}Albumen It can detect in HeLa cell, and not detected in Siha cell, show the Z that this research screening obtains_{HPV18 E7:228}、 Z _{HPV18 E7:242}、Z _{HPV18 E7:310}Specific recognition capability is strong, can specifically bind with HPV18E7, but nonrecognition HPV16E7 albumen.

It follows that the Z filtered out_{HPV18 E7:228}、Z _{HPV18 E7:242}、Z _{HPV18 E7:310}Albumen energy specific recognition is thin The HPV 18E7 albumen naturally expressed in born of the same parents demonstrates the three recombination Affibody albumen and cell filtered out from cellular level The HPV 18E7 of expression has the affinity of specific binding.

[5] bio distribution in cervical cancer cell tumor bearing nude mice and cancer target characteristic

In the experiment of the present embodiment, the Z of screening is selected_{HPV18 E7:228}、Z _{HPV18 E7:242}、Z _{HPV18 E7:310}Albumen conduct Test object, near infrared fluorescent dye DyLight755 NHS Ester(Thermo Fisher company, the U.S., article No. 62278) above-mentioned albumen is marked, and is injected into the Mice Body for carrying Cervical Cancer HeLa Cells transplantation tumor, is carried out Z _HPV18E7The biodistribution research of affibody, and positioning is imaged to study the targeting characteristic of labelled protein, simultaneously to nude mouse It is compared with Zwt albumen.

Specifically:

1. the foundation of cervical carcinoma Xenografts in nude mice model

Respectively by 1x10⁶/ ml, 1x10⁷/ml,4x10⁷The Cervical Cancer HeLa Cells suspension of tri- kinds of various concentrations of/ml and Each 0.2ml of PBS inoculates 4-5 week old Female nude mice, and 21 days or so after inoculation, experimental group nude inoculation position starts grain occur The big lesser tubercle of grain, knurl growth rate is related to cell concentration, 4x10⁷/ ml group knurl increases most fast, 1x10⁷/ ml group knurl increases Take second place, the 30th day tubercle diameter increases to 300mm to two groups of nude mices after inoculation³More than, and knurl is gradually increased over time, And 1x10⁶Also there is 300mm on the 30th day after inoculation in/ml group nude mice³Above knurl, but knurl is not shown as time goes by It writes and increases, control group PBS group whole process does not have nodule growth (see figure 10).During whole process observation, nude mice is without death, the state of mind Can, it is quick on the draw, diet is normal.After inoculation HeLa cell suspension 60 days, mice with tumor is put to death, liver,spleen,kidney, big net is dissected and observed The internal organs such as film, adnexa uteria have no significant change, no transfer.Paraffin section is made in tumor tissues, carries out pathological observation, It is dyed using HE, each experimental group is shown in that the different cell of size shape, kernel hypertrophy, dark cell core, endochylema are rich as the result is shown Rich (see Figure 11).

2. DyLight-755 NHS Ester marks Z_{HPV18 E7}The identification of albumen

It is operated and is identified by DyLight-755 reagent specification.Recombinant protein Dy755- Z after will marking_{HPV18 E7:228}With 16% polyacrylamide gel shading electrophoresis, the gel for cutting suitable size is put in living imaging instrument, exciting light Filter disc is 671-705nm, and transmitting light filter disc is 750longpass, using 8bit and 2X2 mode, between 730-950nm wavelength 5000ms is exposed every time every 10nm and collects image information, carries out image process and analysis with the software that Maesro is carried.As a result it weighs Histone Dy755- Z_{HPV18 E7:228}Swimming lane has exciting light, single fluorescent bands occurs, shows recombinant protein Dy755- Z_{HPV18 E7}It marks successfully.

