CN101210044B

CN101210044B - Polypeptide and application thereof

Info

Publication number: CN101210044B
Application number: CN2007101923447A
Authority: CN
Inventors: 王自正; 俞杨; 杜同信; 瞿卫; 立彦
Original assignee: Individual
Current assignee: Individual
Priority date: 2007-12-25
Filing date: 2007-12-25
Publication date: 2010-06-30
Anticipated expiration: 2027-12-25
Also published as: CN101210044A

Abstract

The invention pertains to the biological engineering field, which discloses an opypeptide and the application thereof. The amino acid sequence of the invention shows as SEQ ID No.1. The opypeptide, nucleotide sequence of encoding the opypeptde and the expression vector thereof can be used for preparing drugs or diagnostic reagents aiming at functioning on thymus gland.

Description

One peptide species and application thereof

Technical field

The invention belongs to bioengineering field, relate to a peptide species and application thereof.

Background technology

Display technique of bacteriophage is a technology that highly multifarious polypeptide or albumen is showed in phage capsid protein surface.This technology links together the genotype and the phenotype of display molecule, is very easy to the functional screening of polypeptide or protein molecular.By the affine screening process of " absorption-wash-out-amplification ", the screening gained molecule in the library can be carried out as many as 10 ⁸Enrichment, and since the library in peptide sequence rich diversity (＞10 ⁹), this screening process is a high flux screening, polypeptide with high degree of specificity and extremely strong avidity that obtains thus or albumen have important use and are worth in the practice of biomedical fundamental research and clinical diagnosis and treatment.Along with the continuous development of phage display, numerous in recent years researchists uses it for the interior screening of body of live body.

Studying data at home and abroad shows that the interior screening of body is the emerging field in the phage display research, have more importantly biological significance: the target protein of vivoexpression, purifying gained all may have very big difference with the intravital native state of machine on space structure and biological function, and the strategy of screening is simulated polypeptide under integrality and the interaction between the blood vessel surface molecule better in the body; Triage techniques might be found new target organ blood vessel surface expression molecule in the body, and estimates the developed by molecule level of organ blood vessel surface objectively by specific binding polypeptide; In screening process, just impose intravital selective pressure, thereby lay the foundation for filtering out polypeptide ligand with potential clinical value, the targeting vector that this polypeptide ligand can be used as tracer agent and medicine is transported to the pathology part with nucleic, chemotherapeutics or gene fragment with therapeutic action, or itself is effectively applied to the diagnosis and the treatment of clinical disease as medicine with regard to having biological effect.

The phage display rondom polypeptide library has embodied important use value aspect the screening in the body of target micromolecule polypeptide.Compare with antibody drug, polypeptide has following advantage at least: 1) compare with the industrialized manufacturing technique of recombinant technology, the cost that polypeptide screens, synthesizes and modifies is the end of than; 2) has tangible high biologic activity with unit mass calculating; 3) at room temperature more stable than antibody; 4) be not easy to produce the immunity rejection, side effect is little; 5) has very strong organ penetrance.

The research group of Pasqualini and Ruoslahti screens tissues such as the lung of mouse, skin, pancreas, intestines, uterus, suprarenal gland, discovery all exists the heterogeneity of corresponding blood vessel endothelium specific molecular to express in these internal organs, this just points out the body lumen of vessels is not a surface molecular consistent pipeline that is evenly distributed, and the internal organs that the distribution of its surface molecular and blood vessel are supplied have very strong dependency.According to this result, the researchist has proposed the notion of " blood vessel password (VascularZip Codes) ", and promptly the blood vessel endothelium of different organs has different molecular markers in certain individuality.Pasqualini research group has filtered out the microvascular endothelial specificity bonded phage polypeptide with the white adipose tissue in the mouse body of heredity obesity, utilizing corresponding synthetic peptide to take apoptosis-induced medicine to local vascular as carrier can make it that apoptosis takes place, white adipose is organized obvious atrophy, the weight loss of obesity mice and significant side effects do not occur.

Normal biological condition has been simulated in screening effectively in the body, and the developed by molecule spectrum of understanding blood vessel is had significant values, and its achievement in research can carry out the transition to the exploitation of clinical diagnosis or medicine easily, and therefore, this field progress rapidly in recent years.

Along with deepening continuously of immunology research, people recognize that thymus gland still has biological function after growing up, play an important role in immunity of organism.Thymus function unusual relevant with immune deficiency, autoimmune disorder, infection, tumour etc.Clinically, some thymus gland relative diseases usually causes the unusual enhancing of thymus gland T cell output function when taking place, thereby causes a series of clinical manifestation (as the myasthenia gravis due to the thymoma).This type of disease can be considered to improve the immunological status of whole body by suppressing the thymus gland output function.TXi Baoshouti is reset resecting loop (TCR rearrangement excisioncircles, TRECs) be that the T cell development is reset when forming TXi Baoshouti α gene in early days, being present in the external annular DNA excision product of cell dyeing, is the specificity marker of T cells.TRECs content in the detection by quantitative peripheral blood can be used for estimating the thymus gland output function, can be used for the assessment of body autoimmune state in the multiple disease treatment process.

