CN104039815A - Polypeptide for use in inhibiting hiv and target site of the polypeptide - Google Patents

Abstract

Disclosed are an HIV-inhibiting polypeptide and a target site thereof. The polypeptide comprises 11 amino acid residues in direct contact with the NHR of the HIV-1, where two of the amino acid residues are located respectively at the N-end and the C-end of the polypeptide. Also disclosed are a target site of the polypeptide and a medicinal use thereof. The polypeptide does not comprise a binding-pocket domain generally recognized as necessary for maintaining a high activity, is capable of inhibiting the HIV, particularly HIV strains having drug resistance against existing fusion inhibitors, and can be used in combating HIV infections.

Description

Polypeptide for use in inhibiting hiv and target site of the polypeptide

Polypeptide and its action target spot technical field for suppressing H I V

The invention belongs to biomedicine field, it is related to the polypeptide and its action target spot for suppressing HIV.Background technology

1 type human immunodeficiency virus（HIV-1) be AIDS pathogen, the current whole world has more than 30,000,000 the infecteds, causes about 2,000,000 people dead every year, and it is annual also increase about 2,000,000 the infecteds newly, the serious health for threatening the mankind.

HIV-1 passes through its envelope glycoprotein（Env) the viral-cellular membrane fusion host cells infected of mediation.Env includes surface subunit gpl20 and cross-film subunit gp41, and three Env formation non-covalent complex are embedded in virus surface.Surface subunit gpl20 is responsible for the molecular recognition during virus infected cell to find and close to target cell, while stable cross-film subunit gp41 functions are played, and machine discharges gp41 to start fusion in due course；Cross-film subunit gp41 is the direct functional molecular of virus-cell membrane fusion.

Have one during virus cell fusion by gp41 N- ends helical region（) and C- ends helical region NHR（CHR) six helical structures formed；The viral-cellular membrane fusion that is formed as of the structure provides energy, most important to virus-cell fusion.The medicine of six spiralizations, which can be prevented, then can effectively suppress AIDS virus-cell membrane fusion so as to prevent virus infection and propagation in vivo, for treating AIDS, referred to as fusion inhibitor.

Crystal structure is shown, in six spirals, and three constitute kernel by the NHR helical structures formed, form three grooves, three antiparallel combinations of CHR are in the trench.External source CHR polypeptides may be incorporated in and six inactive helical structures are formed in NHR target spots, prevent endogenous active six conveyor screw generation, suppresses virus-cell fusion and virus infects, so that as fusion inhibitor.Crystal structure is disclosed in NHR containing a deeper pocket, has key interactions with CHR corresponding function area.This CHR critical binding domain is referred to as pocket land, wherein combining template containing Try-Try-I le (WWI), it is considered to be maintain the key of high activity polypeptide fusion inhibitors（ Chan, D. C. , C. T. Chutkowski, and P. S. Kim, Proceedings of the Nat iona l Academy of Sc iences of the Uni ted States of Amer ica, 1998. 95 (26) : p. 15613-15617. ) 。

Typical C- peptides fusion inhibitor includes C34 (US 6,150,088) and its improves polypeptide, first listing fusion inhibitor T20 (US 5,464,933) and the CP32 found later

( CN1793170, CN1955190 ) .These C- peptides fusion inhibitors prevent virus infection by corresponding NHR targeted integrations；Typical target spot includes N36 (US 6,150,088) and DP107

(US 5,656,480), combines to form six helical structures with C34 and CP32 respectively, is also the popular target spot of small molecule fusion inhibitor containing a common binding pocket in wherein N36 and DP107.

In spite of such as S ifuvi rt ide (CN1334122) of the fusion inhibitor in the fusion inhibitor T20 and other clinical researches of listing, but due to the quick appearance of drug-resistant virus's strain, so that the fusion inhibitor research and development for resistance viral turn into the task of top priority, exploitation is the effective way for solving drug resistance for the medicine of novel targets；The novel targets of determination can be additionally used in the design and exploitation of new small molecule fusion inhibitor.The content of the invention

The present inventor passes through in-depth study and performing creative labour, has unexpectedly obtained being combined very strong polypeptide with NHR, so as to develop the class high activity fusion inhibitor different from existing fusion inhibitor mechanism of action.Such fusion inhibitor（The polypeptide of the present invention）Without pocket land necessary to generally acknowledged holding high activity, its sequence is different from existing fusion inhibitor T20, C34, CP32 etc., inhibition of HIV can be suppressed and be particularly the HIV strains that drug resistance is produced to existing fusion inhibitor, with the potentiality as AntiHIV1 RT activity infection medicine.Thus provide following inventions：One aspect of the present invention is related to one kind（Separation）Polypeptide, its derivative or its officinal salt, wherein, the polypeptide includes 11 amino acid residues directly contacted with HIV -1 NHR, and two N-terminals and C ends respectively positioned at polypeptide in this 11 amino acid residues.

It will be understood by those in the art that term " directly contact " refers in polypeptide and HIV-1 herein In six helical structures of gp41 NHR formation, the space direction of extension of 11 amino acid residues points to NHR, it is pharmaceutically-active critical sites and/or the amino acid residue is directly acted on by hydrophobic effect, electrostatic interaction or hydrogen bond with NHR corresponding residue（Chan, D. C. , C. T. Chutkowski, and P. S. Kim, Proceedings of the Nat iona l Academy of Sc iences of the Uni ted States of Amer ica, 1998. 95 (26)： p. 15613-15617. ) .Such as Fig.l, wherein gray shade partially illustrates these critical sites.

Polypeptide, its derivative or its officinal salt according to any one of the present invention, wherein, the sequence of the polypeptide is all contained in HIV -1 CHR；Preferably, the length of the polypeptide is 36 amino acid residues.

Polypeptide, its derivative or its officinal salt according to any one of the present invention, wherein, the amino acid sequence of the polypeptide includes WMEWDREI (SEQ ID NO:Or WASLWNWF (SEQ ID NO 24): 25 ) ；Preferably, the WMEWDREI or WASLWNWF are neither located at the N-terminal of polypeptide, are not also located at the C-terminal of polypeptide.

Preferably, the sequence of the polypeptide is all contained in HIV -1 CHR respectively；It is highly preferred that the sequence of the polypeptide is all contained in SEQ ID NO hereinafter respectively:In 27；Specifically, included in HIV -1 CHR the 611st -677 amino acid.

Polypeptide, its derivative or its officinal salt according to any one of the present invention, wherein, the amino acid sequence of the polypeptide is respectively such as SEQ ID NO:1 to SEQ ID NO:In 12 shown in any sequence.The table 1 that peptide sequence is seen below.

Table 1:Peptide sequence

Sequence SEQ ID

Amino acid sequence

Number NO:

1 WNASWSNKSLEQ IWNHMTWMEWDRE INNYTSLIHSL 1

2 WSNKSLEQ IWNHMTWMEWDRE I NYTSLIHSLIEES 2

3 KSLEQIWNHMTWMEWDREINNYTSLIHSLIEESQNQ 3

4 QIWN MTWMEWDRE INNYTSLIHSLI EESQNQQEKN 4

5 NNMTWMEWDRE INNYTSLIHSLI EESQNQQEKNEQE 5 6 WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL 6

7 WDREINNYTSLIHSLIEESQNQQEKNEQELLELDKW 7

8 INNYTSLIHSLIEESQNQQEKNEQELLELDKWASLW 8

9 YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF 9

10 IHSL IEESQNQQEKNEQELLELDKWASLWNWF I TW 10

11 L I EESQNQQEKNEQELLELDKWASLWNWF I TNWLW 11

12 SQNQQEKNEQELLELDKWASLWNWFDI TWNLWY I KL 12

Wherein amino acid is that abbreviation has implication well known in the art, for example：W is that tryptophan, N are that asparagine, A are that alanine, S are that serine, K are that lysine, L are that leucine, E are that glutamic acid, Q are that glutamine, I are that isoleucine, H are that histidine, M are that Yue methyllanthionines, T are that threonine, D are that aspartic acid, R are that arginine, Y are that tyrosine, F are phenylalanine.