③ Dy755- Z _{HPV18 E7}In the intracorporal bio distribution of normal nude mice

To analyze Dy755- Z_{HPV18 E7}In the intracorporal metabolism of normal nude mice, induction fiber crops are injected intraperitoneally with 10% chloraldurate It is liquor-saturated, through 50 μ g Dy755- Z of tail vein injection after it enters deep anaesthesia state _HPV18E7:228Albumen is placed in small animal living body Imaging in imager (CRi Maesro 2.10, in-vivo fluorescence imaging system), and use 0.8-1.0 μ l/g chloraldurate maintains narcosis, with guarantee mouse in imaging process in deep anaesthesia, 5 min, 30 min, 1h, 2 h, 4 h, 6 h, 8 h and continuous imaging observation for 24 hours.As a result there is maximum fluorescence signal in visible bilateral renal, by it It is analyzed with the fluorescence signal ratio at leg muscle, i.e. kidney/normal tissue rate (the K/N ratio=[back of kidney ROI Scape signal/normal ROI tissue (muscle) background signal] × 100%.The result shows that Dy755- Z_{HPV18 E7:228}Mainly It is distributed in kidney, the fluorescence signal of kidney gradually increases after injection, and peaks in 2-4 hours, hereafter gradually weaken, and It is completely disappeared in 72 hours fluorescence signals.Show Dy755- Z_{HPV18 E7}Albumen be distributed mainly on the kidney of normal nude mice, i.e., Through kidney excretion, and in excretion (Figure 13 A, B) completely in 72 hours.

④Dy755- Z _{HPV18 E7}In the intracorporal bio distribution of HeLa cell tumor bearing nude mice

When the HeLa tumour of nude mice it is long to 300-500mm3 when take nude mice out of SPF barrier system, with 10% chloraldurate abdomen Chamber injects induced anesthesia, through 50 μ g Dy755- Z of tail vein injection after it enters deep anaesthesia state_{HPV18 E7:228}、 Dy755- Z _{HPV18 E7:242}、Dy755- Z _{HPV18 E7:310}And control Dy755- Zwt albumen, it is placed in small animal living body imaging Imaging in instrument (CRi Maesro 2.10m), and with 0.8-1.0 μ l/g chloraldurate maintenance narcosis, with guarantee mouse at Deep anaesthesia is in as during, in 5 min, 30 min, 1h, 2 h, 4 h, 6 h, 8 h and continuous imaging observation for 24 hours.Observation It was found that Dy755- Z_{HPV18 E7:228}、Dy755- Z _{HPV18 E7:242}、Dy755- Z _{HPV18 E7:310}Albumen tail vein injection 5 minutes Interior near infrared signal is distributed whole body, then gradually assembles to tumour and double kidneys, 1 hour or so, tumor locus and double kidney position letters It number being remarkably reinforced, tumour near infrared signal peaked to 4-6 hours, until 24 hours, double kidneys and tumor locus signal obviously subtract It is weak；And compare Dy755- Z_wt, then tumor locus occurs in the signal that each period of observation does not significantly increase.Tumour with The near infrared signal ratio of normal tissue is consistent with the result of above-mentioned observation, and is peaked with 4-6 hours ratio.Lotus knurl Mouse is in good condition during observation, has no that overt toxicity reacts.

The present embodiment the result shows that, Dylight755 label Dy755- Z_{HPV18 E7:228}、Dy755- Z_{HPV18 E7:242}、Dy755- Z _{HPV18 E7:310}The characteristic that targeting combines HPV18E7 expression positive tumor is all had, and without serious Toxic side effect.Possibility is provided for the cervical carcinoma targeted therapy and molecular imaging of the HPV18 positive.

[6] Z _HPV18E7 Affitoxin is in vitro to cervical cancer cell growth inhibiting effect:

In the experiment of the present embodiment, Z is utilized _HPV18E7Targeting, carry that be changed structure green with cytotoxicity The active fragment PE38KDEL of purulence bacillus exotoxin A (pseudomonas exotoxinA, PEA) is as Cytotoxic molecules. Affibody/PE38KDEL prokaryotic expression carrier is constructed, and is prepared using prokaryotic expression system and has purified affibody/ PE38KDEL albumen, i.e. affitoxin.Affitoxin verifies the inhibiting effect of the growth in vitro of cervical cancer cell.

1) Z is selected_HPV18E7:228And Z_HPV18E7:310As research object, constructed respectively using molecule clone technology building pET21a(+)/Z_HPV18E7:228/ PE38KDEL and pET21a (+)/Z_HPV18E7:310/ PE38KDEL recombinant plasmid；And identification is sequenced.