Summary of the invention

The purpose of this invention is to provide a kind of can with the nucleotide sequence and the expression vector thereof of thymus-specific bonded polypeptide, this polypeptide of encoding.

Another object of the present invention provides the application of aforementioned polypeptides, nucleotide sequence and expression vector thereof.

The objective of the invention is to realize by following technical measures:

One peptide species, its aminoacid sequence is shown in SEQ ID No.1.

The polypeptide expression carrier of aminoacid sequence shown in SEQ ID No.1.Described expression vector is plasmid, phagemid, phage, intestinal bacteria.

The nucleotide sequence of coding SEQ ID No.1.

The expression vector that contains the nucleotide sequence of coding SEQ ID No.1.Described expression vector is plasmid, phagemid, phage, intestinal bacteria.

Described polypeptide is preparing targeting in the medicine of thymus gland or the application in the diagnostic reagent.

Described Nucleotide is preparing targeting in the medicine of thymus gland or the application in the diagnostic reagent.

Described expression vector at targeting in the preparation medicine of thymus gland or the application in the diagnostic reagent.

Beneficial effect of the present invention:

The rondom polypeptide library of this research and utilization phage display carries out the interior screening of body of mouse, separate and thymus gland bonded phage polypeptide, investigate its influence by the quantitative evaluation of TRECs, lay the foundation for developing isotopically labeled targeted polypeptide medicine to the thymus gland output function.Polypeptide provided by the invention, Nucleotide, expression vector can be used for preparing medicine or the diagnostic reagent of targeting in thymus gland.

Description of drawings

Fig. 1. The selection result in the body of phage library.

A: one to take turns the phage titre that obtains after the screening only be 10 ³Pfu/mL, the titre of unit mass is 3.53*10 ²Pfu/g;

B: compare with kidney in contrast, the phage titre of thymus gland is very low.It is less that screening back non-specific binding is taken turns in prompting one, can directly carry out sequential analysis, seeks high-frequency sequence and consistence motif.

Fig. 2. the binding ability analysis of phage polypeptide CHAQGSAEC.(the Cys residue that the both sides of peptide sequence all have a library to carry, the structure of formation disulfide linkage cyclisation, down together)

A: phage polypeptide CHAQGSAEC can combine with the capillary blood vessel of thymus gland; B: with the renal tissue debond of contrast; C: the negative control that adds wild type phage does not dye; D: the negative control that does not add anti-M13 antibody does not dye.

Fig. 3. peptide sequence CHAQGSAEC suppresses the biologic activity of thymus gland output function.

No matter CHAQGSAEC is with the form of phage polypeptide or with the form of free polypeptide, can both suppress the thymus gland output function effectively.

Embodiment

The invention will be further elaborated by the following examples.

Embodiment 1

Main raw: employed phage polypeptide library Ph.D.-C7C buys from NEB company among the present invention.This library seven peptides at random is fused to the combinatorial library that the less important capsid protein of M13 phage (pIII) upward is built into.Different with other NEB displayed polypeptide storehouses is that the rondom polypeptide both sides of being showed respectively have a halfcystine.Under non-reduced condition, these two halfcystines spontaneously form a disulfide linkage, make the polypeptide cyclisation of displaying.Make like this displayed polypeptides with the formal representation that is similar to space structure picture at phage surface, more closely combine with target protein.Contain 1.2 * 10 in the library ⁹Individual stochastic sequence, each sequence have about 200 copies.Be used to increase the coli strain ER2738 of phage also available from NEB company.

Mouse is that the BALB/c mouse in 6-8 week is available from Nantong University's Experimental Animal Center age.

Experimental technique:

Screening in the body of phage library: after mouse is the BALB/c mouse anesthesia in 6-8 week age, go into to be dissolved in 2 * 10 among the 200 μ L TBS by tail vein injection ¹¹The phage polypeptide of individual conversion unit.Circulate after 15 minutes, under the situation of deep anaesthesia, heart perfusion 50mL TBS.Dissect mouse afterwards, get thymus gland, weigh.The purpose internal organs are put into 1mL contain among the proteinase inhibitor TBS of (PMSF 1mM presses down phthalein enzyme 20 μ g/mL, bright peptase 1 μ g/mL), fully grind.Three postoperative infection intestinal bacteria ER2738 of tissue washing with the above-mentioned solution that contains 1%BSA will grind carry out the titre counting.

Peptide sequence is analyzed: picking mono-clonal from＜100 clones' of titre counting the flat board, the genomic dna of recombinant phage is extracted in the amplification back.Check order with M13 (96) primer (primer is known universal primer).Utilize the consistence motif and the amino acid of the ClustalW 1.83 analysis polypeptide of EMBL to form.