Polypeptide 1-12 in table 1 is also referred to as polypeptide 1, the polypeptide of polypeptide 2 in the present invention

12;Or referred to as CHR polypeptides 1, the CHR polypeptides 12 of CHR polypeptides 2.

Polypeptide, its derivative or its officinal salt according to any one of the present invention, wherein, N-terminal connection acetyl group, oligopeptide sequence or the lipophilic group of the polypeptide, and/or

C-terminal connection amide groups, the oligopeptide sequence of such as 1-10 amino acid residue of oligopeptide sequence（Such as EEE, KKK, GQAV [SEQ ID NO: 28]、 GEEE [SEQ ID NO:29] etc.）Or lipophilic group（Fatty acid chain for example containing 3 to 20 carbon atoms, wherein it is preferred that the fatty acid chain containing 8-16 carbon atom）, cholesterol etc..

The N-terminal acetylation of the polypeptide, and/or C-terminal amidatioon.

The embodiment of the present invention 24 and embodiment 25 are proved, polypeptide 1-12 has high AntiHIV1 RT activity fusion inhibitor activity, show HIV-1 gp41 CHR and its flanking sequence continued presence high activity polypeptide, this is a kind of innovation to there are several isolated high activity peptide sequences in generally acknowledged CHR.Another aspect of the present invention is related to a kind of pharmaceutical composition, and it includes polypeptide, its derivative or its officinal salt described in any one of the present invention；Alternatively, described pharmaceutical composition also includes pharmaceutically acceptable carrier or auxiliary material.Preferably, described pharmaceutical composition is injection.

Usual pharmaceutical composition of the present invention contains polypeptide, its derivative or its officinal salt described in 0. 1-90 weight % any one of the present invention.Pharmaceutical composition can be according to side known in the art It is prepared by method.When for this purpose, if it is desired, can be combined polypeptide, its derivative or its officinal salt of the present invention with one or more solids or liquid pharmaceutical excipients and/or assistant agent, be made can as people appropriate administration form or dosage form.

Polypeptide, its derivative or its officinal salt of the present invention or the pharmaceutical composition of the present invention can be administered in a unit, method of administration can be enteron aisle or non-bowel, such as oral, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritonaeum or rectum.Form of administration is such as tablet, glue Nang, dripping pill, aerosol, pill, pulvis, solution, supensoid agent, emulsion, granule, liposome, transdermal agent, buccal tablet, suppository, freeze drying powder injection.Can be ordinary preparation, Slow release formulations, controlled release preparation and various particulate delivery systems.In order to which unit dosage forms for administration is made into tablet, various carriers well known in the art can be widely used.Example on carrier is, such as diluent and absorbent, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white bole, microcrystalline cellulose, alumina silicate；Wetting agent and adhesive, such as water, glycerine, polyethylene glycol, ethanol, propyl alcohol, starch slurry, dextrin, syrup, honey, glucose solution, mucialga of arabic gummy, gelatine size, carboxylic Yue bases sodium cellulosate, lac, Yue bases cellulose, phosphoric acid Potassium, polyvinylpyrrolidone；Disintegrant, such as dry starch, alginate, agar powder, laminaran, sodium acid carbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, dodecyl sodium sulfate, Yue bases cellulose, ethyl cellulose；Disintegration inhibitor, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil and fat；Sorbefacient, such as quaternary ammonium salt, lauryl sodium sulfate；Lubricant, such as talcum powder, silica, cornstarch, stearate, boric acid, atoleine, polyethylene glycol.Tablet can also be further made to coating tablet, such as sugar coated tablet, thin membrane coated tablet, enteric coated tablets, or double-layer tablets and multilayer tablet.In order to which administration unit is made into pill, various carriers well known in the art can be widely used.Example on carrier is, such as diluent and absorbent, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Ge luc i re, kaolin, talcum powder；Adhesive such as Arabic gum, bassora gum, gelatin, ethanol, honey, liquid sugar, rice paste or batter etc.；Disintegrant, such as agar powder, dry starch, alginate, dodecyl sodium sulfate, Yue bases cellulose, ethyl cellulose.In order to which administration unit is made into suppository, various carriers well known in the art can be widely used.Example on carrier is, such as polyethylene glycol, lecithin, cocoa butter, higher alcohol, higher alcohol Ester, gelatin, semi-synthetic glyceride etc..In order to which administration unit is made into glue Nang, polypeptide, its derivative or its officinal salt of the active ingredient present invention are mixed with above-mentioned various carriers, and thus obtained mixture is placed in hard obviously glue Nang or flexible glue Nang.Also micro- Nang agent can be made in polypeptide, its derivative or its officinal salt of the active ingredient present invention, be suspended in aqueous medium formation supensoid agent, can also be fitted into ebonite Nang or injection application is made.In order to which administration unit is made into injection preparation, such as solution, emulsion, freeze drying powder injection and supensoid agent, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-PD, the isooctadecanol of ethoxylation, polyoxygenated isooctadecanol, Polyoxyethylene Sorbitol Fatty Acid Esters etc..In addition, in order to prepare isotonic parenteral solution, appropriate sodium chloride, glucose or glycerine can be added into injection preparation, further, it is also possible to add conventional cosolvent, Slow electuaries, pH adjusting agent etc..

In addition, if desired, colouring agent, preservative, spices, flavouring, sweetener or other materials can also be added into pharmaceutical preparation.

Polypeptide, its derivative or its officinal salt of the present invention or the dosage of the pharmaceutical composition of the present invention depend on many factors, the property and the order of severity for example to be prevented or treated disease, sex, age, body weight and the individual reaction of patient or animal, concrete activity composition used, method of administration and administration number of times etc..Above-mentioned dosage with ingle dose form or can be divided into several, and such as two, three or four dosage forms for administration.

Term " composition " used herein means to include the product of each specified composition comprising specified amount, and any product directly or indirectly produced from the combination of each specified composition of specified amount.

The actual dose level of each active component in pharmaceutical composition of the present invention can be changed, so that the amount of the active component of gained can effectively be directed to specific patient, and composition and administering mode obtain required therapeutic response.Dosage level must be selected according to the patient's condition and medical history of the activity of concrete activity composition, method of administration, the order of severity for treating the patient's condition and patient to be treated.But, the way of this area is, the dosage of active component is since the level required less than therapeutic effect needed for obtaining, gradually incremental dose, until obtaining required effect.Polypeptide, its derivative or its officinal salt that another aspect of the invention is related to described in any one of the present invention are preparing the medicine for the treatment of and/or prevention and/or the infection of auxiliary treatment coating viroid In purposes；Specifically, the coating viroid infection is HIV associated diseases or AIDS.

Another aspect of the invention be related to it is a kind of treat and/or prevention and/or the infection of auxiliary treatment coating viroid method, including the step of give polypeptide described in any one of the present invention of subject's effective dose, its derivative or its officinal salt.Specifically, the coating viroid infection infects associated diseases or AIDS for HIV.

When for above-mentioned treatment and/or prevention or auxiliary treatment, polypeptide of the invention, its derivative or its officinal salt for the treatment of and/or prevention effective dose can be applied in a pure form, or with pharmaceutically acceptable ester or prodrug forms（In the case where there are these forms）Using.Or, the pharmaceutical composition that can be subjected to excipient with one or more medicines with polypeptide, its derivative or its officinal salt containing the present invention is administered.It is to be understood that polypeptide, its derivative or its officinal salt of the present invention or total consumption per day of pharmaceutical composition of the invention must be maked decision by attending physician in reliable medical judgment scope.For any specific patient, depending on specific treatment effective dose level must be according to many factors, the factor includes treated obstacle and the order of severity of the obstacle；The activity of the concrete activity composition used；The concrete composition used；Age, body weight, general health, sex and the diet of patient；Administration time, method of administration and the excretion rate of the concrete activity composition used；Treat the duration；It is applied in combination or medicine used at the same time with the concrete activity composition that is used；And similar factor known to medical field.For example, the way of this area is, the dosage of active component is since the level required less than therapeutic effect needed for obtaining, gradually incremental dose, until obtaining required effect.Generally, the dosage that polypeptide, its derivative or its officinal salt of the present invention is used for mammal particularly people can be between 0. 001-1000 mg/kg body weight/days, for example between 0. 01-100 mg/kg body weight/days, such as between 0. 01-10 mg/kg body weight/days.