2) prokaryotic expression recombinant immunotoxin Z is prepared_HPV18E7:228/ PE38KDEL and Z_HPV18E7:310/ PE38KDEL, 310 albumen of affitoxin 228 and affitoxin, i.e. 310 albumen of affitoxin 228 and affitoxin, passes through respectively SDS-PAGE and Western blot is analyzed and identified；

It 3) is research Z_HPV18E7Affitoxin228, whether 310 polypeptide in-vitro cell growths are inhibited, selection HeLa tumor cell line is as experimental subjects.Affitoxin 228 and 310 albumen of affitoxin are detected using CCK-8 reagent The cells survival rate at time point each in cell growth process is detected, and calculates Z using Grahpad primer5.0 software The IC50 of HPV16E7affibody polypeptide.

Specifically:

①pET21a(+) / Z _{HPV18 E7}The building of/PE38KDEL recombinant plasmid

With pET21a (+)/Z_{HPV18 E7:228}And Z_HPV18E7:310Plasmid is template, through PCR amplification, is utilizedNdeI HeEcoRI carries out double digestion, while pET21a (+)/Z of preservation was originally constructed to laboratory_{HPV18 E7:228}/ PE38KDEL plasmid is double Digestion, and then enzyme connects, and is built into pET21a (+)/Z_{HPV18 E7:228}/ PE38KDEL recombinant plasmid (Figure 14)；The recombination matter of building Grain warpNdeⅠWithEcroⅠAfter digestion, there is single item at 6100 bp and 174 bp in agarose gel electrophoresis in digestion products Band, with pET21a (+)/PE38KDEL and Z_{HPV18 E7:228}Theoretical sequence size match (Figure 15), illustrate this recombinant plasmid It constructs successfully.

②pET21a(+) / Z _{HPV18 E7}Prokaryotic expression, identification and the purifying of/PE38KDEL recombinant plasmid

pET21a(+) / Z _{HPV18 E7:228}/ PE38KDEL and pET21a (+)/Z_{HPV18 E7:310}/ PE38KDEL recombination Plasmid existsE.coli In BL21 (DE3) after IPTG inducing expression, through SDS-PAGE electroresis appraisal, in relative molecular mass (M _r) about 46kDa position occur an obvious band, be consistent with expected theoretical value size, (Figure 16 A).Due to recombinating Z_{HPV18 E7:310}/ PE38KDEL albumen (i.e. Z_{HPV18 E7} Affitoxin C-terminal) contains His label, so being analysed with affinity column After purification, through SDS-PAGE analyze affitoxin228 and 310 albumen occur at the position of about 46kDa 1 it is more single Protein band (Figure 16 B).It is further analyzed and identified by Western Blot, as a result (Figure 16 C) is shown, His-tag mouse monoclonal antibody As primary antibody, it is about that 46kDa locates single band occur in molecular mass, prompts Z_HPV18E7Affitoxin228,310 recombinations Albumen energy specific recognition His-tag serum antibody.Show Z_HPV18E7Affitoxin228,310 recombination purifying proteins are prepared into Function.

3. for research Z _HPV18E7 Affitoxin in-vitro cell growth inhibiting effect selects affitoxin228,310 albumen As research object, and select HeLa tumor cell line as target cell.It prepares cell suspension and counts and be inoculated in 96 hole cells Culture plate, 0.5 × 10⁴/ hole.After culture for 24 hours, 1 μ g/ml cycloheximide and 5% fetal calf serum is added, is then added 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 10 μ g/ml, 1 μ g/ml are arranged in affitoxin228,310 albumen Six concentration groups, while culture medium control is set, using the survival rate of CCK-8 kit detection cell, the 72h after sample-adding is added 10 hole μ l/ of CCK-8 reagent, in CO₂Continue incubation 30min in incubator and is placed on reading at microplate reader A450.Every group is respectively provided with 3 A multiple holes, and carry out 3 repetitions and test.Cell survival (Cell viability) calculation method are as follows: Cell viability (%)=(the OD value of affitoxin group-culture medium group OD value)/(culture medium group control group OD value-blank OD value) × 100%； The IC50 value of cell growth survival is calculated with 5.0 software of GraphPad primer simultaneously.

