CN105859846A - Polypeptide having binding affinity to HPV16 E7 and application thereof - Google Patents

Abstract

The invention relates to polypeptide having binding affinity to HPV16 E7 and an application thereof. The invention firstly reveals the polypeptide having binding affinity to E7protein of HPV16. The invention also provides diagnosis or treatment purposes of the polypeptide as a medicine or a molecular targeted agent.

Description

To HPV16 E7 polypeptide with binding affinity and application thereof

Technical field

The present invention relates to biomedicine field, more particularly it relates to an HPV16 E7 to be had combination parent Polypeptide with power and application thereof.

Background technology

Cervical cancer is mainly relevant to human papillomavirus (HPV) the infection cancer that a kind of whole world is generally acknowledged, mortality rate exists Whole world woman cancer is in second.In prior art, although cancer, prophylaxis epidemic disease based on HPV L1 albumen Seedling commercialization, but therapeutic vaccine based on HPV and E7, be still in the experimentation exploratory stage.And There is the problem that therapeutic vaccine plays a role to be built upon, on body's immunity basis, using tomour specific Property antigen or epi-position excite the strongest antineoplastic specificity immunological effect, with reach remove tumor cell purpose； But it is low often to there is autoimmune function in tumour patient, or immunosuppressant or immunologic escape etc. and make therapeutic vaccine Play a role limited, it is difficult to reach intended therapeutic purposes.

Targeted therapy is the most desired method and strategy in current treatment of human cervical cancer, mainly includes following several types: (1) EGF-R ELISA (EGFR) is the treatment of target spot: EGFR is relevant with intracellular signal transduction, plays Regulate Normocellular growth and differentiation, strengthen invasive ability of tumor cell, promote angiogenesis, suppression tumor cell Apoptosis etc. act on, and become the multiple human tumor diagnosis including cervical cancer and the novel targets for the treatment of.Mesh Before, the anti-tumor medicine of targeting EGFR is broadly divided into two classes: EGFR monoclonal antibody and micromolecular compound Tyrosine kinase antagonist.Wherein monoclonal antibody is mainly HER2 monoclonal antibody, (conspicuous including Herceptin Sai Ting, Herceptin) and Cetuximab etc.；And micromolecular compound mainly include gefitinib, imatinib and Erlotinib etc..(2) treatment with tumor-blood-vessel growth as target spot: growth and the transfer of tumor cell depend on blood vessel Formation, thus angiogenesis inhibitor is also one of focus of neoplasm targeted therapy research.Such as 1. bevacizumab (rhuMAb -VEGF), a kind of Humanized monoclonal antibodies IgG1, suppress angiogenesis by suppression VEGF biological activity, Bevacizumab single therapy or with other chemotherapy drugs in combination application all can reduce tumor-blood-vessel growth.2. Sutent, A kind of multiple receptor tyrosine kinases inhibitor, is hindered by the tyrosine kinase activity of suppression VEGF R thus specificity Disconnected Cellular Signaling Transduction Mediated plays antitumor action.

In a word, molecular targeted therapy can killing tumor cell to greatest extent, be effectively reduced the recurrence and at a distance of tumor The risk of transfer, improves the survival rate of patient and improves total life span of patient.Although based on monoclonal antibody and The targeted molecular treatment of small molecule segment has obvious advantage, but there is also deficiency, including: (1) permeability is not Enough: to limit the solid tumor that antibody penetration volume is big；(2) target site extravasation: cause antibody exist owing to vascular permeability reduces The extravasation of target site；(3) toxic and side effects: the non-specific binding of antibody on normal tissues and produce cross reaction, fall Low Targeting Effect；(4) internal removing: the antibody metabolism of injection improves, and reduces therapeutic effect；(5) the exempting from of antibody Epidemic focus: antibody induction body produces anti-human antibody, makes therapeutic antibodies inactivate.Owing to cross reaction and immunity are compound The formation of thing, the poisonous effect making toxic and side effects produce has become as and develops the main barrier for cancer therapeutic antibody Hinder, produce liver, kidney and nervous system toxicity and make its function reduce.As with HER2 targeting antibodies The cardiac toxicity effect that trastuzumab (Herceptin) is relevant.Radioimmunotherapy treatment is carried out with isotope marked antibodies Also result in bone marrow depression.

Therefore, novel drugs or the new method of magnetic target therapy HPV related neoplasms disease is the most urgently studied in this area, with Improve current clinical status.

Summary of the invention

It is an object of the invention to provide a kind of to HPV16 E7 polypeptide with binding affinity and application thereof.

In a first aspect of the present invention, it is provided that a kind of E7 albumen to HPV 16 (HPV16) has knot Close the polypeptide of affinity, this polypeptide be using the aminoacid sequence of staphylococcal protein A (SPA) Z section (Z domain) as Skeleton, the polypeptide obtained after carrying out 12-20 (preferably 13-16, such as 14,15) amino acid variation.

In a preference, relative to the aminoacid sequence (SEQ ID NO:5) of staphylococcal protein A Z section, institute The E7 albumen to HPV 16 stated has the polypeptide of binding affinity at 9-11,13-14,17-18, 24-25,27-28,32,35,43 there is amino acid mutation.

In another preference, relative to the aminoacid sequence of staphylococcal protein A Z section, described to human papilloma The E7 albumen of virus 16 types has a polypeptide of binding affinity:

9th amino acids sports S or W；

10th amino acids sports F, L or V；

11st amino acids sports E, L or W；

13rd amino acids sports R, I or S；

14th amino acids sports S, L, M or A；

17th amino acids sports W or R；

18th amino acids sports A, W, T or Q；

24th amino acids sports G, R, D or P；

25th amino acids sports D, L, H or G；

27th amino acids sports G, V, A or Q；

28th amino acids sports R, E or L；

32nd amino acids sports L, V or E；

35th amino acids sports G, H, Q or D；

43rd amino acids sports E or K.

In another preference, the described E7 albumen to HPV 16 has the polypeptide of binding affinity Aminoacid sequence such as SEQ ID NO:2-5 arbitrary shown in.

In another preference, the described E7 albumen to HPV 16 has the polypeptide of binding affinity K with the E7 protein-interacting of HPV 16_DValue is 1 × 10^-6M to 1 × 10^-7M。

In another aspect of this invention, it is provided that the targeting molecule of a kind of targeting HPV 16, described target Tropism molecule includes arbitrary described polypeptide, and the conjugate of be connected with this polypeptide (or coupling), described Conjugate includes, but is not limited to: cysteine residues, Polypeptide tags, the medicine of suppression HPV 16, Or detectable (such as fluorescent labeling, enzyme, biotin or radiosiotope).

In a preference, described conjugate is peptide, described conjugate with described to human papilloma virus 16 The E7 albumen of type has the polypeptide of binding affinity and constitutes fused polypeptide.

In another preference, described Polypeptide tags includes but not limited to: His label (such as 6 × His), Myc label, GST label, Flag label.

In another preference, described enzyme includes but not limited to: alkali phosphatase or horseradish peroxidase.

In another preference, the medicine of described suppression HPV 16 includes, but is not limited to: toxin； It is preferred that described toxin is to have suppression HPV 16 virus to infect or the toxin of function of tumor inhibition, Diphtheria toxin, diphtherotoxin, Ricin, Pseudomonas Exotoxin or as described in the functional fragment of toxin；And, described tumor It it is the tumor of the HPV 16 positive.

In another preference, described toxin is Pseudomonas aeruginosa Exotoxin A, or the functional fragment of described toxin is The active fragment PE38KDEL of Pseudomonas aeruginosa Exotoxin A.It is preferred that this Pseudomonas aeruginosa Exotoxin A or its function Property fragment be connected to the described E7 albumen to HPV 16 have binding affinity polypeptide carboxyl end End (C-terminal).

In another preference, described conjugate and the described E7 albumen to HPV 16 have knot The polypeptide closing affinity connects with flexible peptide, and described flexible peptide includes, but is not limited to: (Gly₄Ser)₃。

In another aspect of this invention, it is provided that the polynucleotide of a kind of separation, its coding is arbitrary described to human milk The E7 albumen of head tumor virus 16 type has the polypeptide of binding affinity.

In another aspect of this invention, it is provided that a kind of polynucleotide, its targeting human papilloma virus 16 described in coding The targeting molecule of type, and wherein said conjugate is peptide.

In another aspect of this invention, it is provided that a kind of recombinant vector, this carrier comprises described polynucleotide.

In another aspect of this invention, it is provided that a kind of host cell, this host cell comprises described recombinant vector, or It includes or integrates in genome described polynucleotide.

In another aspect of this invention, it is provided that a kind of prepare arbitrary described E7 to HPV 16 Albumen has the method for the polypeptide of binding affinity, and described method includes: the cell described in (1) cultivation, thus expresses The described E7 albumen to HPV 16 has the polypeptide of binding affinity；(2) isolated and purified (1) acquisition Polypeptide.

In another aspect of this invention, it is provided that the described E7 albumen to HPV 16 have combine affine The polypeptide of power or the purposes of the targeting molecule of described targeting HPV 16, be used for preparing treatment human milk Head tumor virus 16 type catches or the medicine of HPV 16 expression positive tumor；Or

For preparing the detectable that detection HPV 16 virus infects；Or

Catch or HPV 16 expresses positive tumor for preparing diagnosis of human papilloma viral 16 type Diagnostic reagent.

In a preference, in the targeting molecule of described targeting HPV 16, described conjugate It is the suppression medicine of HPV 16 or antitumor drug (such as toxin), described to human papilloma virus 16 The targeting molecule of polypeptide or described targeting HPV 16 that the E7 albumen of type has binding affinity is used Positive tumor is expressed in treatment HPV 16 infection disease or HPV 16.

In another preference, in the targeting molecule of described targeting HPV 16, described conjugate Being detectable (such as fluorescent labeling or enzyme), the described E7 albumen to HPV 16 has combination The polypeptide of affinity or the targeting molecule of described targeting HPV 16 are for diagnosis of human papilloma viral 16 types catch or HPV 16 expresses positive tumor.

In another preference, described HPV 16 is expressed positive tumor and is included: cervical cancer, incidence Tumor or external genitalia tumor etc..

In another preference, described HPV 16 infection disease includes: cervical intraepithelial neoplasia (CIN) Or external genitalia wart etc..

In another aspect of this invention, it is provided that a kind of pharmaceutical composition, it comprises: foregoing to human papilloma virus The E7 albumen of poison 16 types has polypeptide or the targeting of described targeting HPV 16 of binding affinity Molecule；With pharmaceutically acceptable carrier.

In another aspect of this invention, it is provided that one is used for diagnosing or treat HPV 16 infection disease or people The medicine box of positive tumor expressed by human papillomavirus 16 type, and described medicine box includes: described to human papillomavirus The E7 albumen of 16 types has the polypeptide of binding affinity, or the targeting of described targeting HPV 16 Molecule, or described pharmaceutical composition.

In a preference, the described E7 albumen to HPV 16 has the polypeptide of binding affinity Or the targeting molecule of described targeting HPV 16 is effective dose.

In another aspect of this invention, it is provided that one treats HPV 16 infection disease or human papillomavirus Positive tumor expressed by 16 types, has binding affinity including by the described E7 albumen to HPV 16 Polypeptide or the targeting molecule of described targeting HPV 16 give to need the object for the treatment of.

In another aspect of this invention, it is provided that a kind of diagnosis of human papilloma viral 16 type catches or human papillomavirus Positive tumor expressed by 16 types, controls including giving needs by the targeting molecule of described targeting HPV 16 The object treated；In described targeting molecule, described conjugate is detectable (such as fluorescent labeling or enzyme).

The other side of the present invention, due to this disclosure, is apparent to those skilled in the art 's.

Accompanying drawing explanation

Fig. 1, each Z_HPV16E7And Z_wtThe contrast of sequence.The polypeptide Z of the present invention_HPV16E7In adorned aminoacid The most own black matrix in site marks (SEQ ID NO:1-4).

The construction of recombinant plasmid figure (A) of the fused polypeptide produced in Fig. 2, embodiment 1 and aminoacid sequence schematic diagram (B)。

Z_HPV16E7Represent and there is the HPV16E7 binding structural domain selected from SEQ ID NO:1-5 any sequence, 6xHis Representing six groups of ammonia phthalein labellings, HM represents the aminoacid that NdeI (CATATG) translates, and LE represents XhoI (CTCGAG) The aminoacid of translation.

Fig. 3, pET21a (+) the recombiant plasmid electrophoretogram of/affibody.