Polypeptide, its derivative or its officinal salt of the present invention or the pharmaceutical composition of the present invention can effectively prevent and/or treat and/or auxiliary treatment various diseases or illness of the present invention.Another aspect of the invention is related to the polypeptide described in any one of the present invention, its derivative or its officinal salt in preparation or as the purposes in HIV fusion inhibitors or inverase. Another aspect of the invention is related to a kind of method for suppressing HIV in vivo or in vitro, the step of including the use of the polypeptide described in any one of the present invention of effective dose, its derivative or its officinal salt.

Another aspect of the invention is related to a kind of method for the cell fusion for suppressing HIV-1 Env mediation in vivo or in vitro, the step of including the use of the polypeptide described in any one of the present invention of effective dose, its derivative or its officinal salt.Another aspect of the invention is related to one kind（Separation）Polypeptide, its derivative or its officinal salt, wherein, the amino acid sequence of the polypeptide is respectively such as SEQ ID NO:13 to SEQ ID NO:In 23 shown in any sequence.

The table 2 that peptide sequence is seen below.

Table 2:Peptide sequence

Wherein amino acid is that abbreviation has implication well known in the art, for example：W is that tryptophan, N are that asparagine, A are that alanine, S are that serine, K are that lysine, L are that leucine, E are that glutamic acid, Q are that glutamine, I are that isoleucine, H are that histidine, M are that methionine, T are that threonine, D are that aspartic acid, R are that arginine, Y are that tyrosine, F are that phenylalanine, G are sweet ammonia Acid, V are valine.

Polypeptide 13-23 in table 2 is also referred to as polypeptide 13, the polypeptide 23 of polypeptide 14 in the present invention;Or referred to as leg polypeptide 13, the leg polypeptide 23 of leg polypeptide 14.

According to the antiparallel binding characteristic of NHR and CHR in HIV-1 gp41 core crystal structures and gp41, the present inventor devises NHR peptide sequences corresponding with sequence 1-12, the combination target spot for determining polypeptide in sequence 1-12.Specific design is shown in lists peptide sequence in combination with grey font in Fig.2, figure.

NHR:

527/

LAVERYLKDQQLLGIWGIS ( SEQ ID NO: 26 )

CHR is (reversely）：

677n

IYWLW

WTMHNWIQELSKNSWSANW⁶¹¹( SEQ ID NO:27, in order to clearly indicate that and leg combination corresponding relation, the right-hand member of sequence 27 is N-terminal, and sequence direction should being designated as 611 w reads from right-hand member）.

Sequence above（ SEQ ID NO:26 and SEQ ID NO:27) it is that, according to the interaction relationship for having HIV-1 gp41 NHR and CHR that crystal structure speculates, underscore part is the peptide sequence of existing crystal structure.

During the present invention is implemented, polypeptide 1-12 sequences corresponding with polypeptide 13-23 combine to form six helical structures, show that whole HIV-1 gp41 NHR sequences all can be described as the target spot of high activity fusion inhibitor, this is a prominent innovation to the combination target spot that the generally acknowledged NHR pockets formed are high activity fusion inhibitors.

Another aspect of the invention is related to purposes of any one or more polypeptides in screening treatment and/or prevention and/or auxiliary treatment coating viroid infection medicine in polypeptide 13-23；Specifically, the coating viroid infection is HIV associated diseases or AIDS.Specifically, the screening refers to as Sites Screening.

Another aspect of the invention is related to purposes of any one or more polypeptides in screening HIV fusion inhibitors or inverase in polypeptide 13-23.Specifically, the screening refers to as Sites Screening. During the present invention is implemented, according to the interaction between HIV-l gp41 NHR and CHR, the two can target spot and aglucon, therefore regard polypeptide in 1-12 and 13-23 as target spot and the exchange function of inhibitor each other.Specifically, target spot is designed on the basis of polypeptide 1-12, polypeptide 13-23 transform to obtain high activity fusion inhibitor, drug design and exploitation are carried out, in the design spirit for being also contained in the present invention.

Another aspect of the invention is related to the nucleotide sequence of polypeptide any one of the coding present invention.

A kind of nucleic acid construct, it includes the nucleotide sequence described in any one of the present invention；Specifically, the nucleic acid construct is recombinant vector；More specifically, the recombinant vector is recombinant expression carrier.

The invention further relates to the nucleic acid construct comprising nucleotide sequence of the present invention and 1 or multiple regulating and controlling sequence being operatively connected therewith, the regulating and controlling sequence can instruct coded sequence to be expressed in suitable host cell under its consistency condition.Expression is understood to include any step involved in polypeptide production, includes, but are not limited to transcription, posttranscriptional modification, translation, posttranslational modification and secretion.

" nucleic acid construct " is defined as single-stranded or double-stranded nucleic acid molecules in the text, and they are isolated from natural gene, or through modification containing being combined and nucleic acid fragment arranged side by side in non-natural mode.When nucleic acid construct is comprising all regulating and controlling sequences necessary to express coded sequence of the present invention, term nucleic acid construct is synonymous with expression cassette.Term " coded sequence " is defined as directly determining in the text the part of the amino acid sequence of its protein product in nucleotide sequence.The border of coded sequence is typically by the ribosome bind site close to mRNA 5' ends opening code-reading frame upstream（For prokaryotic）With the transcription terminator determination close to mRNA 3' ends opening code-reading frame downstream.Coded sequence can include, but are not limited to DNA, cDNA and recombinant nucleic acid sequence.

The nucleotide sequence for the separation for encoding peptide of the present invention can be operated in many ways, it is expressed the peptide.It may be desirable to or nucleotide sequence must be processed before insertion vector, this depend on expression vector.It is known in the art using the technology of recombinant DNA method modification of nucleic acids sequence.

The terms " control sequence " be defined as including expression peptide of the present invention must or favourable all components.Each regulating and controlling sequence can naturally contain for the nucleotide sequence of coded polypeptide Or it is external.These regulating and controlling sequences include, but not limited to targeting sequencing, polyadenylation sequence, propeptide sequence, promoter, signal sequence and transcription terminator.Bottom line, regulating and controlling sequence will include the termination signal of promoter and transcription and translation.In order to import specific restriction site so as to which the code area of regulating and controlling sequence and the nucleotide sequence of coded polypeptide is attached, the regulating and controlling sequence of belt lacing can be provided.Term " being operatively connected " is defined as such a conformation in the text, and wherein regulating and controlling sequence is located at the appropriate location of the coded sequence of relative DM sequences, so that regulating and controlling sequence instructs the expression of polypeptide.

Regulating and controlling sequence can be suitable promoter sequence, you can be expressed the nucleotide sequence of the host cell identification of nucleotide sequence.Promoter sequence contains the transcription regulating nucleotide sequence of direct polypeptide expression.Promoter can be any nucleotide sequence for having transcriptional activity in selected host cell, include the promoter of mutation, truncation and heterozygosis, can derive from the gene of coding and the homologous or heterologous extracellular or intracellular polypeptide of host cell.

Regulating and controlling sequence can also be suitable transcription terminator, can be recognized to terminate one section of sequence of transcription by host cell.Terminator sequence be operatively connected coded polypeptide nucleotide sequence 3, end.In selected host cell can any terminator of function may be used to the present invention.

Regulating and controlling sequence can also be suitable targeting sequencing, the i.e. highly important mRNA non-translational regions of translation to host cell.Targeting sequencing is operatively connectable to the 5 of the nucleotide sequence of coded polypeptide, end.In selected host cell can any targeting sequencing of function be used equally for the present invention.

Regulating and controlling sequence can also be signal peptide coding region, and the area encodes one section of amino acid sequence for being connected in polypeptide aminoterminal, and coded polypeptide can be guided to enter cell secretory pathway.The 5 of nucleic acid sequence encoding area, the signal peptide coding region that end may naturally containing translation frame as one man with the coding domain segment Nature Link of secrete polypeptide.Or, the 5 of code area, it is external signal peptide coding region to coded sequence that end, which can be contained,.When coded sequence does not contain signal peptide coding region under normal circumstances, it may be necessary to add extraneous signal peptide-coding region.Or, natural signal peptide coding region can be simply replaced with external signal peptide coding region to strengthen polypeptide secretion.But, polypeptide signal peptide coding region of the secretory pathway of host cell used in after any energy lead schedule reaches may be used to the present invention.