As a result such as Figure 17, the IC50 value of HeLa cell growth survival is calculated through 5.0 software of GraphPad primer, The IC50 of affitoxin228,310 albumen is respectively 0.11 Μ m (Figure 17 A) and 0.05 μM (Figure 17 B).Figure 18 shows, The albumen of affitoxin 228 and 310 has stronger lethal effect to the cervical cancer cell HeLa of the expression HPV18 E7 positive, After acting on 72h, cells survival rate declines with the raising of dose concentration, has dose dependent.Affitoxin228, Between six dosage groups of 310 albumen (200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 10 μ g/ml, 1 μ g/ml), Have a significant difference between 200 μ g/ml and 100 μ g/ml dosage groups there was no significant difference to HeLa cell growth rate (p > 0.05), but 200 μ g/ml and 100 μ g/ml and other dosage groups to HeLa cell growth rate have significant difference (p < 0.05).

The above result shows that affitoxin 228,310 has the special of external HPV18E7 targeted inhibition cell growth Property.

[7] Z is studied_HPV18E7 Tumor-inhibiting action of the affitoxin 228,310 to cervical cancer cell model of nude mice bearing tumor:

To study Z _HPV18E7 Affitoxin228, the interior tumor-inhibiting action to tumor bearing nude mice of 310 proteosomes are thin by cervical carcinoma Born of the same parents' strain HeLa cell number is adjusted to 1x10⁷/ ml is inoculated at the nearly right fore of nude mice back side of 4-5 week old, every 0.2ml, to swollen Tumor grows 100 ~ 200 mm³When size, be divided into 3 groups, i.e. 228 albumen of affitoxin, 310 albumen of affitoxin and PBS control group, albumen is according to the dosage of 5mg/kg, and every nude mice is through tail vein injection 0.2ml counter sample.Injection 1 in every 3 days It is secondary, totally 6 times.Observation mouse life in every 3 days simultaneously and the state of mind etc., measure tumor size and photograph to record, observe to 45 days. Mouse is put to death later removes tumor tissues, tumor size of weighing.As can be seen from Figure 19, each group nude mice is after injecting albumen, with when Between passage, PBS group knurl is gradually increased, and amplification is obvious, is greater than 1000mm in the 33rd day knurl³, and inject After 228 albumen of affitoxin and 310 albumen of affitoxin 6 times, 310 egg of 228 albumen of affitoxin and affitoxin White group of knurl increasess slowly, and knurl is always less than 300mm during observation³Until 45 days.During whole observation in 45 days, nude mice Reaction can, diet is normal, and changes of weight is unobvious.Nude mice is put to death later, the tumor tissues of removing are taken pictures (Figure 20 A) and are weighed, 310 groups of knurls of 228 albumen of affitoxin and affitoxin are minimum, average weight be respectively 0.16 ± 0.05g and 0.29 ± 0.04g, and PBS group tumor weight reaches 0.92 ± 0.08g, examines through t, 228 albumen of affitoxin and affitoxin 310 Group knurl with PBS group compared with, difference statistically significant (P < 0.5) (such as Figure 20 B), and 228 albumen of affitoxin with 310 groups of affitoxin to compare difference statistically significant (P > 0.5).

The above results show that affitoxin228 and affitoxin310 albumen all has HPV18 E7 targeting tumor suppression and makees With.And toxic side effect is unobvious.

SEQUENCE LISTING

<110>Wenzhou Medical University

<120>there is polypeptide and its application of binding affinity to HPV18 E7 albumen

<130> 2016

<160> 6

<170> PatentIn version 3.3

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aacctgaacc accaccaggc ccccgctttc tccggttctc tgccagacga cccgtctcag 120

tctgctgact tcctggtgga agctaaaaaa ctgaacgacg ctcaggctcc gaaa 174

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aacctgaacc cagaacagga actagctttc atccgctctc tggtagacga cccgtctcag 120

tctgctgagc tcctggctga agctaaaaaa ctgaacgacg ctcaggctcc gaaa 174

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gttgacaaca aattcaacaa agaagccctc cgcgctcggg gcgaaatcgc gtggctgccg 60

aacctgaacg aggagcagga cgaggctttc atcccatctc tgcacgacga cccgtctcag 120

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Val Asp Asn Lys Phe Asn Lys Glu Pro Val Leu Ala Ser Gln Glu Ile