The phage polypeptide bonded is identified: the mono-clonal phage polypeptide CHAQGSAEC tail vein injection that the frequency of occurrences is the highest is gone into the BALB/c mouse of anesthesia, circulate and after 15 minutes mouse is anaesthetized once more, heart perfusion 50mLTBS, results mouse thymus, make frozen section, carry out immunohistochemical staining.Acetone is fixed 30 minutes for 4 ℃, and PBS washing 5 minutes is through 3%H ₂O ₂Handle after 15 minutes, with 5%BSA sealing 45 minutes.Add anti-M13 antibody and spend the night for 4 ℃, PBST washing back adds two of horseradish peroxidase-labeled and resists, and hatches 1 hour for 37 ℃, adds the DAB colour developing with PBST washing back.Hematorylin is redyed after the alcohol gradient is dewatered, the transparent back of dimethylbenzene neutral gum mounting, last sem observation.

Biological function analysis in the body of polypeptide: with mouse is that the BALB/c mouse in 6-8 week goes into 2 * 10 by tail vein injection age ¹¹The synthetic free peptide C HAQGSAEC of the phage polypeptide CHAQGSAEC that contains the consistence motif of individual conversion unit or 100 μ M.Got peripheral blood after circulating respectively 15 minutes, 1 hour, 3 hours, 6 hours and 24 hours, extract whole blood DNA, use the TRECs primer: upstream primer: 5 '-CAGAGGGGTGCCTCTGTCA-3 ' (SEQ IDNo.2), downstream primer: 5 '-GGACCCCTCACAAAGTGTCG-3 ' (SEQ ID No.3) and SYBR GreenI carry out quantitative fluorescent PCR, by the method (2 of relative quantification ^{-Δ Δ Ct}) estimate the influence of polypeptide to the thymus gland output function.

The result:

Utilize phage polypeptide library Ph.D.-C7C to carry out screening in the body, one to take turns the phage titre that obtains after the screening only be 10 ³Pfu/mL, the titre of unit mass is 3.53*10 ²Pfu/g, compare with kidney in contrast titre very low (Figure 1A, B).It is big that results suggest one is taken turns the possibility that occurs the consistence motif after the screening, so the picking mono-clonal checks order.

The highest phage polypeptide sequence of the visible frequency of occurrences that checks order is CHAQGSAEC, accounts for 50% of positive colony, and this results suggest thymus-specific bonded sequence has obtained enrichment.Select phage polypeptide CHAQGSAEC, identify its binding ability by immunohistochemical methods.Confirm that it can combine (Fig. 2 A) with the capillary blood vessel of thymus gland, with the renal tissue debond (Fig. 2 B) of contrast, the negative control that adds wild type phage and do not add anti-M13 antibody do not dye (Fig. 2 C, D).The result shows that phage polypeptide CHAQGSAEC can combine with thymus-specific.

After the free peptide C HAQGSAEC of phage polypeptide CHAQGSAEC and synthetic injects in the body, utilize whole blood DNA to carry out the expression that quantitative fluorescent PCR is estimated TRECs.This polypeptide the different periods all can be in various degree the expression amount (Fig. 3) of inhibition TRECs.No matter results suggest CHAQGSAEC is with the form of phage polypeptide or with the form of free polypeptide, can both suppress the thymus gland output function effectively.

Conclusion:

Utilize phage-displayed polypeptides library (NEB Ph.D.-C7C) to carry out screening in the mouse body, only isolate after taking turns screening and thymus gland bonded phage polypeptide, there is consistence motif GSA in the sequencing result prompting.The sequence C HAQGSAEC that the frequency of occurrences is the highest is injected in the mouse body, finds that it can combine with thymus-specific, and no matter it be with the form of phage polypeptide or with the form of free polypeptide, can both suppress the thymus gland output function effectively.

The peptide coding that the application provides, the nucleotide sequence of this polypeptide and their expression vector can be used for preparing medicine or the diagnostic reagent of targeting in thymus gland, and its preparation method adopts technology well known in the art to get final product.

Sequence table

＜110〉king is from positive Yu Yang Du upright man of virtue and ability of letter Qu Wei together

＜120〉peptide species and application thereof

<160>3

<210>1

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉mouse thymus specific binding polypeptide, and have the biologic activity that suppresses thymus gland T cell output function

<400>1

His?Ala?Gln?Gly?Ser?Ala?Glu

1 5

<210>2

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉upstream primer

<400>2

cagaggggtg?cctctgtca?19

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer

<400>3

ggacccctca?caaagtgtcg?20

Claims

1. a peptide species, its aminoacid sequence is CHAQGSAEC.

2. expression vector, this expression vector is expressed the described polypeptide of claim 1.

3. nucleic acid, the described polypeptide of its coding claim 1.

4. expression vector, this expression vector contains the described nucleic acid of claim 3 and expresses the described polypeptide of claim 1.

5. according to claim 2 or 4 described expression vectors, it is characterized in that described expression vector is plasmid, phagemid, phage.

6. the described polypeptide of claim 1 is in the application of preparation targeting in the medicine of thymus gland.

7. the described nucleic acid of claim 3 is in the application of preparation targeting in the medicine of thymus gland.

8. claim 2 or 4 described expression vectors are in the application of preparation targeting in the medicine of thymus gland.