Regulating and controlling sequence can also be peptide former code area, and area coding is positioned at the one of amino terminus Section amino acid sequence.Gained polypeptide is referred to as proenzyme or propolypeptide.Propolypeptide is usually not active, can cut peptide former by catalysis or self catalysis from propolypeptide and be converted into ripe active peptides.

When the amino terminal of polypeptide is to have signal peptide to have peptide former zone again, peptide former zone is close to the amino terminal of polypeptide, and signal peptide area is then close to the amino terminal of peptide former zone.

The regulating and controlling sequence that addition can adjust expression of polypeptides according to the growing state of host cell may be also what is needed.The example of regulator control system is that those can be to chemical or physical stimulus thing（It is included in the case of having regulating compound）React, so that the system of open or close gene expression.The other examples of regulating and controlling sequence are that those can make the regulating and controlling sequence of gene magnification.In these examples, the nucleotide sequence of coded polypeptide should be operatively connected with regulating and controlling sequence together with.

The invention further relates to the recombinant expression carrier comprising nucleotide sequence of the present invention, promoter and transcription and translation termination signal.Above-mentioned various nucleic acid and regulating and controlling sequence can be joined together to Prepare restructuring expression vector, the carrier can include 1 or multiple convenient restriction site, to be inserted in these sites or to replace the nucleotide sequence of coded polypeptide.Or, can be by by nucleotide sequence or the nucleic acid construct comprising the sequence inserts appropriate expression vector and expresses nucleotide sequence of the present invention.When preparing expression vector, coded sequence can be made to be located in carrier to be operatively connected with appropriate expression regulation sequence.

Recombinant expression carrier can be any carrier for being convenient for recombinating DM operations and express nucleic acid sequence（Such as plasmid or virus）.The selection of carrier generally depends on the compatibility for the host cell that carrier will be imported with it.Carrier can be linear or closed circular form plasmid.

Carrier can be autonomous replicating vector（It is present in extrachromosomal complete structure, can be replicated independently of chromosome）, such as plasmid, extra-chromosomal element, minute chromosome or artificial chromosome.Carrier can include any mechanism for ensureing self-replacation.Or, carrier be one when import host cell when, the carrier that would be integrated into genome and replicated together with the chromosome being incorporated into.In addition, single carrier or plasmid can be applied, or the overall two or more carriers or plasmid for including all DNA by host cell gene group is imported, or transposons.

It is preferred that carrier of the present invention contains the 1 or multiple selected marker for being easy to select transformed cells.Selected marker is such a gene, and its product assigns resistance, the resistance of heavy metal to biocide or virus, or assigns auxotroph prototrophy etc..The example of bacterial selectable markers Such as the resistance marker of the da l genes of bacillus subtilis or bacillus licheniformis, or antibiotic such as ampicillin, kanamycins, chloramphenicol or tetracycline.

It is preferred that carrier of the present invention, which is included, can make carrier stable integration into host cell gene group, or ensure that carrier carries out the element of autonomous replication in cell independently of cellular genome.

For carrying out the situation of autonomous replication, carrier can also include replication orgin, carrier is independently replicated in target host cell.Replication orgin, which can be carried, makes its turn into the mutation of responsive to temperature type in host cell.

More than 1 nucleotide sequence of the present invention copied can be inserted to host cell to improve the yield of the gene outcome.The copy number increase of the nucleotide sequence can be by least 1 additional copies Insertion Into Host Cell genome by the sequence, or an amplifiable selected marker is inserted together with the nucleotide sequence, by cultivating cell in the presence of appropriate selectable agent, the selected marker containing amplification copy is picked out, so as to the cell containing additional copies nucleotide sequence.

It is well-known to those skilled in the art for connecting operation of the above-mentioned each element to build recombinant expression carrier of the present invention（See, for example, Sambrook etc., Molecular Cloning: A Laboratory room handbook, the second edition, CSH Press, Cold SpringHarbor , New-York about, 1989).Another aspect of the invention is related to a kind of recombinant host cell, the nucleic acid construct described in its any one of the present invention.

The invention further relates to include the recombinant host cell for the nucleotide sequence of the present invention that can be used to recombinant production polypeptide.Can be by the vector introduction host cell of the nucleotide sequence comprising the present invention, so that the carrier is maintained with the external carrier format of the dyeing of above-mentioned chromosomal integrant or self-replacation.Term " host cell " cover it is any due to duplication during the mutation that occurs and the offspring different from parental cell.The selection of host cell is heavily dependent on polypeptide coding genes and its source.

Host cell can be prokaryotic or eukaryotic, such as bacterium（Such as Bacillus coli cells）Or yeast cells.Can be by technology well known to those skilled in the art by vector introduction host cell.

The polypeptide of the present invention can be synthesized with artificial chemistry, can also carry out protein expression by recombinant host cell, it may for example comprise：（A) under conditions of suitable for the generation peptide, culture contains There is the host cell of nucleic acid construct, the nucleic acid construct includes the nucleotide sequence of coding for said peptides；With（B) peptide is reclaimed.

In preparation method of the present invention, cell is cultivated in the nutrient medium that appropriate polypeptides are produced with means known in the art.For example, under conditions of expression of polypeptides and/or separation is allowed, Shaking culture, laboratory or industrial fermentation tank middle and small scale or large scale fermentation can be passed through in suitable culture medium（Including it is continuous, in batches, batch charging or solid state fermentation）To cultivate cell.In the suitable culture medium comprising carbon and nitrogen source and inorganic salts, cultivated using step known in the art.Suitable culture medium can provide or be referred to disclosed constitute (for example, described in the catalogue of American type culture collection by supplier）To prepare.If polypeptide is secreted into culture medium, polypeptide directly can be reclaimed from culture medium.If polypeptide is not secreted, it can be reclaimed from cell lysate.

Polypeptide produced by being reclaimed with means known in the art.For example, routine operation can be passed through（Include, but are not limited to centrifuge, filter, extract, be spray-dried, evaporate or precipitate）Polypeptide is reclaimed from culture medium.

Polypeptide of the present invention can be purified by various operations known in the art, these operations include, but are not limited to chromatography（For example, ion-exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing and size exclusion chromatography）, electrophoresis（For example, preparative isoelectric focusing）, differential solubility（Such as ammonium sulfate precipitation）, SDS-PAGE or extracting（See, for example, protein purification, J. C. Janson and Lars Ryden are compiled, VCH Publ i shers, New York, and 1989).In the present invention,

Term " polypeptide " has general sense known to those skilled in the art, and the also derivative including polypeptide, trim etc..

Term " leg " or " HIV-1 legs " specifically refer to HIV-1 gp41 NHR;Term " CHR " or " HIV-1 CHR " specifically refer to HIV-1 gp41 CHR.

Term " HIV fusion inhibitors " includes but is not limited to：Suppress HIV (such as suppressing HIV propagation, infection, propagate）Or suppress the medicine or reagent of the fusion of HIV-1 Env mediated cells. Term " effective dose " includes to realize that treatment, prevention, mitigation and/or Slow solve the dosage of disease of the present invention or illness in subject.

Term " subject " can refer to patient or polypeptide, its derivative or its officinal salt described in other receiving any one of the present invention or the pharmaceutical composition described in any one of the present invention to treat, prevent, mitigate and/or Slow solves the animal of disease of the present invention or illness, particularly mammal, such as people, dog, monkey, ox, horse.

Term " disease and/or illness " refers to a kind of condition of the subject, and the condition is relevant with disease of the present invention and/or illness.The beneficial effect of invention

The polypeptide of the present invention is to infect polypeptide with the different new AntiHIV1 RT activity of the sequence such as existing fusion inhibitor T20, C34, CP32, with good AntiHIV1 RT activity fusion-activity, inhibition of HIV can be suppressed and be particularly the HIV strains that drug resistance is produced to existing fusion inhibitor.Brief description of the drawings

Fig. 1：The CHR peptide sequences of the present invention, wherein, dash area is the amino acid residue directly contacted with NHR in CHR, is pharmaceutically-active critical sites.