M1:DNA marker；1-5 be respectively pET21a (+)/Z_HPV16E7:127、pET21a(+)/Z_HPV16E7:301、 pET21a(+)/Z_HPV16E7:384、pET21a(+)/Z_HPV16E7:745And pET21a (+)/Z_wtRecombiant plasmid.

Fig. 4, Affibody recombiant protein prokaryotic expression (A) and the SDS-PAGE electrophoretic analysis of purification (B).

In A: M: albumen marker；1-2 be respectively BL21 (DE3) bacterial strain and pET21 (+) empty carrier transfection BL21 (DE3) bacterial strain, 3-8 be respectively pET21a (+)/Z_HPV16E7:127、pET21a(+)/Z_HPV16E7:301、 pET21a(+)/Z_HPV16E7:384、pET21a(+)/Z_HPV16E7:745And pET21a (+)/Z_wtTransfected Recombinant Plasmid BL21 (DE3) bacterial strain.

In B: M: albumen marker；1-5 is respectively Z after purification_{HPV16 E7:127}, Z_{HPV16 E7:301}, Z_{HPV16 E7:384}, Z_{HPV16 E7:745}And Z_wtAffibody recombination fusion protein.

The SDS-PAGE electrophoresis of Fig. 5, HPV16E7 recombiant protein and Western Blot analyze.

M:marker；1.E.coli.BL21 (DE3) bacterial strain；2. recombiant plasmid pET21a (+)/HPV16E7 albumen table Reach；HPV16E7 recombination fusion protein the most after purification.A:SDS-PAGE；B:Western Blot analyzes, and one Resist for His monoclonal antibody (1:10000)；C:Western Blot analyzes, and one resists for cervical cancer patient serum (1:500).

Fig. 6, Z_{HPV16 E7}Binding peptide SPR detection on ProteOn XPR36 instrument.

A, B, C, D, E are respectively Z_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}And Z_wt Albumen and the affinity analysis of target protein HPV16E7.

Fig. 7, Z_{HPV16 E7}The cellular immunofluorescence method of Binding peptide and HPV16 E7 native protein affinity is identified.

A:Z_{HPV16 E7:127}The indirect immunofluorescene assay of Binding peptide；B:Z_{HPV16 E7:301}The immunity indirectly of albumen is glimmering Light detects；C:Z_{HPV16 E7:384}The indirect immunofluorescence of albumen；D:Z_{HPV16 E7:745}The indirect immunofluorescence of albumen Detection；E:HPV16E7 rabbit antibody is an anti-indirect immunofluorescene assay.

The Z of Fig. 8, Dylight755 labelling_{HPV16 E7:384}Binding peptide SDS-PAGE electrophoresis and fluorescence analysis.

A is Z_{HPV16 E7:384}Polypeptide SDS-PAGE electrophoretic analysis；B is the Z of labelling Dylight755_{HPV16 E7:384} The fluorescence analysis of polypeptide SDS-PAGE electrophoresis.

The Z of Fig. 9, Dylight755 labelling_{HPV16 E7:384}Polypeptide bio distribution in tumor bearing nude mice and cancer target Imaging analysis.A is the Z of Dylight755 labelling_{HPV16 E7:384}The each time point of polypeptide respectively load Siha, Caski, Fluorescence imaging in HeLa229 and A375 cell tumor bearing nude mice body；B be each time point respectively load Siha, Average fluorescence signal in Caski, HeLa229 and A375 cell (according to top-down order) tumor bearing nude mice body Intensity.

The Z of Figure 10, Dylight755 labelling_{HPV16 E7:384}Polypeptide imaging in each internal organs of tumor bearing nude mice and tumor tissues Analyze.A1-D1 is the Z at injection Dylight755 labelling_{HPV16 E7:384}After polypeptide 24h, naked in load tumor Fluorescence distribution in Mus each Main Tissues internal organs；A2-D2 is the average fluorescence signal of corresponding tumor tissues and each internal organs Intensity.

Figure 11, four kinds of Z_{HPV16 E7}The IC50 of Siha cell is analyzed by Binding peptide.A, B, C, D are respectively Z_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}And Z_{HPV16 E7:745}The IC50 of Siha cell is analyzed by albumen.

Figure 12, variable concentrations Z_{HPV16 E7}The Binding peptide growth inhibited effect to Siha cell.A, B, C, D divide Wei the Z of variable concentrations_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}And Z_{HPV16 E7；745}Albumen is thin to Siha The growth inhibited effect of born of the same parents.

Figure 13, pET21a (+)/Z_{HPV16 E7}Affitoxin construction of recombinant plasmid figure.

Figure 14, Z_{HPV16 E7}Affitoxin protein SDS-PAGE electrophoresis and WB identify.

A:1, BL21 (DE3) bacterial strain；2, pET21a (+) BL21 (DE3) of vector；3-7 is respectively PET21a (+)/HPV16 E7affitoxin127,301,384,745 and Z_wtAffitoxin Transfected Recombinant Plasmid BL21 (DE3) bacterial strain；

B:Z_{HPV16 E7}Affitoxin127,301,384,745 and Z_wtAffitoxin purifying protein SDS-PAGE； 1-5 is respectively Z_{HPV16 E7}Affitoxin127,301,384,745 and Z_wtaffitoxin；

C: for Z_{HPV16 E7}Affitoxin127,301,384,745 Western blot analyze (one resists for His monoclonal antibody), and 1 is BL21 (DE3) bacterial strain, 2 be pET21a (+) BL21 (DE3) of vector, 3-6 is respectively Z_{HPV16 E7} affitoxin127,301,384,745；

D: for Z_wtAffitoxin albumen Western blot analyzes (one resists for His monoclonal antibody), and 1 is BL21 (DE3) bacterial strain, 2 be pET21a (+) BL21 (DE3) of vector, 3 is Z_wtAffitoxin albumen.

E:HPV16 E7-affitoxin albumen Western blot analyzes (one resists for rabbit anti-SPA-Z multi-resistance), and 1 is BL21 (DE3) bacterial strain 2 be pET21a (+) carrier transfection BL21 (DE3), 3-6 is respectively HPV16 E7 AffitoxinE127,301,384,745.

F: for Z_wtAffitoxin albumen Western blot analyzes (one resists for rabbit anti-SPA-Z multi-resistance), and 1 is BL21 (DE3) Bacterial strain, 2 be pET21a (+) BL21 (DE3) of vector, 3 is Z_wtAffitoxin albumen.

Figure 15, Z_{HPV16 E7}The cellular immunofluorescence of affitoxin Yu HPV16 E7 native protein affinity is identified.A: Z_{HPV16 E7}Affitoxin127 albumen；B:Z_{HPV16 E7}Affitoxin301 albumen；C:Z_{HPV16 E7} affitoxin384 Albumen；D:Z_{HPV16 E7}Affitoxin 745 albumen.

Figure 16, Z_{HPV16 E7}The SPR detection on ProteOn XPR36 instrument of the affitoxin albumen.A、B、C、 D, E are respectively Z_{HPV16 E7}Affitoxin127,301,384,745 and Z_wtAffitoxin protein and target protein The affinity analysis of HPV16E7.

Figure 17, Z_{HPV16 E7}The IC50 of Caski cell (A) and Siha cell (B) is analyzed by affitoxin384.

Figure 18, Z_{HPV16 E7}The affitoxin384 impact on four kinds of growth of tumour cell.

Figure 19, variable concentrations Z_HPV16E7Affitoxin384 is to Siha (A) and the growth inhibited effect of Caski (B) cell.

Z in Figure 20, blood plasma_HPV16E7: 384 (B) and Z_HPV16E7Affitoxin384 (A) half-life is detected.

Figure 21, Z_{HPV16 E7}The affitoxin384 tumor-inhibiting action effect to Balb/c tumor bearing nude mice.

A: five groups of mices while inoculation Siha, the Z of tail vein injection 4mg/kg concentration_HPV16E7 affitoxin384 Or Z_HPV16E7: 384, PE38KDEL albumen, Z_wtAffitoxin albumen and PBS, every injection in 3 days once, altogether 6 times, in figure shown in arrow.

B: five groups of mices, after inoculation Siha, treat that tumor length is to 100～200mm³During size, tail vein injection 4mg/kg The Z of concentration_HPV16E7Affitoxin384 or Z_HPV16E7: 384, PE38KDEL albumen, Z_wtAffitoxin albumen and PBS, every injection in 3 days once, totally 6 times, in figure shown in arrow.

Detailed description of the invention

The present inventor, through in-depth study, discloses a kind of E7 to HPV 16 (HPV16) first Albumen has the polypeptide of binding affinity；Present invention also offers this polypeptide as medicine or the diagnosis of molecular targeted reagent Or therapeutic use.

As used herein, described " having the polypeptide of binding affinity to HPV16 E7 " refers to staphylococcus A The aminoacid sequence of albumen Z section is as skeleton, the polypeptide obtained after carrying out 10-20 amino acid variation, and this polypeptide Can specific binding HPV16 E7, have few or there is no non-specific binding.

As used herein, described " polypeptide of the present invention ", " HPV16 E7 is had the polypeptide of binding affinity ", " HPV16 E7 Binding peptide ", " Z_HPV16E7Affibody polypeptide ", " Z_HPV16E7 affibody”、“Z_HPV16E7”、 " affibody albumen ", " affibody recombiant protein ", " Z_{HPV16 E7}Recombiant protein " use can be exchanged.

As used herein, described " targeting molecule " refer to by the present invention HPV16 E7 is had combine affine The polypeptide of power be connected with other functional conjugate after obtain, can be with the molecule of targeting HPV16 E7.Described idol Connection thing can be cysteine residues, Polypeptide tags, and the medicine of suppression HPV 16, enzyme maybe can detect Label etc..

As used herein, described " fused polypeptide " is the subordinate concept of described " targeting molecule ", refers to this The polypeptide that HPV16 E7 is had binding affinity of invention and other functional peptide (such as toxin protein or functional Protein fragments) connect after obtain, can be with the molecule of targeting HPV16 E7.

HPV16 type infects the main type being to cause cervical cancer.After HPV infection host cell, its viral gene is integrated To host chromosome, and then make host cell undergo mutation and develop into tumor, its carcinogenic Chief molecule it One is the HPV gene E7 albumen that district (E) encodes in early days, and E7 albumen is mainly by its N-terminal conserved sequence LXCXE is combined with cancer suppressor protein pRb, makes the transcription factor E2F being combined with Rb under normal condition dissociate out, And inactivate pRb albumen, the E2F dissociated can activated gene transcribe, promote numerous cell proliferation related because of Transcribe, cause cell hyperproliferation vicious transformation.High-risk HPV E7 albumen is the tumor of cervical cancer caused by HPV Immunogenic peptide, continue and stably in cervical cancer and Precancerous Lesion thereof in high expressed, and in the normal tissue Do not exist.Considering above-mentioned factor, the present inventor selects HPV16 E7 as target antigen.The present inventor is with Portugal Z domain (the Z of grape coccus A albumen_wt, SEQ ID NO:1) as support, its surface amino groups acid residue is simulated Antibody combining site carries out random mutation, builds mutated library by display technique of bacteriophage, makees with HPV16 E7 For target antigen, this library is carried out affine screening, through substantial amounts of screening operation, be finally obtained for HPV16 E7 There is the polypeptide of high affinity.

The polypeptide of the present invention is using the aminoacid sequence of staphylococcal protein A Z domain as skeleton, carries out 14-20 The polypeptide obtained after individual (preferably 14) amino acid variation.As the optimal way of the present invention, the present invention is many Peptide is relative to the aminoacid sequence (SEQ ID NO:5) of staphylococcal protein A Z section, at 9-11,13-14,17-18, 24-25,27-28,32,35,43 there is amino acid mutation.It is highly preferred that the polypeptide of the present invention has SEQ Aminoacid sequence shown in ID NO:2-5 is arbitrary.

Present invention also contemplates that and add volume at aminoacid sequence either end or the two ends of described HPV16 E7 Binding peptide Outer amino acid residue and the polypeptide that formed.These extra amino acid residues can be when polypeptide combines HPV16 E7 Work, but also can be equally used for other purpose, such as relating to the production of this polypeptide, purification, stable, coupling or inspection One or more surveyed.These extra amino acid residues can include that one or more adds for chemical coupling purpose Amino acid residue.As added first of polypeptide chain or last position, i.e. add one and half at N or C-terminal Cystine residue etc..This extra amino acid residue can also include " a mark for peptide purification or detection Note ", such as the six histidine peptide (His interacted with traget antibody₆) labelling, " myc " labelling or " flag " mark Note.Additionally, other alternative well known to those skilled in the art is also contained in the present invention.