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Ser Pro Leu Pro Asn Leu Asn His His Gln Ala Pro Ala Phe Ser Gly

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Ser Leu Pro Asp Asp Pro Ser Gln Ser Ala Asp Phe Leu Val Glu Ala

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20 25 30

Ser Leu Val 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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Ala Trp Leu Pro Asn Leu Asn Glu Glu Gln Asp Glu Ala Phe Ile Pro

20 25 30

Ser Leu His Asp Asp Pro Ser Gln Ser Ala Glu Leu Leu Ala Glu Ala

35 40 45

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50 55

Claims (12)

1. the polypeptide that the E7 albumen of a kind of pair of HPV 18 has binding affinity, it is characterised in that: the polypeptide is Using Z sections of staphylococcal protein A of amino acid sequence as skeleton, the polypeptide obtained after 12-20 amino acid variation is carried out,

Relative to Z sections of amino acid sequence of staphylococcal protein A, the polypeptide is in 9-11,13-14,17-18,24-25,27- 28,32,35,43 upper generation amino acid mutations, the polypeptide are selected from following amino acid sequence: SEQ ID NO:6.

2. having the polypeptide of binding affinity, feature to the E7 albumen of HPV 18 as described in claim 1 Be: the KD value of the E7 protein-interacting of the polypeptide and HPV 18 is 1 × 10^-6M to 3 × 10^-7M。

3. a kind of targeting molecule for targeting HPV 18, which is characterized in that the targeting molecule includes power Benefit requires any polypeptide of 1-2, and the conjugate being connected with the polypeptide, the conjugate include: cysteine Residue, Polypeptide tags inhibit the drug or detectable marker of HPV 18.

4. the targeting molecule of targeting HPV 18 as claimed in claim 3, which is characterized in that the inhibition The drug of HPV 18 includes: toxin；The toxin be have inhibit HPV 18 virus infection or The toxin of function of tumor inhibition, the toxin are the functionality of diphtheria toxin, ricin (WA), Pseudomonas Exotoxin or the toxin Segment；And the tumour is the tumour of the HPV 18 positive.

5. a kind of isolated polynucleotides, any E7 egg to HPV 18 of coding claim 1-2 The white polypeptide with binding affinity.

6. a kind of polynucleotides encode the targeting molecule that HPV 18 is targeted described in claim 3 or 4, And wherein the conjugate is peptide.

7. a kind of recombinant vector, which is characterized in that the carrier includes polynucleotides described in claim 5 or 6.

8. a kind of host cell, which is characterized in that the host cell includes recombinant vector as claimed in claim 7, or it includes Have or genome in be integrated with polynucleotides described in claim 5 or 6.

9. a kind of any E7 albumen to HPV 18 of claim 1-2 for preparing has binding affinity The method of polypeptide, which is characterized in that the described method includes:

(1) cell according to any one of claims 8 is cultivated, so that it is any described to human papilloma virus 18 to express claim 1-2 The E7 albumen of type has the polypeptide of binding affinity；

(2) polypeptide of (1) acquisition is isolated and purified.

10. a kind of E7 albumen to HPV 18 as described in claim 1-2 is any has binding affinity The purposes of the targeting molecule of polypeptide or targeting HPV 18 as described in claim 3 or 4, is used to prepare treatment The drug of HPV 18 infection disease or HPV 18 expression positive tumor；Or it is used to prepare detection people The detection reagent of 18 type virus infection of papillomavirus；Or it is used to prepare 18 type of diagnosis of human papilloma viral infectious disease or human milk The diagnostic reagent of head 18 type of tumor virus expression positive tumor.

11. a kind of pharmaceutical composition, which is characterized in that it includes:

E7 albumen claimed in claims 1-2 to HPV 18 has the polypeptide or claim of binding affinity The targeting molecule of HPV 18 is targeted described in 3 or 4；And pharmaceutically acceptable carrier.

12. one kind is positive swollen for diagnosing or treating HPV 18 infection disease or HPV 18 expression The medicine box of tumor, which is characterized in that include: described in claim 1-2 any in the medicine box to HPV 18 E7 albumen have binding affinity polypeptide or claim 3 or 4 described in target HPV 18 targeting Pharmaceutical composition described in molecule or claim 11.