Fig. 2：The design of NHR target sequences.Dash area is the CHR polypeptides functioned.According to the antiparallel feature of the two combination, CHR peptide sequences are represented with inverting sequence.

Fig. 3:The CD spectrums and N-PAGE results of CHR polypeptides 2 and many peptide interactions of NHR are characterized, wherein：

Fig. 3 (A) are that the polypeptide 2 and CD that polypeptide 22 interacts is composed, wherein square represents that the CD spectrum Round circles of polypeptide 2 represent the CD spectrums of polypeptide 22, upper triangle 2/22 represents polypeptide 22 and the mixed CD of polypeptide 2 spectrums, and lower triangle 2+22 represents the CD languages superposition that polypeptide 22 and polypeptide 2 are individually scanned；

Fig. 3 (B) polypeptides 2 are composed with the CD that polypeptide 21 interacts, and the implication of legend therein does similar understanding with Fig. 3 (A)；

Fig. 3 (C) polypeptides 2 are composed with the CD that polypeptide 20 interacts, and the implication of legend therein does similar understanding with Fig. 3 (A)； Fig.3 (D) polypeptide 2 is composed with the CD that polypeptide 19 interacts, and the implication of legend therein does similar understanding with Fig.3 (A)；

Fig.3 (E) polypeptide 2 is composed with the CD that polypeptide 17 interacts, and the implication of legend therein does similar understanding with Fig.3 (A)；

The N-PAGE results of Fig.3 (F) polypeptide 2 and many peptide interactions of NHR.

Fig.4:The CD spectrums and N-PAGE results of CHR polypeptides 3 and many peptide interactions of NHR are characterized, wherein：

Fig.4 (A) is that the polypeptide 3 and CD that polypeptide 21 interacts is composed, wherein square represents that the CD spectrum Round circles of polypeptide 3 represent the CD spectrums of polypeptide 21, upper triangle 2/21 represents polypeptide 21 and the mixed CD of polypeptide 3 spectrums, and lower triangle 2+21 represents the CD languages superposition that polypeptide 21 and polypeptide 3 are individually scanned；

Fig.4 (B) polypeptide 3 is composed with the CD that polypeptide 20 interacts, and the implication of legend therein does similar understanding with Fig.4 (A)；

Fig.4 (C) polypeptide 3 is composed with the CD that polypeptide 19 interacts, and the implication of legend therein does similar understanding with Fig.4 (A)；

Fig.4 (D) polypeptide 3 is composed with the CD that polypeptide 18 interacts, and the implication of legend therein does similar understanding with Fig.4 (A)；

Fig.4 (E) polypeptide 3 is composed with the CD that polypeptide 17 interacts, and the implication of legend therein does similar understanding with Fig.4 (A)；

The N-PAGE results of Fig.4 (F) polypeptide 3 and many peptide interactions of NHR.

Fig.5:The CD spectrums and N-PAGE results of CHR polypeptides 4 and many peptide interactions of NHR are characterized, wherein：

Fig.5 (A) is that the polypeptide 4 and CD that polypeptide 21 interacts is composed, wherein square represents that the CD spectrum Round circles of polypeptide 4 represent the CD spectrums of polypeptide 21, upper triangle 2/21 represents polypeptide 21 and the mixed CD of polypeptide 4 spectrums, and lower triangle 2+21 represents the CD languages superposition that polypeptide 21 and polypeptide 4 are individually scanned；

Fig.5 (B) polypeptide 4 is composed with the CD that polypeptide 20 interacts, and the implication of legend therein does similar understanding with Fig.5 (A)；

The CD that Fig.5 (C) polypeptide 4 and polypeptide 19 interact composes, and legend therein contains Justice does similar understanding with Fig.5 (A)；

Fig.5 (D) polypeptide 4 is composed with the CD that polypeptide 18 interacts, and the implication of legend therein does similar understanding with Fig.5 (A)；

Fig.5 (E) polypeptide 4 is composed with the CD that polypeptide 17 interacts, and the implication of legend therein does similar understanding with Fig.5 (A)；

The N-PAGE results of Fig.5 (F) polypeptide 4 and many peptide interactions of NHR.

Fig.6:The CD spectrums and N-PAGE results of CHR polypeptides 5 and many peptide interactions of NHR are characterized, wherein：

Fig.6 (A) is that the polypeptide 5 and CD that polypeptide 21 interacts is composed, wherein square represents that the CD spectrum Round circles of polypeptide 5 represent the CD spectrums of polypeptide 21, upper triangle 2/21 represents polypeptide 21 and the mixed CD of polypeptide 5 spectrums, and lower triangle 2+21 represents the CD languages superposition that polypeptide 21 and polypeptide 5 are individually scanned；

Fig.6 (B) polypeptide 5 is composed with the CD that polypeptide 20 interacts, and the implication of legend therein does similar understanding with Fig.6 (A)；

Fig.6 (C) polypeptide 5 is composed with the CD that polypeptide 19 interacts, and the implication of legend therein does similar understanding with Fig.6 (A)；

Fig.6 (D) polypeptide 5 is composed with the CD that polypeptide 18 interacts, and the implication of legend therein does similar understanding with Fig.6 (A)；

Fig.6 (E) polypeptide 5 is composed with the CD that polypeptide 17 interacts, and the implication of legend therein does similar understanding with Fig.6 (A)；

The N-PAGE results of Fig.6 (F) polypeptide 5 and many peptide interactions of NHR.

Fig.7:The CD spectrums and N-PAGE results of CHR polypeptides 6 and many peptide interactions of NHR are characterized, wherein：

Fig.7 (A) is that the polypeptide 6 and CD that polypeptide 19 interacts is composed, wherein square represents that the CD spectrum Round circles of polypeptide 6 represent the CD spectrums of polypeptide 19, upper triangle 6 eight 9 represents polypeptide 6 and the mixed CD of polypeptide 19 spectrums, and lower triangle 6+19 represents the CD languages superposition that polypeptide 6 and polypeptide 19 are individually scanned；

Fig.7 (B) polypeptide 6 is composed with the CD that polypeptide 18 interacts, and the implication of legend therein does similar understanding with Fig.7 (A)； Fig.7 (C) polypeptide 6 is composed with the CD that polypeptide 17 interacts, and the implication of legend therein does similar understanding with Fig.7 (A)；

Fig.7 (D) polypeptide 6 is composed with the CD that polypeptide 16 interacts, and the implication of legend therein does similar understanding with Fig.7 (A)；

Fig.7 (E) polypeptide 6 is composed with the CD that polypeptide 15 interacts, and the implication of legend therein does similar understanding with Fig.7 (A)；

The N-PAGE results of Fig.7 (F) polypeptide 6 and many peptide interactions of NHR.