Described " extra amino acid residue " also may make up one or more polypeptide domain with expectation function, as With first, combined function that HPV16E7 binding structural domain is identical, or other combined function, or a kind of enzyme Promote function, or a kind of fluorescent functional, or a combination thereof.

On the basis of the present invention is also contained in described HPV16 E7 Binding peptide, modified so that increase its alkalescence bar The polypeptide of the stability under part.This stability includes taking with for alkalescence condition less sensitive amino acid residue fixed point Any Radix Asparagi phthalein amine residue that generation occurs in not having the sequence modified.Due to affinity column between different reactions Stand highly basic frequently to process to carry out eluting, this characteristic that alkali reduces sensitivity, be conducive to using the present invention Polypeptide as the affinity ligand in affinity chromatograph, it is possible to extend affinity chromatography matrices service life.

On the basis of the present invention is also contained in the HPV16 E7 Binding peptide of the present invention, carry out acquisition after other is modified many Peptide.These are modified (the most not changing primary structure) form and include: the chemically derived form of inner or in vitro polypeptide is such as Acetylation or carboxylated.Modify and also include glycosylation, as those or are processed further step in the synthesis of polypeptide and processing Polypeptide that is glycosylation modified and that produce is carried out in Zhou.This modification can carry out glycosylated enzyme by being exposed to by polypeptide (such as glycosylase or the deglycosylating enzyme of mammal) and complete.It is residual that modified forms also includes having phosphorylated amino acid The sequence of base (such as phosphotyrosine, phosphoserine, phosphothreonine).Also include being modified thus improve it and resist Proteolytic enzyme performance or optimize the polypeptide of solubility property.

The HPV16 E7 Binding peptide of the present invention can be connected with conjugate, thus constitute functional targeting and divide Son, this connection can be passed through chemical bond (including peptide bond) and be connected or absorption；Described chemical bond is covalent bond or non-covalent Key.As a kind of optimal way, bonded by peptide, thus form fused polypeptide.

Can be joined directly together between described HPV16 E7 Binding peptide and conjugate and connect, or pass through polypeptide linker (connection peptides) connects.Described connexon such as includes 1-30 aminoacid；Preferably 1-20 aminoacid.Even The setting connecing peptide has no substantial effect on the activity of each polypeptide in fusion protein.Preferably, it is possible to use flexible peptide (Gly₄Ser)₃It is attached.Other connection peptides well known to those skilled in the art also apply be applicable to the present invention.

In " allos " fused polypeptide, described HPV16 E7 Binding peptide constitutes first domain or first Part, second and other parts there is other function in addition to combining HPV16 E7, these expected results are also Within the scope of the invention.Second and other parts of this fused polypeptide may comprise its in addition to HPV16 E7 Its target molecule has the binding structural domain of affinity.This binding structural domain is likely to relevant to SPA domain, but There is replacement sudden change 1 to about 20 position.Result is that fused polypeptide has at least one HPV16 E7 to combine knot Structure territory has the domain of affinity with at least one and other target molecule described.This extend the present invention polypeptide should With, as treatment preparation or as capture, detection or separation agent.

Other of fused polypeptide second of the present invention and other parts selects to include one or more portion for therapeutic application Point.In therapeutic is applied, it is many that other molecule can also covalently or non-covalently be coupled to the present invention by other method On peptide.As the preferred embodiment of the present invention, the Pseudomonas aeruginosa extracellular toxin PE38KDEL of transformation is passed through Flexible peptide is connected to the C-end of HPV16 E7 Binding peptide, constitutes fusion protein.Non-limitative example includes with this Invention polypeptide guiding effect enzyme (such as carboxypeptidase) and carry out " ADEPT " (antibody-mediated enzyme prodrug treatment, Antibody-directed enzyme prodrug therapy) enzyme；Including in order to raise immune effector lymphocyte and The protein of other component；Including cytokine, such as IL-2, IFN γ, IL-12, TNF α, IP10；Including coagulant Blood factor, such as tissue factor, the von Willebrand factor；Including toxin, as ricin A, calcheamicin, Maytansinoid；Including toxic small molecule, such as auristatin analog, amycin etc..Meanwhile, in order to more Convenient incorporation radionuclide (as⁶⁸Ga、⁷⁶Br、¹¹¹In、⁹⁹Tc、¹²⁴I、¹²⁵I) for diagnosis or radionuclide (as⁹⁰Y、¹³¹I、²¹¹At) it is used for treating, it may be considered that above-mentioned extra aminoacid (the particularly six histidine labellings enumerated And cysteine), its objective is radioisotopic intercalating agent is coupled to peptide sequence.

Present invention also contemplates that and on described HPV16 E7 Binding peptide, connect a detectable (such as fluorescence mark Note, biotin or radiosiotope), thus can the specificity of polypeptide based on the present invention, it is achieved detection HPV16 Infect or the purpose of HPV16 infection relevant disease.

" HPV16 E7 binding affinity " refers to can be such as by utilizing surface plasma resonance (surface Plasmon resonance) technology is such asDevice carries out a kind of polypeptide nature detected.HPV16 E7 combines parent Can be detected by an experiment with power, wherein HPV16 E7 is fixed on the induction chip of this device, so After by the sample containing candidate polypeptide by this chip.Or, it is also possible to polypeptide to be detected is fixed on this device On induction chip, then by the sample containing HPV16 E7 by this chip.Those skilled in the art can utilize institute The sensed image obtained sets up at least one observation measurements method of the HPV16 E7 binding affinity of polypeptide.If needed Want method for quantitative measuring, such as in order to set up certain K between interaction_DValue, it is possible to use surface plasma is common Method of slight.Such as, associated value can utilize2000 devices (Biocore AB) are measured.HPV16 E7 It is fixed on the induction chip of this device, and affinity polypeptide sample to be detected is prepared by serial dilution and with at random Order is injected.Then K can be calculated from result_DValue.In an embodiment of the present invention, the K of described polypeptide_D Value reaches 1 × 10^-6M to 1 × 10^-7M。

Present invention also offers the HPV16 E7 Binding peptide of code book invention or targeting molecule or fused polypeptide point From nucleic acid, it is also possible to be its complementary strand.Described nucleic acid can be with complete sequence synthetic, it is also possible to PCR amplification Method obtains respectively.

Present invention also offers and comprise the carrier encoding described nucleic acid molecules.Described carrier also can comprise and described nucleic acid The expression regulation sequence that the series of operations of molecule is connected, in order to the expression of described fusion protein.It is as used herein, " it is operatively connected " or " being operably coupled to " refers to some part of such a situation, i.e. linear DNA molecule The activity of same linear DNA molecule other parts can be affected.Such as, if promoter controls with coded sequence Transcribe, then it is operably coupled to coded sequence exactly.

In the present invention, any suitable carrier can use, more such as antibacterial, fungus, yeast and the food in one's mouth The clone of breast zooblast and the carrier of expression, such as Pouwels etc., cloning vehicle: described in laboratory manual.

Additionally, the reconstitution cell containing described nucleotide sequence is also included in the present invention.Term " host cell " includes Prokaryotic cell and eukaryotic cell.Conventional prokaryotic host cell includes escherichia coli, bacillus subtilis etc.；Can be such as big Coli cell (E.coli), such as escherichia coli HMS174 (DE3) or BL21 (DE3).Conventional eucaryon host is thin Born of the same parents include yeast cells, insect cell and mammalian cell.

The method of the HPV16 E7 Binding peptide or targeting molecule or fused polypeptide that produce the present invention has been included in this most In invention.Described method includes the reconstitution cell cultivating the code nucleic acid containing corresponding polypeptide, it is thus achieved that product polypeptide.Can It is substantially uniform character by the above-mentioned peptide purification prepared, such as in single bar on SDS-PAGE electrophoresis Band.

Based on wanting information and the technical merit of current recombinant expression protein of express polypeptide, in conjunction with the present invention disclose interior Hold, the polypeptide of the easily prepared present invention of those skilled in the art.Such as, the plasmid of the most adorned Z domain is expressed Can serve as parent material.Use known technology, required replacement sudden change can be introduced in this plasmid to obtain this The expression vector of invention.

When using chemistry polypeptide synthesis method to prepare the polypeptide of the present invention or targeting molecule or fusion protein, above-mentioned many Amino acid residue that any naturally-produced amino acid residue in peptide can be produced by any corresponding, non-natural or Its derivant is replaced, as long as the function of product polypeptide is not compromised substantially.

The invention still further relates to described HPV16 E7 Binding peptide or targeting molecule or fused polypeptide at different aspect Application, including being applied to treatment, diagnosing and/or detect.

The HPV16 E7 Binding peptide of the present invention can be as the HPV16 E7 antibody a kind of substitute in different application. As nonrestrictive example, it can be used for treating what feature was characterized with HPV16 E7 expression or HPV16 infection Disease, such as tumor (such as cervical cancer, tumor of head and neck) etc..Believe with suppression cell by combining intracellular HPV16 E7 Number conduction, for the internal of relevant disease and in-vitro diagnosis.The polypeptide of the present invention can be as a kind of detectable, one Capture agent or separation agent, but also one can be directly used as and treat preparation or other is treated preparation targeting The means of HPV16 E7 albumen.The method of the polypeptide of the external use present invention can be carried out by different way, as trace drips Determine plate, protein arrays, biosensor surface and tissue slice etc..Special in order to make polypeptide of the present invention be applicable to Purposes, in the case of without departing from the scope of the present invention, can modify the polypeptide of the present invention and/or add. Being discussed in more detail below these modify and add, it may be included in the extra amino comprised in same polypeptide chain Acid, or labelling and/or treatment preparation, its chemical modification or otherwise combine the polypeptide of the present invention.It addition, Present invention also contemplates that the fragment remaining this polypeptide combining HPV16 E7 ability.

The HPV16 E7 Binding peptide of the present invention can be as treatment preparation, and its therapeutical effect is based on below at least one Mechanism: (i) strengthens Chemotherapy, uses the polypeptide of the present invention and current and chemotherapeutic treatment synergism in the future.Block The destruction to antioncogene pRb of the HPV16 E7 albumen of intracellular；(ii) propagation of tumor cell is suppressed.May In the intracellular following mechanism that exists: when Binding peptide enters cell interior with HPV16 E7 protein binding, block The combination of HPV16 E7 Yu pRb, thus blocked growth and proliferation of cell signal.

The HPV16 E7 binding characteristic of polypeptide of the present invention and produce targeting molecule (including fusion protein) with this polypeptide And/or the stability of the binding molecule of labelling means that this polypeptide can be used for other active substance target tumor portion Position, these tumors include the cell expressing HPV16 E7.Therefore, another aspect of the present invention there is provided institute herein The HPV16 E7 Binding peptide stated and the application of a kind of material coupling with active anticancer, to transport described material To the cell of expression HPV16 E7, produce damage or the apoptosis of target cell.

The material of this active anticancer may be by merging or being coupled on HPV16 E7 Binding peptide by chemical bond Protein, as selected from the effect applied for " ADEPT " (antibody-directed enzyme prodrug therapy) Answer enzyme；For raising the albumen of immune effector lymphocyte and other component；Cytokine, as IL-2, IFN γ, IL-12, TNF α a, IP 10 etc.；Clot-promoting factor, such as tissue factor, the von Willebrand factor etc.；Toxin, Such as ricin A, Pseudomonas exotoxin, calcheamicin, maytansinoid etc..Or, described Active substance is also likely to be cytotoxic drug, as auristatin analog or amycin or radiosiotope (as⁹⁰Y、¹³¹I、²¹¹At etc.), this isotope can with HPV16 E7 Binding peptide directly in conjunction with, or by a kind of huge legendary turtle Mixture, intercalating agent DOTA or DTPA as the well-known and be combined with HPV16 E7 Binding peptide.

In related fields, present invention also offers a kind of guiding by the material with active anticancer in vivo and express HPV16 The method of E7 cell, including be administered to patient's described active substance as herein described with HPV16 E7 Binding peptide Conjugate.This conjugate is suitably described above.

Present invention additionally comprises the HPV16 E7 albumen in the polypeptide being combined with the HPV16 E7 detection sample described in use. Such as, this detection can be used to diagnostic characteristic is the disease event expressing HPV16 E7.Detection HPV16 E7's Existence can also be carried out in vivo in vitro.Preferably selecting of in-vivo diagnostic is to use positron emission x-ray to break Layer photography, PET.Detected sample can e.g. biological fluid sample or tissue sample.Present is universal Method is that this method goes for being combined with HPV16 E7 of the present invention with for the antibody with HPV16 E7 Polypeptide, this method is the existence of histochemical method's detection and HPV16 E7, is used for identifying fresh, freezing or good fortune Your Malin fixes, in paraffin-embedded tissue sample with the expression of HPV16 E7 albumen.In order to detect HPV16 E7, The polypeptide of the present invention can also serve as a part for fusion protein, and wherein other domain is reporter enzyme or luciferase.Or, It can also be by one or more fluorescent reagents and/or labelled with radioisotope, optionally through intercalating agent labelling. Suitably radiosiotope includes⁶⁸Ga、⁷⁶ Br、¹¹¹In、⁹⁹Tc、¹²⁴I and¹²⁵I etc..

Present invention additionally comprises and be applied to described HPV16 E7 Binding peptide detect HPV16 in biological fluid sample E7.This method comprises the following steps: (1) provides the biological fluid sample of detected patient, and (2) are by described herein HPV16 E7 Binding peptide under conditions of described polypeptide can be made to be combined with HPV16 E7 any present in sample Adding in sample, (3) remove uncombined polypeptide, and the polypeptide that (4) detection combines.The polypeptide of the combination being detected Amount and sample present in HPV16 E7 amount relevant.In step (2), HPV16 E7 Binding peptide can be to appoint What suitable form joins in sample, including the most such situation, when HPV16 E7 Binding peptide is fixed on On a kind of solid support, contacted the sample with by it, or HPV16 E7 Binding peptide exists in solution.

Other application of described HPV16 E7 Binding peptide also includes: the method for HPV16 E7 in detection sample, Comprise the steps: that (1) provides a kind of and suspects containing the tissue sample of HPV16 E7, such as frozen section or use Fu Er The tissue slice of Malin's embedding, (2) add the HPV16 E7 Binding peptide of the present invention under optimum conditions to described sample In, described condition is combined for benefiting any HPV16 E7 present in described polypeptide and sample, and (3) remove unconjugated Polypeptide, and the polypeptide that (4) detection combines.HPV16 E7 present in the amount of the polypeptide of the combination detected and sample Amount is relevant.

Present invention also offers the test kit that HPV16 E7 in a diagnostic bank tissue samples expresses, including with reporter enzyme (as Alkali phosphatase or horseradish peroxidase) merge, the HPV16 E7 Binding peptide of the present invention, detection enzymatic activity Reagent and positive and negative control tissue cut into slices.

Present invention also offers the test kit that in a diagnostic bank tissue samples, HPV16 E7 expresses, examine including by antibody The HPV16 E7 Binding peptide of the present invention merged with labelling (such as flag labelling or myc labelling) surveyed, one special Resist in the one of labelling, be specific to anti-and, the reagent of detection enzymatic activity anti-with the two of reporter enzyme coupling, and positive and cloudy Property control tissue sections.

One field of diagnostic application is exactly to detect cancerous cell or its aggregation in vivo.The invention provides one to carry out The test kit of this diagnosis, this test kit includes that being marked with a sequestration thing, present invention HPV16 E7 combines many Peptide, (nonrestrictive example is a kind of diagnosis radiosiotope⁶⁸Ga、⁷⁶Br、¹¹¹In、⁹⁹Tc、¹²⁴I and¹²⁵I Deng), and for analyzing the reagent of doping efficiency.

As it has been described above, present invention encompasses the HPV16 E7 Binding peptide of the present invention by active substance targeted expression The application of the most certain form of cancer cell of cell of HPV16 E7.Present invention also offers one for this purpose Test kit, this test kit includes the HPV16 E7 Binding peptide of the present invention with a sequestration substance markers, a treatment (nonrestrictive example is property radiosiotope⁹⁰Y、¹³¹I、²¹¹At), and for analyzing the reagent of doping efficiency.

The present invention also provides for a kind of pharmaceutical composition, and it comprises: effective dose of the present invention to human papillomavirus The E7 albumen of 16 types has polypeptide or the targeting molecule of targeting HPV 16 of binding affinity, and Pharmaceutically acceptable carrier.

As used herein, the composition of " pharmaceutically acceptable " applies to people and/or mammal and nothing is the worst Side reaction (such as toxicity), i.e. has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " Refer to the carrier for Therapeutic Administration, including various excipient and diluent.This term refers to so some medicament carriers: Themselves it is not necessary active component, and there is no undue toxicity after using.Suitably carrier is that this area is general Known to logical technical staff.At Remington ' s Pharmaceutical Sciences (Mack Pub.Co., N.J. 1991) absolutely proving about pharmaceutically acceptable carrier can be found in.The most pharmaceutically acceptable load Body can contain liquid, such as water, saline, glycerol and sorbitol.It addition, these carriers there is likely to be complementary Material, such as lubricant, fluidizer, wetting agent or emulsifying agent, pH buffer substance and stabilizer, such as albumin etc..

Described compositions can be made the various dosage form being suitable for mammal administration, and described dosage form includes but not limited to: Injection, capsule, tablet, Emulsion, suppository.

In use, it is that the E7 albumen to HPV 16 of the present invention of safe and effective amount is had The polypeptide of binding affinity or targeting molecule are applied to mammal (such as people), and wherein this safe and effective amount is typically at least About 1 microgram/kg body weight, and in most of the cases it is no more than about 10 mg/kg body weight, preferably this dosage It it is about 1 microgram/kg body weight-about 1 mg/kg body weight.Certainly, concrete dosage is it is also contemplated that route of administration, patient The factors such as health status, within the scope of these are all skilled practitioners technical ability.

Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate this Bright rather than limit the scope of the present invention.In the following example, the experimental technique of unreceipted actual conditions, generally presses More solito condition such as J. Pehanorm Brooker etc. is write, Molecular Cloning: A Laboratory guide, the third edition, Science Press, and 2002 Described in condition, or according to the condition proposed by manufacturer.

Embodiment 1, the library construction of HPV16E7 Binding peptide and screening study

Build the random combinatorial libraries of phage display HPV16 E7 Binding peptide, the most many different SPA domains The library of related polypeptide, screens HPV16 E7 Binding peptide from this library, and is identified its affinity.

1, the structure of the random combine phage display library of HPV16 E7 Binding peptide and qualification

Aminoacid sequence according to wild type SPA-Z and structure (Nilsson B etc., Protein Eng.1987；1(2): 107-113), for the coded sequence design random primer that its three helical structure districts are corresponding, PCR method is utilized to expand Obtain the SPA coded sequence that may result in random amino acid sudden change, named SPA-N.

2, the structure of pCANTAB5E/SPA-N recombiant plasmid

Select to carry out the expression of affine body with M13 bacteriophage systems (purchased from Beijing Bao Kewei Shi An biotech firm), therefore With pCANTAB5E (purchased from Beijing Bao Kewei Shi An biotech firm) as carrier, by Sfi I and Not I by acquisition SPA-N coding sequence, in the Sfi I/Not I site of pCANTAB5E carrier, builds PCANTAB5E/SPA-N recombiant plasmid, converts to competence E.coli TG1 cell, is coated with 2YT-A flat board, 37 DEG C of overnight incubation.It is primary storehouse, is labeled as affibody primary storehouse standby.Grow on result random picking flat board 28 monoclonal bacterium colonies, extraction plasmid Sfi I and Not I double digestion are accredited as positive colony, through order-checking and Analyze its randomness.

Result: according to sequencing result, send 28 clones of order-checking have sequencing result for 26 clones, Qi Zhonglian Being connected into merit 21 clone, randomness is entirely different, therefore recombination fraction is 21/26=88%；Multiformity is 21/21=100%. Meanwhile, the bacterium solution cultivated after taking above-mentioned conversion, with 2 × YT culture fluid doubling dilution (1:10,1:10²...), coating SOB-AG flat board, calculates the single clump count on flat board, calculates storage capacity.Storage capacity is added up by increasing connection conversion number of times, Repeatedly connecting makes clone's number reach 2.4 × 10 after converting⁵Individual Z protein variant (affibody molecule), its 9th, 10, 11,13,14,17,18,24,25,27,28,32,35 and 43 have random amino acid residue.

3, the screening of HPV16 E7 Binding peptide and titer determination

The HPV16 E7 albumen of purification is coated 96 hole ELISA Plate, through closing, add phage library (primary storehouse) and hatching, Add E.coli TG137 DEG C, jog incubation；Take 100 μ l, do gradient doubling dilution by 2*YT culture medium, take Diluent 100 μ l is coated with SOB-AG flat board, and 30 DEG C overnight, and counting combines phage-infect clump count, calculates HPV16 E7 combines phage titre；Result flat board visible colonies, titre is 10²；Now complete first round elutriation, another part Bacterium solution adds 10¹⁰Helper phage M13KO7 (purchased from Beijing Bao Kewei Shi An biotech firm) and kanamycin were cultivated At night, after being centrifuged, take supernatant through 0.22 μm membrane filtration, it is thus achieved that the phage literary composition after screening that HPV16 E7 molecule is affine Storehouse, for one-level affibody storehouse.Repeat above-mentioned 4 and take turns enrichment isolation, obtain the HPV16 affine screening of E7 molecule respectively After phage library, be two grades, titre is respectively 1 × 10⁶；On the basis of two grades of storehouses, repeat above-mentioned 4 take turns richness Collection screening, is three grades of libraries, and titre is 1 × 10⁸.Setting simultaneously is not added with the blank of phage and makees Synchronous Screening.

4, preparation and the ELISA of HPV16 E7 Binding peptide monoclonal phage identifies

ELISA combines the phage of affibody molecule for screening expression HPV16 E7.HPV by 10 μ g/ml 16E7 albumen is coated 96 hole ELISA Plate with 200 μ l/ holes, and 4 DEG C overnight；PBS washs, and closes 2h with 2% defatted milk powder； Washing, the phage obtained after taking four-wheel screening and isopyknic 3% defatted milk powder mixing, 200 μ l/ holes, 37 DEG C, 2h.Washing, adds the HRP/anti-M13 ELIAS secondary antibody (the anti-M13 of rabbit, Abcam#ab6188) of 1:10000 dilution, 200 μ l/ holes, 37 DEG C, 1h；Washing, addition OPD nitrite ion 200 μ l/ hole, 37 DEG C, 15min；2M H₂SO₄50μl/ Hole, terminates reaction；Put microplate reader (ELx800TM, BIO-TEK, Winooski, USA) and read OD490 value.

In four-wheel panning rounds, select the affibody molecule of conjugated antigen, select circulation through this four-wheel, further Activity is combined to analyze its HPV16 E7, with the ELISA of the OD490 higher than 0.5 with Phage-ELISA detection Value is selection standard, and the phage of identification code HPV16 E7 Binding peptide is selected above this ELISA signal value 150 clones, and carry out DNA sequence analysis with 12 clones without ELISA value of other random choose.

5, the Sequence Detection of HPV16 E7 affine body molecule and screening

Totally 150 monoclonals send Chinese Shanghai Sheng Gong company to check order, and sequencing primer is CATATGGTTGACAAC AAATTCAACAAAGAA(SEQ ID NO:12).Sequencing result with DNA STAR software analysis to standard sequence Row SPA-Z Yu SPA-N analyzes randomness and the multiformity of its three helical regions further.Result is the most just obtaining 78 Really cloned sequence, has partial sequence to repeat completely, obtains 65 right-on clones of sequence after annexing repetitive sequence.

It is analyzed according to DNA sequencing result, in 65 clones that above-mentioned order-checking is correct, selects and HPV16 E7 4 monoclonal phage that protein binding activity is the strongest (i.e. present the monoclonal phagocytosis of HPV16 E7 affine body molecule Body) DNA sequence (respectively Z_HPV16E7:127、Z_HPV16E7:301、Z_HPV16E7:384、Z_HPV16E7:745) as target mesh Mark is studied, and aminoacid sequence is SEQ ID NO:1,2,3,4, such as Fig. 1, their coded sequence such as SEQ ID NO:6-9).Next step combines the molecular cloning of affine body and expression and Function detection for HPV16 E7.