Fig.8:The CD spectrums and N-PAGE results of CHR polypeptides 3 and many peptide interactions of NHR are characterized, wherein：

Fig.8 (A) is that the polypeptide 7 and CD that polypeptide 17 interacts is composed, wherein square represents that the CD spectrum Round circles of polypeptide 7 represent the CD spectrums of polypeptide 17, upper triangle 7 eight 7 represents polypeptide 7 and the mixed CD of polypeptide 17 spectrums, and lower triangle 7+17 represents the CD languages superposition that polypeptide 7 and polypeptide 17 are individually scanned；

Fig.8 (B) polypeptide 7 is composed with the CD that polypeptide 16 interacts, and the implication of legend therein does similar understanding with Fig.8 (A)；

Fig.8 (C) polypeptide 7 is composed with the CD that polypeptide 15 interacts, and the implication of legend therein does similar understanding with Fig.8 (A)；

The N-PAGE results of Fig.8 (D) polypeptide 7 and many peptide interactions of NHR.Embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, it will be appreciated by those skilled in the art that the following example is merely to illustrate the present invention, and be should not be taken as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.The abbreviation used in the present invention has following meaning：

AIDS (Acquired Immure Deficiency Syndrome) AIDS, Immune Deficiency Syndrome. Ala (Alanine, A) alanine

Arg (Arginine, R) arginine

Asn (Asparagine) Dong Ugly amine

Asp (Aspart icacid, D) asparagus fern atmosphere acid

DCM (Dichlorome thane) dichloro Yue alkane

The Yue base Yue acid amides of DMF (N, N-Dimethyl malonate) two

Env (Envelope glycoprotein) envelope glycoprotein

ESI-MS (Electronic spray ion mass spectroscopy) EFI shield language

Fmoc (Fluorenylmethoxycarbonyl) medicine Yue oxygen takes advantage of base

Gly (Glycine, G) glycine

Gin (Glutamine, Q) Gu Ugly amine

Glu (Glutamic acid, E) paddy atmosphere acid

6-HB (six-helix bundle) six conveyor screws

HBTU 2- (1H-1- hydroxybenzotriazoles）- 1,1,3,3- tetra- Yue base hexafluorophosphoric acids His (Histidine, H) histidine

1_ bases are stupid and three nitrogen are high by HoBt (1-Hydroxyl benzot iazole anhydrous)

The repetitive sequence of NHR (N-terminal heptad repeat) N-end seven

The repetitive sequence of CHR (C-terminal heptad repeat) C-end seven

HIV (Human immunodeficiency virus) human immunodeficiency virus

The type human immunodeficiency viruses of HIV-1 1

HPLC (high performance liquid chromatography) high performance liquid chromatography

Lie (Isoleucine, I) isoleucine

The bright atmosphere acid of Leu (Leucine, L)

Met (Methionine, M) Yue dredges propylhomoserin

Nal nor-leucines

Lys (Lysine, K) lysine

Phe (Phenylalanine, F) phenylpropyl alcohols atmosphere acid Ser (Ser ine, S) serine

TFA (Tr if luoroacet ic acid) trifluoroacetic acid

Thr (Threonie, T) threonine

Tyr (Tyros ine, Y) bribes atmosphere acid

Va l (Va l ine, V) solid-phase synthesized carrier Rink amide resins used in valine embodiment are Tianjin Nankai synthesis responsibility Co., Ltd product；The natural amino acid and the alpha-non-natural amino acid of D types that HBTU, H0Bt, DIEA and Fmoc are protected are Shanghai gill biochemical corp and Chengdu promise new technology responsible company product.N- Yue base pyrrolidones（NMP) it is ACR0S Products；TFA is Beijing Bo Mai Science and Technology Ltd.s product；DMF, DCM are Samsung of South Korea product；Trifluoroacetic acid aqueous solution is Fi sher Products.Other reagents are domestic analysis net product such as without explanation.Embodiment 1:The preparation of polypeptide 1

Using the Fmoc solid-phase peptide synthesis of standard.All equal C- ends of peptide sequence are amidatioon, and N- ends are acetylation（Those skilled in the art know that these modifications do not have fundamental effect to polypeptide active）.From Rink Amide resins, peptides are extended from C- ends to N- ends.Condensing agent is HBTU/H0Bt/DIEA.Deprotection agent is piperidines/DMF solution.Decomposition agent is TFA, freezes and preserves after thick peptide water dissolving.Isolated and purified with medium pressure liquid chromatography method or HPLC, pure peptide content ＞ 95%.Base shield Assisted Laser Desorption flight time mass spectrum（MALDI-T0F-MS polypeptide molecular weight) is determined.

Microwave Peptide systhesis condition is as follows：

Amino acid：0. 2 M DMF solution；Activator：0. 45M HBTU/HOBt DMF solution；Activate alkali：2M DIEA nmp solution；Deprotection agent：The DMF solution of 20% v/v piperidines；Closed reagent：The DMF solution of 20% v/v acetic anhydrides.

The 5g of Rink Amide resins 0. (0. 25 mmo l) is weighed to insert in the reactor of CEM microwave Peptide synthesizers; then after preparing amino acid, activator, activation alkali, deprotecting regent, closed reagent by above-mentioned condition, synthesized with CEM microwave full-automatic Peptide synthesizers.After the completion of peptide resin washed with DMF after 3 times with anhydrous Yue alcohol shrink, room temperature in vacuo dry, obtain peptide The 05g of resin 2.

The cracking of peptide resin：It is put into after above-mentioned synthetic peptide resin is weighed in 250ml eggplant-shape bottles, ice bath, electromagnetic agitation.The amount for adding 10ml by lg peptide resins configures lysate（Volume ratio：Trifluoroacetic acid：Dithioglycol：Between phenol：Water=82. 8: 10: 5:2. 5) TFA needs advance ice bath cooling 30min or is put in advance；Used in water tank.The lysate prepared is added in the peptide resin under condition of ice bath, electromagnetic agitation, resin becomes orange red, and 30min is reacted under condition of ice bath, ice bath is then removed, reaction 90min is further continued at room temperature completes reaction.The lower addition cold diethyl ether 200ml into reactor is stirred vigorously, white precipitate is separated out, continues to stir 30min;Precipitate is filtered out with G4 core suction funnel, with cold diethyl ether cyclic washing 3 times, is dried.Distilled water 50ml is added, acetonitrile 5ml makes solid fully dissolve, suction filtration, filtrate freezes to obtain the thick 03g of peptide 1.

The thick peptide of gained is purified with medium-pressure or high pressure chromatogram.Chromatographic column is C18 posts, and eluent is acetonitrile, water and a small amount of acetic acid.Concrete operation step：Thick peptide lg is weighed, add water 20ml, acetonitrile 5ml dissolves, centrifuge 10min under 3000 revs/min, take supernatant loading.Chromatographic column is balanced with the glacial acetic acid solution 200ml of 15% acetonitrile/water/0. 1% in advance, continues to be balanced with the same eluents of 200ml after loading, efficient liquid phase detection eluent composition.Ethane nitrile content is stepped up according to testing result, until purified polypeptide peak is eluted out.Merge with component eluent, rotary evaporation removes most of solvent, freezes to obtain pure peptide, HPLC detection levels ＞ 90%

Pure peptide determines its molecular weight through MALDI-TOF-MS matter languages（The table 3 seen below)

Table 3:The molecular weight and purity of polypeptide

The corresponding experiment numbers of polypeptide sequence number determine molecular weight purity %

1 LKC058 4563. 4 98. 8

2 LKC121 4540. 6 83. 3

3 LKC087 4523. 9 95. 9

4 LKC060 4565. 7 98. 4

5 LKC088 4524. 8 96. 9

6 LKC025 4532. 8 97. 6

7 LKC090 96. 3

8 LKC062 4386. 6 88. 7 9 LKC012 4492. 2 91. 9

10 LKC091 4543. 5 96. 8

11 LKC064 4619. 2 97. 4

12 LKC065 4652. 9 98. 6

13 LKC063 4107. 6 98. 0

14 LKC118 4090. 0 93. 1

15 LKC048 4385. 9 90. 0

16 LKC061 4365. 0 96. 5

17 LKC094 4420. 8 95. 8

18 LKC026 95. 4

19 LKC119 94. 1

20 LKC059 4540. 3 96. 6

21 LKC120 4427. 6 83. 6

22 LKC132 4328. 5 97. 9

The embodiment 2-23 of 23 LKC057 4500. 1 96. 5:Polypeptide 2-23 preparation

Prepared with reference to the method in embodiment 1, except the sequence of specific amino acid is distinguished as Table 1 and Table 2 below.Its molecular weight and purity are as shown in table 3 above.Polypeptide sample used in the following examples 24-25 is the polypeptide 1-23 synthesized by embodiment 1-23

Embodiment 24:The anti-HIV-1 fusion-activity detection of polypeptide

The Cell-Cell Fusion model that the present inventor is mediated with HIV-1 Env carries out determination of activity to the polypeptide of design.Target cell is TZM-b l cells（U.S. NIH AIDS reagent and object of reference project are provided, 8129) catalog number (Cat.No.) is, its surface expression CD4 T-cell receptors and chemotactic factor (CF) auxiliary receptor CCR 5 and CXCR4, it can be recognized by HIV-1 Env, it is simultaneously intracellular also to transcribe luciferase reporter gene, but the promoter without the gene, therefore the luciferase background expression quantity of separate cell is very low.Effector cell is HL2/3 cells（U.S. NIH AIDS reagent and reference Items mesh is provided, and catalog number (Cat.No.) is 1294) its surface expression HIV-1 Ε η ν, by Ε η ν attack target cells, completes cell fusion, while the intracellular promoter for also transcribing luciferase reporter gene.Two kinds of cells first in the DMEM containing 10% hyclone dual anti-containing ammonia benzyl/streptomysin, are containing 5% C0 under 37 degree₂Incubator in individually cultivate.Two kinds of cells are attached cell, and passage is harvested after cell pancreatin/EDTA digestion.Cell is counted with cell counting count board.