The present invention utilizes display technique of bacteriophage, the affibody random peptide library that will build, and is illustrated in phage surface, With HPV16 E7 albumen as target proteins, carry out the screening of the affine body of HPV16 E7.Eluriate through four-wheel, often take turns All pass through: affine-washing-amplification, and the concentration of target protein HPV16 E7 albumen drops along with the increase eluriating number of times Low, 50 μ g/ml, 30 μ g/ml, 15 μ g/ml, 10 μ g/ml it is thus possible to preferably filter out the affine body that affinity is high, And along with a large amount of amplifications often taken turns, affine body is often taken turns and is all had 10²Selective enrichment.Sieve further through ELISA Select the OD higher (A of value reading₄₉₀> 0.5) and monoclonal order-checking, by the most correct HPV16 E7 affibody of order-checking Sequence is analyzed by NTI software comparison, and 4 monoclonals that then random picking affinity is higher carry out next step Research, the amino acid sequence analysis of these 4 clones and the wild type comparison of standard, three helical structure Variable Areas go out Show about 13-14 amino acid whose change..

Embodiment 2, HPV16E7 Binding peptide construction of recombinant plasmid and prokaryotic protein expression and purification

As front have selected 4 clone (affibody Z in Fig. 1 with higher ELISA reading_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}), in order to the affibody molecule of screening is carried out Function detection, it is carried out Construction of recombinant plasmid, the expression of protokaryon albumen and qualification thereof, and prepare purifying protein.

1.pET21a the construction of recombinant plasmid of (+)/affibody and qualification

With reference to affibody gene order (GenBank:GY324633.1) design PCR primer, forward primer 5 ' GGGAATTCCATATGGTTGACAACAAATTCAACAAAGAA 3 ' (SEQ ID NO:13, lower stroke Line represents Ned I restriction enzyme site), downstream primer 5 ' CCGGAATTCCGTTTCGGAGCCTGAGCGT 3 ' (SEQ ID NO:14, underscore represents Xho I restriction enzyme site)；Order-checking correct level Four storehouse monoclonal with screening affibody Z_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}For template, expanded by PCR Affibody genes of interest (SEQ ID NO:6-9), simultaneously by affibody Z_wt(SEQ ID NO:1) is through protokaryon codon After optimization, complete sequence (SEQ ID NO:10) synthesizes as negative control.The genes of interest expanded by PCR is through Nde I With Xho I be cloned into pET21a (+) carrier, build pET21a (+)/Z_{HPV16 E7}Recombiant plasmid, and through order-checking mirror Fixed (Fig. 2, Fig. 3).

2. protokaryon protein production

By in recombinant plasmid transformed to escherichia coli (E.coli) BL21 (DE3), 37 DEG C, cultivate 16h；Add 0.8mM different Propyl dithiocarbamate-β-D-Thiogalactopyranoside (IPTG) (Merck & Co., Inc., Germany) IPTG inducing culture 6h express with The Z of His label_HPV16E7Affibody and Z_wtAffibody albumen.The recombiant protein expressed after induction, uses nickel chela Close affinity chromatograph colloid (Ni-NTA Agarose) (QIAGEN company, the U.S.) affinity chromatography purification warp SDS-PAGE Analysis and Identification.

As a result, utilize Protocols in Molecular Biology successfully construct pET21a (+)/Z_{HPV16 E7}Recombiant plasmid, and use former Nuclear expression system is prepared for the Z of purification_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}And Z_WT Affibody recombination fusion protein, confirms the dense dye of Coomassie brilliant blue occur through SDS-PAGE electrophoretic analysis (Fig. 4) Band molecular mass about 7.8kDa, with expection Z_{HPV16 E7}The molecular mass of affibody polypeptide is in the same size.The present invention Selection pET21a (+) carrier, the initial enzyme site utilizing its multiple clone site in design is NdeI (CATATG), Its codon ATG is aminoacid (M) start codon of destination protein translation, so utilizes prokaryotic expression system table The albumen reached is the destination protein Z of total length_{HPV16 E7}And without carrier protein fragment, it is to avoid carrier protein is to experiment The interference of result.

Embodiment 3, Z_{HPV16 E7}Affibody polypeptide and the combination of HPV16 E7 albumen

For identifying Z_{HPV16 E7}Affibody polypeptide and the protein bound specificity of HPV16 E7, utilize surface plasma altogether Four Z of technology of shaking (SPR) Analysis and Screening_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}Molecule and It compares Z_wtAffibody is specific binding with target protein HPV16 E7's.

The preparation of HPV16 E7 albumen: expand HPV16 E7 gene from cervical cancer tissues by PCR method, clone To pET21a (+) carrier, build pET21a (+)/HPV16 E7 recombiant plasmid, and qualification of checking order；To recombinate matter Grain converts to e. coli bl21 (DE3), expresses recombiant protein, use Ni-NTA affinity chromatograph after IPTG induces Method purification also (sees the restructuring of Wang Bingbing etc., HPVl6 type E7 through SDS-PAGE and Western blot Analysis and Identification The expression of albumen and the preparation of polyclonal antibody thereof；Cell and molecular immunology magazine, 2014 (2): 167-170).Knot Really, it is successfully prepared HPV16 E7 purifying protein and polyclonal antibody thereof, and has carried out specificity verification (Fig. 5 A-C).

Z_{HPV16 E7}The biosensor analysis of affibody polypeptide: in ProteOn XPR36 system instrument (Bio-Rad company) Carry out HPV16 E7 albumen and Z_{HPV16 E7}The affinity analysis of the interphase interaction of affibody polypeptide, i.e. utilizes table Face plasma resonance technology (SPR) analyzes the above-mentioned Z with His label_{HPV 16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、 Z_{HPV16 E7:745}And Z_wtInteraction between affibody molecule (as comparison) and HPV16 E7.According to manipulator Volume, fixes HPV16 E7 recombiant protein by being coupled to GLH chip on different flow cells, carries out and sieve The affinity between polypeptide is selected to measure.6th flow cell surface is activated and inactivates using right as blank during injection According to.Affibody molecule carries out six different gradient concentrations dilutions respectively and is combined, i.e. with HPV16 E7 recombiant protein Z_{HPV16 E7:127}Concentration is 1.0nM, 2.0nM, 4.0nM, 8.0nM, 16.0nM, 32.0nM, 64.0nM, Z_{HPV16 E7:301}Concentration is 0.7nM, 1.4nM, 2.8nM, 5.6nM, 11.2nM, 22.4nM, 44.8nM, Z_{HPV16 E7:384}Concentration is 0.6nM, 1.2nM, 2.4nM, 4.8nM, 9.6nM, 19.2nM, 38.4nM, Z_{HPV16 E7:745}Concentration be 1.1nM, 2.2nM, 4.45nM, 9.1nM, 18.3nM, 36.5nM, 73.2nM and Z_wtAffibody molecular concentration is 1.0nM, 2.0nM, 4.0nM, 8.0nM, 16.0nM, 32.0nM, 64.0nM, All of analysis is 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 flow velocity 30 μ l / min random order is injected, and (solves with HCl (BIO-RAD article No.: #176-2250100mM HCl) washing 6min subsequently From), utilize ProteOn Manager^TMThe 1:1 Langmuir binding model of software (BIO-RAD) analyzes binding curve (sense Should scheme).

Using surface plasma resonance technology (SPR) detection, analyze the result being repeated twice detection, affinity balance is dissociated Constant KD average, Z_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}And Z_wtAffibody molecule Respectively for 1.91 × 10^-6mol/L、1.18×10^-7mol/L、1.73×10^-6mol/L、5.02×10^-5Mol/L and 4.63×10^-2mol/L.As a result, the Z of purification_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}Albumen Molecule and HPV16 E7 recombiant protein have the ability of interaction, as Fig. 6 A-E shows, relatively Z_wtAffibody divides Sub-dissociation constant KD value differs 1000 to 10000 times.Through the Z that screening obtains_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}All can be combined with HPV16E7 recombiant protein, in conjunction with affinity reached nmol/L Level level, the meanwhile Z of wild type_wtAffibody molecule and HPV16 E7 recombiant protein almost without adhesion, Show that filter out 4 affibody molecules and HPV16 E7 recombiant protein have higher special affinity, simultaneously Show the Z of protokaryon abduction delivering_{HPV16 E7}Affibody molecule and HPV16 E7 recombiant protein are respectively provided with biological activity.

Therefore, the Z of the present invention_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}Protein molecular and HPV16 E7 protein target molecule has and be combined with each other and identification ability.Z is demonstrated from protein level_{HPV16 E7:127}、Z_{HPV16 E7:301}、 Z_{HPV16 E7:384}、Z_{HPV16 E7:745}Affinity between molecule and HPV16E7 target protein.

Embodiment 4, Z_{HPV16 E7}Affibody polypeptide and the combination expressing HPV16 E7 albuminous cell

Z for checking screening further_{HPV16 E7}Affibody polypeptide and the affinity of HPV16E7 target protein, utilize table Reach the cervical cancer cell of HPV16 E7 as object of study, i.e. Human cervical cancer cell lines Siha (ATCC:HTB-35, Express the cervical cancer cell of HPV16 E7), (ATCC:CRL-1550 expresses HPV16 and the palace of 18 E7 to Caski Neck cancer cell) and HeLa229 (cervical cancer cell of ATCC:CCL-2 HPV 18 positive), and utilize melanoma thin Born of the same parents system A375 (melanoma cell that ATCC:CRL-1619, HPV are negative), as comparison, verifies Z further_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}Combination between protein molecular and HPV16 E7 protein molecular.

Cell is cultivated: Human cervical cancer cell lines Caski, Siha, HeLa229 and K-1735 A375 cell Be incubated at RPMI 1640 culture medium (10% hyclone, 2.05mM L-paddy ammonia phthalein amine and 100IU/ml penicillin and 100 μ g/ml streptomycins).Cell contains 5%C0 at 37 DEG C₂Incubator in cultivate to 24h, cell state is good Shi Jinhang Immunofluorescence test.

Cellular immunofluorescence detects: put into by the coverslip of sterilizing in six orifice plates, by cultivate to the Caski of 24h, Siha, It is 1 × 10 that HeLa229, A375 cell adjusts number⁶/ hole, 5%CO₂, 37 DEG C cultivate 24h to cell monolayer.Add Enter final concentration of 50 μ g/ml Z_{HPV16 E7}Affibody polypeptide in above-mentioned containing in 10%FBS culture medium, 5%CO₂、 Cultivate 6h, sucking-off culture fluid, wash with pre-cooling PBS for 37 DEG C；2% paraformaldehyde is used to fix cell monolayer 10min, PBST washs 3 times, adds 0.3%Triton X-100 punching 10min, adds 10%FBS+1640 training after washing Support 37 DEG C of base and close 1h, washing；Be separately added into mouse-anti His monoclonal antibody (ABR company, the U.S., 1:2000), Rabbit anti-SPA serum (1:500) and HPV16E7 rabbit anteserum antibody (1:500), put 37 DEG C of 1h, add FITC-after washing Goat anti-rabbit igg two anti-(Shanghai Lian Ke biotech company, China) and PI (Suo Laibao company, Beijing) 2 μ l/ hole 1h, Lucifuge, adds coverslip and with buffering glycerol mounting, confocal fluorescent microscope (Leica TCS SP2 after washing Microscope Germany) observe and make film (400 ×).

With the Z of purification_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}Hatch HPV16 E7 albumen table After reaching positive cervical cancer Caski, Siha cell 6h, respectively with His monoclonal antibody, SPA rabbit anteserum analysis restructuring The identification of the HPV16 E7 recombiant protein that affibody albumen is expressed with cell strain and binding ability, arranged simultaneously The Humanmachine tumour A-375 cell strain of HPV16 E7 protein expression feminine gender and the cervical cancer of the HPV18 E7 positive HeLa229 cell strain, as comparison, meanwhile arranges HPV16 E7 rabbit anteserum antibody as positive control.

Result shows, Z_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}Siha, Caski that albumen is hatched The nuclear membrane of cell strain and the point-like of the multiple strong green of cytoplasm kernel Zhou Kejian or lumps fluorescin (are schemed 7A-D), consistent with the result of HPV16 E7 rabbit anteserum antibody incubation (Fig. 7 E), and HeLa229 cell and A375 Cell strain is showed no obvious fluorescence agglomerate (Fig. 7 A-E).Show, Z_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、 Z_{HPV16 E7:745}Natural expression HPV16 E7 albumen in recombiant protein energy specific recognition cell, prepared by the present invention Z_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}HPV 16 E7 that recombiant protein and living cells are expressed Albumen has the strongest specific bond ability.

The above results demonstrates Z from cellular level further_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745} Recombiant protein and HPV16 E7 albumen have the strongest targeting binding specificity and affinity.

Embodiment 5, Z_{HPV16 E7}Affibody polypeptide bio distribution in cervical cancer cell tumor bearing nude mice and cancer target

In the experiment of the present embodiment, select the Z in above-mentioned affibody molecule_{HPV16 E7:384}Affibody polypeptide conduct Detection object, with near infrared fluorescent dye DyLight755 NHS Ester (Thermo Fisher company, the U.S., goods Numbers 62278) labelling Z_HPV16E7:384Affibody polypeptide, and be injected into carrying Siha, Caski, HeLa229 And in the Mice Body of A375 transplantation tumor, carry out Z_{HPV16 E7:384}Affibody polypeptide biodistribution research, and to naked Mice imaging location is to study the targeting characteristic of labeling polypeptide.

The preparation of animal tumor model: select 6-7 week old BALB/c-nu mice (to be purchased from Shanghai Si Laike laboratory animal to have Limit responsible company, quality certification SCXK (Shanghai) 2012-0002), body weight is 15-18g).To cultivate to exponential phase, After Siha, Caski, HeLa229 and A375 EDTA (pancreatin) digestion that growth conditions is good, with containing 10% blood Clear cell culture fluid carries out blowing and beating, collecting, and 1000 turns/min of room temperature is centrifuged 3min, by centrifuge cell with without blood Clear culture fluid is resuspended and counts, and is configured to 1 × 10⁷/ ml, take 0.2ml in back nearly right forearm or lower limb nearly abdomen stock Ditch inoculated with subcutaneous injections nude mice.Every 3 days observe the mental status of mice, energy, reaction, diet, body weight and Subcutaneous vaccination region outward appearance and sense of touch, and with electronic cursor kind of calliper tumorous size diameter.Treat tumor length extremely 300-500mm³For Z_{HPV16 E7}The bio distribution of affibody polypeptide and imaging research.

Result shows, nude mice by subcutaneous inoculates all visible significantly tumor growth of above-mentioned cell, and 22 nude mices all become tumor. After 3 weeks, longest diameter of tumor reaches 300-500mm³Time start experiment.

Z_{HPV16 E7:384}Polypeptide near infrared fluorescent dye Dylight755 labelling and qualification: to specifications step carry out by The labelling of Dylight755 is also identified.I.e. take 91 μ g DyLight755 NHS-Ester dyestuffs and be dissolved into 273 μ l DMF After organic solvent, add the Z after dialysis_{HPV16 E7:384}In affibody albumen (300 μ g/ml, altogether 1ml) solution, keep away Light, reacts 1h, reacted solution lucifuge is dialysed under the conditions of 4 DEG C, (phosphate delays to change dialysis solution every half an hour Rush liquid, pH7.2-7.4), collect Dy755-Z after 2h_HPV16E7:384Fluorescin, it is 100 μ g/ml that protein concentration is surveyed in sampling, SDS-PAGE electrophoresis is used to identify.Take Dy755 labelling respectively Z_HPV16E7:384(Dy755-Z_HPV16E7:384) 1 μ g, 0.5 μ g and 0.1 μ g, it is diluted to 10 μ l with phosphate buffer, with Time take 10 μ l Dy755-DMF dyestuffs and do negative control, be separately added in 10 μ l albumen loading buffer, Yu Bing Electrophoresis under the conditions of upper lucifuge, and gel is put in living imaging instrument (CRi Maesro 2.10, in-vivo fluorescence Imaging system) in, exciting light filter disc is 671-705nm, and launching light filter disc is 750 longpass, uses 8bit With 2 × 2 patterns, expose 5000ms with 730-950nm wavelength interval 10nm every time and collect image information, use Maesro Software carries out image process and analysis.Identified Dy755-Z_HPV16E7:384It is sub-packed in brown centrifuge tube ,-20 DEG C of guarantors Deposit standby.

Result shows, labelled protein Dy755-Z_HPV16E7:384Through SDS-PAGE electrophoretic analysis, 1.0 μ g, 0.5 μ g and The swimming lane of 0.1 μ g is to occur single stainable bands at 7.8 kDa at relative molecular mass, but Dylight755 dyestuff Without band (Fig. 8 A), scan through small animal living body imager, all occur single at corresponding 1.0 μ g and 0.5 μ g position Fluorescent bands (Fig. 8 B).Show, near infrared fluorescent dye Dylight755 labelling Z_{HPV16 E7:384}Affibody polypeptide Success.

Z_{HPV16 E7:384}Affibody polypeptide bio distribution in the nude mice of tumor cell xenograft and cancer target Effect: treat that the tumor length of nude mice is to 300-500mm³Time take nude mice out of SPF barrier system, use 10% chloral hydrate Lumbar injection induced anesthesia, through tail vein injection 10 μ g Dy755-Z after it enters deep anaesthesia state_HPV16E7:384 Fluorescin, is placed in small animal living body imager (CRi Maesro 2.10, in-vivo fluorescence imaging System) imaging in, and maintain narcotism with 0.8-1.0 μ l/g chloral hydrate, to ensure that mice is located in imaging process In deep anaesthesia, 5min, 10min, 15min, 30min, 45min, 1h, 1.5h, 2h, 4h, 6h, 8h and 24h continuous imaging observes (Fig. 9 A-B).And put to death after 24h and dissect mice, take tumor tissues, liver, The tissue internal organs such as spleen, kidney and brain are placed in imaging system.The exciting light filter disc of imaging is 671-705nm, sends out Penetrating light filter disc is 750 longpass, uses 8bit and 2 × 2 patterns, and 730-950nm wavelength interval 10nm exposes every time Light 5000ms collects image information, and carries out image process and analysis with Maesro software, and display background is spontaneous respectively Fluorescence and target fluorescent signal, then measure its fluorescent value, and background auto-fluorescence is set to black, and target fluorescent is believed Number it is set to redness, finally by superimposed for two kinds of colors.The data obtained GraphPAD software processes.

As a result, Siha and Caski tumor bearing nude mice is at tail vein injection 50 μ g Dy755-Z_HPV16E7:384Fluorescin 10min After, i.e. there is obvious fluorescence signal in tumor locus, and 1h-1.5h fluorescence signal is the most complete, corresponding with tumor size, 8 During h, the fluorescence imaging of tumor substantially diminishes, and still has fluorescence imaging during 24h.Fluorescence in addition to kidney, in tumor tissues Intensity starts to raise when 30min, is about all remarkably higher than the fluorescence intensity of other each internal organs to 6h, although to 24h Fluorescence intensity has weakened, but still higher than other each internal organs.During the observation of HeLa229 and A-375 matched group the most not The fluorescence signal being clearly distinguishable from background fluorescence detected.The experimental group position corresponding with nude mice of control group liver spleen exists Dy755-Z after intravenous injection_HPV16E7:384Visible significantly fluorescence signal after fluorescin, along with dyestuff is in Mice Body Constantly metabolism, signal is gradually lowered, and has weakened disappearance to 24h；And kidney Dy755-after intravenous injection Z_HPV16E7:38410min after fluorescin is until 24h, it is seen that the strongest fluorescence signal (Fig. 9).Analysis tumor tissues is glimmering Light signal strength finds, Siha and Caski cell tumor-bearing mice is the highest, the most gradually in 30min left-right signal intensity Reduce, respectively to 6h and 8h left-right signal intensity the most substantially (Fig. 9).

At tail vein injection 50 μ g Dy755-Z_HPV16E7:384Dissect mice during fluorescin 24h, take experimental group and right The vital tissue organs such as tumor, heart, lung, spleen, liver and the kidney according to group carry out imaging, find tumor, kidney In the tissue such as dirty, the heart, brain, intestinal, lung, muscle and skeleton, fluorescence signal distribution is basic with living imaging result with intensity Consistent (Figure 10 A-D), and in Siha and Caski tumor-bearing mice, the fluorescence intensity of tumor tissues is each dirty with other The fluorescence intensity of device has a significant difference (p < 0.05), and tumor tissues in HeLa229 and A375 tumor-bearing mice Fluorescence intensity there was no significant difference (p > 0.05) with the fluorescence intensity of other each internal organs.Tumor-bearing mice is state during observing Well, have no that overt toxicity reacts.

Result shows, labelling Dylight755-Z_HPV16E7:384Polypeptide has targeting and combines HPV16E7 expression positive tumor Characteristic, and there is no serious toxic and side effects.

Embodiment 6, Z_{HPV16 E7}The biological function research of affibody polypeptide

For research Z_{HPV16 E7}Affibody polypeptide in-vitro cell growth is the most inhibited, and the present invention selects Siha Tumor cell line is as experimental subject.Utilize the cell of each time point in CCK-8 reagent detection cell growth process raw The rate of depositing detects, and utilizes Grahpad primer5.0 computed in software Z_HPV16E7The IC50 of affibody polypeptide.

Preparation Siha tumor cell suspension is also inoculated in 96 porocyte culture plates, and 0.5 × 10⁴/ hole.After cultivating 24h, add Enter 1 μ g/ml cycloheximide, 5% hyclone, be then respectively adding 4 i.e. Z of affibody albumen_{HPV16 E7:127}、 Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}, be respectively provided with 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 10 μ g/ml, 1 μ g/ml five concentration group, and with Z_wtAffibody is matched group, sets culture medium comparison simultaneously, uses CCK-8 examination The survival rate of agent box (Cell Counting Kit-8, GC-0030, CN) detection cell, i.e. adding after Binding peptide 0, 1,3,6,12,24,36,48 and 72h, add CCK-8 reagent, 10 μ l/ holes, at CO₂Continue in incubator Hatch 30min and be placed on reading at microplate reader A450.Often group is all provided with 3 multiple holes, and carries out 3 times repeating experiment. Cell survival (Cell viability) computational methods are: Cell viability (%)=(OD value-cultivation of affitoxin384 group Basis set OD value)/(SPA-Z_wtMatched group OD value-cultivate basis set OD value) × 100%；Use GraphPad primer simultaneously 5.0 softwares calculate the IC50 value of cell growth survival.

As a result, Z_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}The polypeptide life to Siha tumor cell Long all have inhibitory action (Figure 12 A-D), its IC50 is 2.882 ± 0.661 μMs respectively, 3.876 ± 0.125 μMs, 2.725 ± 0.358 μM and 1.835 ± 0.233 μMs (Figure 11 A-D).Display, Z_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、 Z_{HPV16 E7:745}It is respectively provided with inhibitory action to expressing cervical cancer cell Siha positive for HPV16 E7, and it presses down in 6h Cell growth effect processed shows as the strongest, to 72h, with Z_wtStill significant difference (p < 0.001) is had with culture medium comparison； Show, Z simultaneously_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}The polypeptide suppression to Siha cell Effect has concentration dependent, and the cells survival rate between 100 μ g/ml, 50 μ g/ml and 10 μ g/ml groups has significantly Sex differernce (p < 0.001), simultaneously three groups of dosage groups and Z_wtMore also have aobvious with the cells survival rate of medium controls Write sex differernce (p < 0.001).

Result above shows, Z_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}Polypeptide has suppression Siha The characteristic of cell growth, demonstrates Z further_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745}Have External HPV16 E7 targeting binding specificity.

Embodiment 7, Z_{HPV16 E7}The protective effect to cervical cancer cell tumor bearing nude mice of the affitoxin albumen

In the present embodiment, Z is utilized_{HPV16 E7}Targeting, carry the green pus bar through changing structure with cytotoxicity The active fragment PE38KDEL of bacterium exotoxin A (pseudomonas exotoxinA, PEA) is as Cytotoxic molecules (Gao J, et al.Mol Cancer Ther, 2008,7 (10): 3399-3407).Build Z_{HPV16 E7}/ PE38KDEL protokaryon Expression vector, and utilize prokaryotic expression system preparation purification Z_HPV16E7/ PE38KDEL albumen, i.e. affitoxin, Specific binding to affitoxin and target protein HPV16 E7, and targeting carries out and verifies, and entered one Step be expelled to carry Siha (ATCC:HTB-35 expresses the cervical cancer cell of HPV16), Caski (ATCC: CRL-1550, expresses the cervical cancer cell of HPV16 and 18), (ATCC:CCL-2 HPV 18 is positive for HeLa229 Cervical cancer cell) and the mice of A-375 (ATCC:CRL-1619, HPV16 negative melanoma) transplantation tumor Internal, carry out tumor protection and Experiment on therapy.Use Z simultaneously_wtAffitoxin compares.