TZM-bl target cells are adjusted to concentration with culture medium for 750,000/ml, added with every μ 1 of hole 50 in 96 porocyte culture plates（3.75 ten thousand/hole）, 5% C0₂, 37 degree are cultivated 24 hours down.

By polypeptide or positive control sample（T20 or C34) it is dissolved in phosphoric acid Slow and rushes in solution physiological saline (PBS), or add appropriate DMS0 and make abundant dissolving, peptide concentration is determined at 280nm with ultraviolet spectrometer.Then polypeptide solution is diluted to appropriate concentration, the proportional diluted in 96 hole elisa Plates (Corning).

The HL2/3 effector cell for preparing 1,500,000/ml.

In the TZM-bl cells that the inhibitor of the proportional diluted in the holes of 20 μ 1/ is added to the previous day culture, the HL2/3 effector cell prepared in the holes of 50 μ 1/ is then added；A wherein row for 96 porocyte culture plates substitutes inhibitor with PBS to be used to determine saturation fusion signal, and another row substitutes HL2/3 cells with DMEM culture mediums to be used to determine background signal. 5% C0₂, cultivate down for 37 degree and be allowed within 6-8 hours fully fusion.

By the kit of luciferase reporter gene（Promega) taken out from refrigerator, 5x cell pyrolysis liquids are diluted to lx lysates with distilled water according to consumption, placed at room temperature；Substrate is dissolved with substrate Slow fliud flushings, is placed at room temperature；Simultaneously by ELIASA（Molcular Devices M5 multi-function microplate readers）Testing conditions set standby.

The cell merged is taken out, culture medium is discarded, is washed twice with the hole PBS of 200 μ 1/, cleaning fluid is removed as far as possible；Then the lysate for equilibrating to room temperature is added with the holes of 50 μ 1/, gently shaking lower reaction 5min makes cell fully crack；Lysate is added into 96 hole chemiluminescence detection ELISA Plate plates with the holes of 40 μ 1/（Corning tried one's best in), during sample-adding and avoid introducing bubble；Substrate is rapidly added in chemiluminescence ELISA Plate with the holes of 40 μ 1/ under lucifuge, chemiluminescence is determined on ELIASA immediately.

The ratio for merging signal and background signal according to saturation determines having for target cell and effector cell Effect fusion, ratio ＞ 5 shows effective integration.Its semi-inhibit agent concentration is determined according to the concentration of sample-chemiluminescence signal curve（ IC₅.）, the IC of positive control sample₅.Value should be stablized in certain limit；Signal should be close to background signal under preferable suppression curve middle and high concentration inhibitor, and signal should merge signal close to saturation under least concentration inhibitor.

Polypeptide 1-12 cell fusion inhibitory activity is listed in table 4, and positive control T20 IC50 is 2 ± 05 nM, is consistent with the blunt road of document 4（Wild, C. T. , et al. , Proceedings of the National Academy of Sciences of the United States of America, 1994. 91 (21): p. 9770-9774· ) .

Table 4:The cell fusion inhibitory activity of polypeptide

The result of table 4 shows that polypeptide 1-12 is respectively provided with HIV-1 fusion inhibitory activity, particularly Wherein 2-10 IC₅.<46nM, has reached preclinical activity level, the potentiality with the drug candidate as new fusion inhibitor.Embodiment 25:The chromatograms of Round bis-（) and native polyacrylamide gel electrophoresis CD（N-PAGE polypeptide 2 and polypeptide 13-23 interaction) is characterized

1. experiment purpose

The chromatograms of Yong Round bis-（The technique study CHR polypeptides 2 and NHR polypeptides 13-23 interaction such as CD).

2. experiment material

Polypeptide 2,13-23.

The color Pass instrument of Round bis- is Bio log i c MOS450 spectrometers.

3. experimental method

CHR polypeptides will be determined to be dissolved in PBS, NHR polypeptides are dissolved in distilled water, and concentration is determined according to UV absorption under 280nm；Then 20 μ Μ polypeptide solution is prepared.

Prepare the polypeptide sample to be detected：By NHR polypeptides and CHR polypeptides with 1:The two biased sample of 1 volume ratio mixture；If independent polypeptide sample, solution 1 is rushed by 20 μ Μ sample with corresponding Slow:1 mixing.Sample places 30min under 37 degree makes abundant reaction.Above-mentioned experimental procedure can ensure that the peptide concentration in sample is consistent.

It will be determined on the chromatographs of sample Round bis- prepared, instrument scanning wavelength scope is 190-260nm, wavelength interval is 0. 5nm, and sweep speed is 150nm/min, scans 3 times and is averaged.Solution example scanning is first rushed with Slow and obtains blank, sample signal is then scanned, blank signal is deducted from sample signal and obtains CD signals.

By comparing before the mixing of CHR and NHR polypeptides and mixed CD signal intensities determine the interaction of the two.CD signal intensities show that both have interaction.

The present inventor also determines six spiral stabilities of polypeptide 2 and 13-23 formation by CD temperature scannings.Specific method is as follows：Sample is diluted to 1 μ Μ, sample cell is added, CD instrumentation programs are set to temperature scanning, Detection wavelength 220nm, 20-90 degrees Celsius of scanning range, the lower progress program temperature scanning of stirring and inspection obtain CD signals and vary with temperature curve.Once differentiation is calculated according to curve, Tm values are determined according to the peak value of once differentiation curve（It is shown in Table 5), and sample Thermal transition temperature.

The present inventor's native polyacrylamide gel electrophoresis（N-PAGE) the interaction of research polypeptide 2 and polypeptide 13-23, electrophoresis apparatus is used using BG-Power3500（Beijing Baijing Biotechnology Co., Ltd.）Complete.Experimental method is as follows：

By 200 μ Μ NHR polypeptides and CHR polypeptide solutions with 1:1 volume ratio is mixed, and is for biased sample；If independent polypeptide sample, solution 1 is rushed by 200 μ Μ sample with corresponding Slow:1 mixing.Sample places 30min under 37 degree makes abundant reaction.After end, isometric N-PAGE 2X loading Slow fliud flushings are added in each sample（Invitrogen companies）, it is stand-by after mixing.

Prepare separation gel（20%) :30% acrylamide solution 10 ml, 4X separation gel Slow fliud flushings 3.75 ml, water 1.24 ml, 10% AP (cross the sour ammoniums of υ）Solution 100 μ, TEMED

(Ν, Ν ,-four Yue base ethylenediamines）10 μ, concussion mixing.Carefully by above-mentioned solution, between layer glass plate, upper strata leaves l-2cm space to record concentration glue, reuses syringe and is carefully added into water-saturated n-butanol.Gel completed polymerization at 30 minutes or so.

Prepare concentration glue（4%) :N-butanol layer is discarded, gel upper strata is carefully cleaned with deionized water, water is blotted with filter paper.30% acrylamide solution 1 ml, 4X concentration glue Slow fliud flushings 2 ml, water 4.8ml are added, the μ of 10% AP solution 100 μ, TEMED 10, concussion mixing is added.Glass plate is filled with above-mentioned solution, loading comb is inserted.