Z_{HPV16 E7}The preparation of affitoxin albumen and qualification: select Z_{HPV16 E7}Affibody is as the target of HPV16 E7 To molecule, utilize flexible peptide (Gly₄Ser)₃Connect PE38 (KDEL) at its C-terminal, flexibility PEPC end is connected warp The complete sequence (SEQ ID NO:11) of the PE38 (KDEL) that protokaryon is codon optimized, the complete sequence of PE38 (KDEL) DNA synthesis and through EcoRI and XhoI site be cloned into pET21a (+) carrier, build pET21a (+)/PE38 (KDEL) Carrier, further by molecular cloning method by Z_{HPV16 E7:127}、Z_{HPV16 E7:301}、Z_{HPV16 E7:384}、Z_{HPV16 E7:745} And Z_wtCoded sequence through NedI and EcoRI be cloned into pET21a (+)/PE38 (KDEL) vector construction recombiant plasmid is also Order-checking is identified, is converted further to e. coli bl21 (DE3), expresses recombiant protein after IPTG induces, Purify with Ni-NTA affinity chromatography and through SDS-PAGE and Western Blot Analysis and Identification.

As a result, successfully construct pET21a (+)/Z_{HPV16 E7}Affitoxin127,301,384,745 and pET21a (+)/Z_wt Affitoxin recombiant plasmid (Figure 13)；This recombiant plasmid is at prokaryotic expression the Z that obtains purification_HPV16E7 affitoxin127、Z_HPV16E7 affitoxin301、Z_HPV16E7 affitoxin384、Z_HPV16E7Affitoxin745 and Z_wt Affitoxin recombiant protein, SDS-PAGE electrophoresis points out the molecular mass of this albumen to be about 45 kDa (Figure 14 A, B)； Western blot result (Figure 14 C, D) shows, His monoclonal antibody and rabbit SPA-Z multi-resistance resist as one, in molecular mass It is about 45kDa and all locates that single band occurs, point out Z_HPV16E7 affitoxin127、Z_HPV16E7 affitoxin301、 Z_HPV16E7 affitoxin384、Z_HPV16E7Affitoxin745 and Z_wtAffitoxin recombiant protein energy specific recognition rabbit SPA-Z polyclonal serum antibody.Result shows, Z_HPV16E7 affitoxin127、Z_HPV16E7 affitoxin301、Z_HPV16E7 affitoxin384、Z_HPV16E7Affitoxin745 and Z_wtAffitoxin restructuring purifying protein is successfully prepared.

Z_HPV16E7Affitoxin albumen is studied with target protein binding characteristic: for checking Z_HPV16E7Affitoxin albumen with The characteristic that HPV16 E7 combines, uses SPR and cellular immunofluorescence method to verify further.Select Siha (HPV16+), Caski (HPV16+, HPV18+), HeLa229 (HPV16-, HPV18+) and melanoma Cell A375 is as negative control, and method is with embodiment 4.Will cultivate the Caski to 24h, Siha, HeLa229, A375 cell, adjusting cell number is 1 × 10⁶/ hole, 24h is to cell monolayer in cultivation, adds final concentration of 50 μ g/ml Z_{HPV16 E7}affitoxin127、Z_{HPV16 E7}affitoxin301、Z_{HPV16 E7}affitoxin384、Z_{HPV16 E7} Affitoxin745 and Z_wtAffitoxin albumen, 5%CO₂, cultivate 6h for 37 DEG C, after fixing, washing, 0.3%Triton X-100 punches, and adds 10%FBS+1640 culture medium 37 DEG C and closes 1h, washs 3 times with PBS；It is separately added into Mouse-anti His monoclonal antibody (ABR company, the U.S., 1:2000), rabbit anti-SPA serum (1:500) and rabbit are anti- PE38KDEL serum (1:5000), puts 37 DEG C of 1h, adds anti-(the Shanghai Lian Kesheng of FITC-goat anti-rabbit igg two after washing Thing technology company, China) and PI (Suo Laibao company, Beijing) 2 μ l/ hole 1h, lucifuge, add coverslip after washing and use Buffering glycerol mounting, (40 ×) are observed and made film to confocal fluorescent microscope (Leica TCS SP2 microscope Germany). Meanwhile, employing is not added with Z_{HPV16 E7} affitoxin127、Z_{HPV16 E7} affitoxin301、Z_{HPV16 E7} affitoxin384、 Z_{HPV16 E7}Affitoxin745 albumen, is that one anti-(1:5000) is as positive control using rabbit HPV16 E7 serum antibody.

As a result, detect through cellular immunofluorescence, show at confocal fluorescent basis of microscopic observation, Z_{HPV16 E7} affitoxin127、Z_{HPV16 E7}affitoxin301、Z_{HPV16 E7}affitoxin384、Z_{HPV16 E7}Affitoxin745 albumen The nuclear membrane of Siha, Caski cell strain hatched and the point-like of the multiple green of cytoplasm kernel Zhou Kejian or lumps Fluorescin, consistent with the result of HPV16 E7 rabbit anteserum antibody incubation, and HeLa229 cell and A375 are thin Born of the same parents' strain is showed no obvious fluorescence agglomerate (Figure 15 A-D), Z simultaneously_wtSiha that affitoxin albumen is hatched, Caski, All there is not fluorescence agglomerate in HeLa229 and A375 cell strain.Show, Z_{HPV16 E7}affitoxin127、Z_{HPV16 E7} affitoxin301、Z_{HPV16 E7} affitoxin384、Z_{HPV16 E7}Affitoxin745 recombiant protein identification HPV16 E7, And the ability of natural expression HPV16 E7 albumen keeps not in nonrecognition HPV18 E7, i.e. specific recognition living cells Become, and the natural HPV16 E7 albumen expressed with living cells has the strongest binding ability.The present embodiment is further from carefully Born of the same parents level verification Z_{HPV16 E7} affitoxin127、Z_{HPV16 E7} affitoxin301、Z_{HPV16 E7} affitoxin384、Z_{HPV16 E7}Affitoxin745 recombiant protein has the HPV16 E7 albumen expressed with living cells and has the strongest affinity.

Z is carried out further at ProteOn XPR36 system instrument (Bio-Rad company)_{HPV16 E7}Affitoxin albumen and The affinity analysis of the interphase interaction of HPV16E7, i.e. utilizes surface plasma resonance technology (SPR) to analyze above-mentioned The Z of His label_HPV16E7 affitoxin127、Z_{HPV16 E7} affitoxin301、Z_{HPV16 E7} affitoxin384、Z_{HPV16 E7} Affitoxin745 and Z_wtInteraction between affitoxin molecule (as negative control) and HPV16 E7.Experiment Step is with embodiment 3.

As a result, use surface plasma resonance technology (SPR) analyze record affinity dissociation constant KD value respectively be 1.36×10^-6mol/L、1.76×10^-6mol/L、1.45×10^-6mol/L、3.90×10^-6mol/L.It is repeated twice detection. Result shows, the Z of purification_HPV16E7 affitoxin 127、affitoxin 301、affitoxin 384、affitoxin 745 Albumen is (with His₆Label) there is the ability of interaction, such as Figure 16 A-D and table 2 with HPV16E7 recombiant protein Shown in, 4 affitoxin albumen all can be combined with HPV16 E7 recombiant protein, in conjunction with affinity the most reach Nmol/L level level, and the Z of wild type_wtAffitoxin and HPV16 E7 recombiant protein is almost without adhesion, table Bright four the affibody albumen filtered out and HPV16E7 recombiant protein have higher special affinity.

Result shows, the Z of the present invention_HPV16E7 affitoxin 127、affitoxin 301、affitoxin 384、affitoxin 745 Protein molecular is (with His₆Label) have be combined with each other and identification ability with HPV16E7 protein target molecule.Further Z is demonstrated from protein level_HPV16E7 affitoxin 127、Z_HPV16E7 affitoxin 301、Z_HPV16E7 affitoxin 384、 Z_HPV16E7Affinity between affitoxin 745 and HPV16E7 target protein.

Z_HPV16E7Affitoxin in-vitro cell growth inhibitory action: for research Z_HPV16E7Affitoxin in-vitro cell growth Inhibitory action, selects Z_HPV16E7Affitoxin384 albumen is as object of study.Select equally Caski, Siha, Tetra-kinds of tumor cell lines of HeLa229 and A375 are as target cell.Prepare above-mentioned four kinds of cell suspension and counting is inoculated in 96 porocyte culture plates, 0.5 × 10⁴/ hole.After cultivating 24h, add 1 μ g/ml cycloheximide and 5% hyclone, It is subsequently adding Z_HPV16E7Affitoxin384 albumen, arrange 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 10 μ g/ml, 1 μ g/ml five concentration group, with SPA-Z_wtFor matched group (PE38KDEL), five same concentration groups are set, simultaneously If culture medium comparison, use CCK-8 kit detection cell survival rate, after sample-adding 1h, 3h, 6h, 12h, 24h, 48h and 72h, add CCK-8 reagent 10 μ l/ hole, at CO₂Continue to hatch 30min in incubator to be placed on Reading at microplate reader A450.Often group is respectively provided with 3 multiple holes, and carries out 3 times repeating experiment.Cell survival (Cell Viability) computational methods are: Cell viability (%)=(Z_HPV16E7The OD value of affitoxin384 group-cultivate is basis set OD value)/(SPA-Z_wtMatched group OD value-cultivate basis set OD value) × 100%；Simultaneously with GraphPad primer 5.0 Software calculates the IC50 value of cell growth survival.

Result such as Figure 17, calculates the IC50 of Siha and Caski cell growth survival through GraphPad primer 5.0 software Value, respectively 0.28 μM and 0.24 μM.Figure 18 shows, Z_HPV16E7Affitoxin 384 is to expressing HPV16 E7 Positive cervical cancer cell Siha and Caski has stronger lethal effect, and to expressing the palace of the HPV18E7 positive Melanoma cell A375 of neck cancer HeLa229 cell and expression HPV feminine gender is all without obvious lethal effect.Figure 19 displays, Z_HPV16E7After affitoxin 384 acts on Siha and Caski 72h, its cells survival rate and compared with control cells Between compare, compare zero difference (p > 0.05) between Siha with Caski cells survival rate, Siha and Caski cell with A significant difference (p < 0.05) is more all had between HeLa229 cells survival rate, and Siha and Caski cell and A375 More also there is between cells survival rate significant difference (p < 0.05), and HeLa229 Yu A375 cells survival rate it Between compare and there was no significant difference (p > 0.05).Z_HPV16E7Three dosage groups (the 100 μ g/ml, 10 μ g/ml of affitoxin 384 And 1 μ g/ml) between Siha and Caski cell growth rate all had significant difference (p < 0.05), and 1 μ g/ml with Difference that between PE38KDEL and medium controls, there are no significant (p < 0.05).Z_HPV16E7Affitoxin 384 is with right Act on Siha according to albumen PE38KDEL, its cells survival rate relatively have significant difference (p < 0.05)；And Z_HPV16E7Affitoxin384 and PE38KDEL be equal zero difference (p > 0.05) between compareing with culture medium.Z_HPV16E7 Affitoxin 384 and reference protein PE38KDEL acts on Caski, its cells survival rate relatively have significance poor Different (p < 0.05)；And Z_HPV16E7Affitoxin384 and PE38KDEL be equal zero difference between compareing with culture medium (p>0.05).Result above further demonstrates that, Z_HPV16E7Affitoxin 384 have external HPV16E7 targeting combine and Kill the specificity of cell.

Z_HPV16E7The pharmacokinetics of affitoxin384 albumen: select 7 week old BALB/C-nu Female nude mice, 5 Nude mice tail vein injection 5mg/kg Z_HPV16E7Affitoxin384, respectively 1min, 5min, 15min, 30min, Tail venous blood sampling 100 μ l when 60min, 120min, after 5h, anesthetized mice end end takes blood.Separately take 5 nude mice tails quiet Arteries and veins injection 4mg/kg Z_{HPV16 E7:384}, 3 nude mice tail vein injection PBS 100 μ l as comparison, method and take blood The time point of specimen is ibid.After blood is put 37 DEG C of water bath 20min, 1500r, 4 DEG C of centrifugal 10min, collect blood Clearly-80 degree subpackages preserve.Use affitoxin and the affibody concentration in ELISA method detection serum.Will The HPV16E7 albumen of 10 μ g/ml is coated 96 hole ELISA Plate 4 DEG C overnight, adds 1:50 dilute serum, hatches for 37 DEG C 2h, adds 3% defatted milk powder, after 37 DEG C are closed 2h, adds the SPA-Z rabbit anteserum prepared this room as two after washing Anti-, 100 μ l/ holes, 37 DEG C of 1h, add goat anti-rabbit igg-HRP (1:5000) 100 μ l/ hole, 37 DEG C of 1h after washing；Wash Wash, add TMB nitrite ion, 100 μ l/ holes, 37 DEG C of lucifuge colour developing 30min；Put microplate reader 450nm wavelength to read Absorbance.Repeat 3 multiple holes.It is not added with affibody, and difference after arranging to be coated HPV16E7 albumen simultaneously It is one anti-as positive and negative control using the rabbit anteserum (1:1000) of anti-16E7 and SPA-Z rabbit anteserum (1:2000).Take 0,0.1 μ g/ml, 1 μ g/ml, 10 μ g/ml, affibody and the affitoxin albumen of 100 μ g/ml, examines through ELISA Standard curve is made in survey.

As a result, with the HPV16E7 albumen of purification as envelope antigen, use Z in ELISA method detection serum_{HPV16 E7} Affibody and Z_{HPV16 E7}Affitoxin concentration, draws standard curve.Z_HPV16E7Affibody and Z_HPV16E7 affitoxin Computing formula be respectively y=0.1295x-0.11065, R²=0.9942；Y=0.2378x-0.141, R²=0.9782.? In 1min, 5min, 15min, 30min, 60min, 120min and 300min blood after nude mice tail vein injection Content be respectively 122.59 μ g/ml, 82.82 μ g/ml, 54.25 μ g/ml, 48.07 μ g/ml, 45.37 μ g/ml, 34.17 μ g/ml, 28.49 μ g/ml, 20.66 μ g/ml and 112.28 μ g/ml, 91.36 μ g/ml, 67.28 μ g/ml, 51.09 μ g/ml, 42.05 μ g/ml, 32.49 μ g/ml, 29.54 μ g/ml, 24.14 μ g/ml, according to Graphpad prisimer5.0 Draw curve (Figure 20 A, B) to calculate, Z_{HPV16 E7}Affibody and Z_{HPV16 E7}The half-life of affitoxin is respectively 6.231 ± 0.717min and 8.95 ± 0.389min, Z_{HPV16 E7}Affitoxin compares Z_{HPV16 E7}The half-life of affibody About many 2 minutes.

Z_HPV16E7The median lethal component analysis LD50 of affitoxin albumen: for research Z_{HPV16 E7}Affitoxin384 albumen Acute Toxicity, the selection female Mus of BALB/c is as experimental subject, and calculates its median lethal dose(LD 50).Select 4w age The female Mus of BALB/c, 18-20g, it is divided into 9 dosage groups, often group 5-7 is only, is shown in Table 3.Every mouse tail vein is only Single administration, injects the Z of 200 μ l corresponding dosage_{HPV16 E7}Affitoxin384 albumen.Observation index after administration: outward appearance, Sign, body weight and each dosage group mouse diing time, continuous observation to 14 day, and use Graphpad Primer5.0 Method calculates LD50, detects in triplicate.Result Z_{HPV16 E7}Affitoxin384 albumen causes the LD50 that mice half is dead For: 15.577 ± 3.738mg/kg.

Table 3, Z_HPV16E7The Acute Toxicity of affitoxin384 albumen

Dosage, mg/kg

25

20

15

10

5

2.5

1

0.5

0.25

Mortality rate (1)

5/5

4/5

3/5

1/5

0/6

0/7

0/7

0/7

0/7

Mortality rate (2)

5/5

2/5

0/5

0/5

0/6

0/7

0/7

0/7

0/7

Mortality rate (3)

5/5

3/5

2/5

0/5

0/6

0/7

0/7

0/7

0/7

Z_{HPV16 E7}The protective effect of affitoxin albumen: for detection Z_{HPV16 E7}Affitoxin384 albumen is to tumor cell The protective effect of oncogenic function, the present embodiment uses Siha cell positive for HPV16 E7 to prepare BALB/c nude mice Tumor model, concrete grammar with embodiment 5, i.e. select 4-5 week old BALB/c-nu mice, body weight is 12-15g, Raise in Wenzhou Medical University's barrier system in experiment animal center in colleges (SPF level), be divided into into 5 groups, i.e. Z_{HPV16 E7}Affitoxin384 protein groups, arranges Z simultaneously_HPV16E7:384、PE38KDEL、Z_wtAffitoxin and PBS conduct Matched group.Often group 10～12.Affitoxin384.0mg/kg, arranges PE38KDEL (mg/kg) as right simultaneously According to group.By cultivation 48h to exponential phase Siha cell, it is configured to required cell number i.e. 1x10⁷/ ml, takes 0.2ml In the back of nude mice nearly left forearm subcutaneous injection, each histone (being 4mg/kg) is diluted in the PBS of 0.2ml simultaneously In, by group through tail vein injection 0.2ml correspondence albumen while carrying out tumor cell inoculation, PBS group is only injected The PBS of 0.2ml.Injection in every 3 days 1 time, totally 6 times.Within every 3 days, observe mice life and the mental status etc., measure Tumor size Taking Pictures recording, observed to 60 days.Putting to death mice, detection mice serum surveys liver function simultaneously.

Meanwhile, for detection Z_{HPV16 E7}The affitoxin384 albumen therapeutical effect to Siha tumor-bearing mice, alternative selects 4-5 Week old BALB/c-nu mice, inoculates 1x10⁷/ ml Siha cell, treats tumor growth 100～200mm³During size, Being divided into above-mentioned same 5 groups, every nude mice is through tail vein injection 4mg/kg albumen 0.2ml counter sample altogether.Often Injection in 3 days 1 time, totally 6 times.Within the most every 3 days, observe mice life and the mental status etc., measure tumor size and clap According to record, observe to 60 days.Putting to death mice, detection mice serum surveys liver function simultaneously.

Shown in result as Figure 21 A, Z_{HPV16 E7}Affitoxin384 albumen has certain guarantor to tumor cell oncogenic function Protect effect, Z_{HPV16 E7}The volume size of affitoxin384 albumen dosage group tumor and matched group Z_HPV16E7:384、 PE38KDEL、Z_wtAffitoxin albumen and PBS group began to have significant difference (p < 0.01) at the 36th day respectively, And Z_HPV16E7: 384 and PE38KDEL, Z_wtAffitoxin albumen and PBS group began to also begin to out at the 42nd day respectively Existing significant difference (p < 0.01), but PE38KDEL, Z_wtZero difference (p > 0.05) between affitoxin albumen and PBS group. Prolongation over time, gross tumor volume size is between each group, and obvious difference becomes big, until 60 days.Liver merit Transaminase's index can be detected the most normal.

Z_{HPV16 E7}The therapeutical effect result of Siha tumor-bearing mice is shown in Figure 21 B by affitoxin384 albumen.Z_{HPV16 E7} Affitoxin384 protein groups and Z_HPV16E7:384、PE38KDEL、Z_wtAffitoxin albumen and PBS control group compare, Began to have significant difference (p < 0.01) at the 30th or 36 day respectively to the inhibitory action of tumor growth, and Z_HPV16E7: 384 and Z_wtIt is aobvious that affitoxin albumen, PE38KDEL albumen and PBS group compare appearance of also beginning in 36 days Write sex differernce (p < 0.01), Z_wtCompare without significance between affitoxin albumen, PE38KDEL albumen and PBS group Difference (p > 0.05).Prolongation over time, gross tumor volume size is between each group, and obvious difference becomes big, until 60 days.Liver function detection transaminase's index is the most normal.

The above results shows, Z_{HPV16 E7}Affitoxin384 and Z_HPV16E7: 384 albumen are respectively provided with protection or suppression tumor The growth of cell, but with Z_{HPV16 E7}Affitoxin384 act as by force.I.e. Z_{HPV16 E7}Affitoxin384 albumen And Z_HPV16E7: 384 have HPV16 E7 targeting tumor-inhibiting action.And toxic and side effects is inconspicuous.

The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document by individually It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, this area skill The present invention can be made various changes or modifications by art personnel, and these equivalent form of values fall within right appended by the application equally and want Seek book limited range.

Claims (14)

1. the E7 albumen to HPV 16 has the polypeptide of binding affinity, it is characterised in that should Polypeptide is using the aminoacid sequence of staphylococcal protein A Z section as skeleton, obtains after carrying out 12-20 amino acid variation The polypeptide obtained.

2. the E7 albumen to HPV 16 as claimed in claim 1 has the polypeptide of binding affinity, It is characterized in that, relative to the aminoacid sequence of staphylococcal protein A Z section, this polypeptide at 9-11,13-14, 17-18,24-25,27-28,32,35,43 there is amino acid mutation.

3. the E7 albumen to HPV 16 as claimed in claim 2 has the polypeptide of binding affinity, It is characterized in that, relative to the aminoacid sequence of staphylococcal protein A Z section, this polypeptide

9th amino acids sports S or W；

10th amino acids sports F, L or V；

11st amino acids sports E, L or W；

13rd amino acids sports R, I or S；

14th amino acids sports S, L, M or A；

17th amino acids sports W or R；

18th amino acids sports A, W, T or Q；

24th amino acids sports G, R, D or P；

25th amino acids sports D, L, H or G；

27th amino acids sports G, V, A or Q；

28th amino acids sports R, E or L；

32nd amino acids sports L, V or E；

35th amino acids sports G, H, Q or D；

43rd amino acids sports E or K.

4. the E7 albumen to HPV 16 as claimed in claim 1 has the polypeptide of binding affinity, It is characterized in that, the K of the E7 protein-interacting of this polypeptide and HPV 16_DValue is 1 × 10^-6M is extremely 1×10^-7M。

5. the targeting molecule of a targeting HPV 16, it is characterised in that described targeting molecule Including the arbitrary described polypeptide of claim 1-4, and the conjugate of be connected with this polypeptide (or coupling), described Conjugate includes: cysteine residues, Polypeptide tags, the medicine of suppression HPV 16, maybe can detect Label.

6. the targeting molecule of targeting HPV 16 as claimed in claim 5, it is characterised in that institute The medicine of the suppression HPV 16 stated includes: toxin；It is preferred that described toxin is to have suppression human milk Head tumor virus 16 type virus infects or the toxin of function of tumor inhibition, such as diphtheria toxin, diphtherotoxin, Ricin, bacillus pyocyaneus Extracellular toxin or the functional fragment of described toxin；And, described tumor is the tumor that HPV 16 is positive.

7. the polynucleotide separated, its coding claim 1-4 is arbitrary described to HPV 16 E7 albumen there is the polypeptide of binding affinity.

8. polynucleotide, the targeting of its coding targeting HPV 16 described in claim 5 or 6 Property molecule, and wherein said conjugate is peptide.

9. a recombinant vector, it is characterised in that this carrier comprises the polynucleotide described in claim 7 or 8.

10. a host cell, it is characterised in that this host cell comprises the recombinant vector described in claim 9, Or it includes or integrates the polynucleotide having the right described in requirement 7 or 8 in genome.

Prepare the arbitrary described E7 albumen to HPV 16 of claim 1-4 for 11. 1 kinds and there is combination The method of the polypeptide of affinity, it is characterised in that described method includes:

(1) cultivate cell described in claim 10, thus it is arbitrary described to human papilloma to express claim 1-4 The E7 albumen of virus 16 types has the polypeptide of binding affinity；

(2) isolated and purified (1) polypeptide obtained.

The arbitrary described E7 albumen to HPV 16 of 12. claim 1-4 has binding affinity The purposes of the targeting molecule of the targeting HPV 16 described in polypeptide or claim 5 or 6, is used for making Standby treatment HPV 16 infection disease or HPV 16 express the medicine of positive tumor；Or

For preparing the detectable that detection HPV 16 virus infects；Or

Catch or HPV 16 expresses positive tumor for preparing diagnosis of human papilloma viral 16 type Diagnostic reagent.

13. 1 kinds of pharmaceutical compositions, it is characterised in that it comprises:

The E7 albumen to HPV 16 described in claim 1-4 has polypeptide or the power of binding affinity Profit requires the targeting molecule of the targeting HPV 16 described in 5 or 6；With

Pharmaceutically acceptable carrier.

14. 1 kinds are used for diagnosing or treat HPV 16 infection disease or HPV 16 expression The medicine box of positive tumor, it is characterised in that described medicine box includes: claim 1-4 is arbitrary described to human milk The E7 albumen of head tumor virus 16 type has the polypeptide of binding affinity, or the targeting human milk described in claim 5 or 6 The targeting molecule of head tumor virus 16 type, or the pharmaceutical composition described in claim 13.