Electrophoresis：Loading comb is extracted after waiting gel polymerisation, illustrates to install according to electrophoretic apparatus, upper groove is negative pole, lower groove is positive pole.10X Tris-Gly electrophoresis Slow fliud flushings are diluted to IX, electric capacity groove is filled.Voltage about 150V, electric current is about 25mA, prerunning 20min, is powered off.Loading groove is cleaned using Slow fliud flushings, matched somebody with somebody sample solution Slow is slowly injected into sample-adding trench bottom using sample loading gun or micro syringe, turned on the power, start electrophoresis, after about 2 hours, completing electrophoresis, (band of visual bromophenol blue is forward position, is stopped after the certain distance of loading hole）.

Dyeing：Gel is removed, is washed with distilled water three times, each 5 minutes.BioRadG250 dyeing liquors covering gel is added, is dyed 1 hour.Dyeing liquor is discarded, distilled water is added and decolourizes three times, each 10 minutes.Use the gel after flat bed scanner or gel imaging system scanning dyeing.

The interaction for determining NHR polypeptides and CHR polypeptides is brought by the dyeing strip of comparative electrophoresis. 3. experimental result

(1) such as Fig.3A-E, all there are CD signal intensities after being mixed with NHR polypeptides 22,21,20,19,18,17 in CHR polypeptides 2, it was demonstrated that the interaction between them.

(2) Tm values are as shown in table 5.

Table 5:The Tm values of six spirals of polypeptide 2 and 13-23 formation

(3) result of native polyacrylamide gel electrophoresis is shown（Fig.3 F) ：Each mixed solution of polypeptide 17,18,19,20,21,22 and polypeptide 2 shows the new band different from the independent solution of polypeptide 2 in electrophoresis, it was demonstrated that there is clearly interaction between polypeptide 17,18,19,20,21,22 and polypeptide 2.Embodiment 26:The chromatograms of Round bis-（) and native polyacrylamide gel electrophoresis CD（- PAGE) characterize polypeptide 3 and polypeptide 13-23 interaction

Tested with reference to the method in embodiment 25.

The result of embodiment 26 shows that CHR polypeptides 3 CD signal intensities all occur after being mixed with NHR polypeptides 21,20,19,18,17（Fig.4 A-E), it was demonstrated that the interaction between them.

The heat endurance of polypeptide 3 and six spirals of formation of polypeptide 21,20,19,18,17（Tm values）It is shown in Table 6.

Table 6:The Tm values of polypeptide 2 and six spirals of NHR polypeptides formation

Polypeptide sequence number experiment numbers Tm (°C)

17 LKC094 44.0

18 LKC026 54.0 19 LKC119 52. 9

20 LKC059 65. 9

21 LKC120 59. 7

22 LKC132 36. 1

The N-PAGE results that polypeptide 3 and 13-23 interacts are shown（Fig. 4 F), each mixed solution of polypeptide 17,18,19,20,21 and polypeptide 3 shows the new band different from the independent solution of polypeptide 3 in electrophoresis, it was demonstrated that there is clearly interaction between polypeptide 17,18,19,20,21 and polypeptide 3.Embodiment 27:The chromatograms of Round bis-（) and native polyacrylamide gel electrophoresis CD（- PAGE) characterize polypeptide 4 and polypeptide 13-23 interaction

Tested with reference to the method in embodiment 25.

The result of embodiment 27 shows that CHR polypeptides 4 CD signal intensities all occur after being mixed with NHR polypeptides 21,20,19,18,17（F i g. 5 A-E), it was demonstrated that the interaction between them.

The heat endurance of polypeptide 4 and six spirals of formation of polypeptide 21,20,19,18,17（Tm values）It is shown in Table 7.

Table 7:The Tm values of polypeptide 4 and six spirals of NHR polypeptides formation

The N-PAGE results of polypeptide 4 and many peptide interactions of NHR are shown（The F of F ig. 5), each mixed solution of polypeptide 17,18,19,20,21 and polypeptide 4 shows the new band different from the independent solution of polypeptide 4 in electrophoresis, it was demonstrated that there is clearly interaction between polypeptide 17,18,19,20,21 and polypeptide 4. Embodiment 28:The chromatograms of Round bis-（) and native polyacrylamide gel electrophoresis CD（N-PAGE polypeptide 5 and polypeptide 13-23 interaction) is characterized

Tested with reference to the method in embodiment 25.

The result of embodiment 28 shows that CHR polypeptides 5 CD signal intensities all occur after being mixed with NHR polypeptides 21,20,19,18,17（Fig.6 A-E), it was demonstrated that the interaction between them.

The heat endurance of polypeptide 5 and six spirals of formation of polypeptide 21,20,19,18,17（Tm values）It is shown in Table 8.

Table 8:The Tm values of polypeptide 5 and six spirals of NHR polypeptides formation

The N-PAGE results of polypeptide 5 and many peptide interactions of NHR are shown（Fig.6 F), each mixed solution of polypeptide 17,18,19,20,21 and polypeptide 5 shows the new band different from the independent solution of polypeptide 5 in electrophoresis, it was demonstrated that there is clearly interaction between polypeptide 17,18,19,20,21 and polypeptide 5.Embodiment 29:The chromatograms of Round bis-（) and native polyacrylamide gel electrophoresis CD（- PAGE) characterize polypeptide 6 and polypeptide 13-23 interaction

Tested with reference to the method in embodiment 25.

The result of embodiment 29 shows that CHR polypeptides 6 CD signal intensities all occur after being mixed with NHR polypeptides 19,18,17,16,15（Fig.7 A-E), it was demonstrated that the interaction between them.

The heat endurance of polypeptide 6 and six spirals of formation of polypeptide 19,18,17,16,15（Tm values）It is shown in Table 9. Table 9:The Tm values of polypeptide 6 and six spirals of NHR polypeptides formation

The N-PAGE results of polypeptide 6 and many peptide interactions of NHR are shown（The F of F ig. 7), each mixed solution of polypeptide 15,16,17,18,19 and polypeptide 6 shows the new band different from the independent solution of polypeptide 6 in electrophoresis, it was demonstrated that there is clearly interaction between polypeptide 15,16,17,18,19 and polypeptide 6.Embodiment 30:The chromatograms of Round bis-（) and native polyacrylamide gel electrophoresis CD（N-PAGE polypeptide 7 and polypeptide 13-23 interaction) is characterized

Tested with reference to the method in embodiment 25.

The result of embodiment 30 shows that CHR polypeptides 7 CD signal intensities all occur after being mixed with NHR polypeptides 17,16,15（Fi g. 8 A-C), it was demonstrated that the interaction between them.

The heat endurance of polypeptide 7 and six spirals of formation of polypeptide 15,16,17（Tm values）It is shown in Table

10。

Table 10:The Tm values of polypeptide 7 and six spirals of NHR polypeptides formation

The N-PAGE results of polypeptide 7 and many peptide interactions of NHR are shown（The D of F ig. 8), each mixed solution of polypeptide 15,16,17 and polypeptide 7 shows the new band different from the independent solution of polypeptide 7 in electrophoresis, it was demonstrated that there is clearly interaction between polypeptide 15,16,17 and polypeptide 7. Although the embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all teachings, various modifications and replacement can be carried out to those details, these change within protection scope of the present invention.The four corner of the present invention is provided by appended claims and its any equivalent.

Claims (1)

1. Claim

   1. a kind of polypeptide of separation, its derivative or its officinal salt, wherein, the polypeptide includes 11 amino acid residues for directly being contacted with HIV -1 HR, and two in this 11 amino acid residues are respectively positioned at the N-terminal and C-terminal of polypeptide.

   2. polypeptide according to claim 1, its derivative or its officinal salt, wherein, the sequence of the polypeptide is all contained in HI V -1 CHR respectively；Preferably, the length of the polypeptide is 36 amino acid residues.

   3. polypeptide according to claim 1 or 2, its derivative or its officinal salt, wherein, the amino acid sequence of the polypeptide includes WMEWDREI or WASLWNWF;Preferably, the WMEWDREI or WASLWNWF are neither located at the N-terminal of polypeptide, are not also located at the C ends of polypeptide.

   4. polypeptide according to claim 1, its derivative or its officinal salt, wherein, the amino acid sequence of the polypeptide is respectively such as SEQ ID NO:1 to SEQ ID NO:In 12 shown in any sequence.

   5. a kind of polypeptide of separation, its derivative or its officinal salt, wherein, the amino acid sequence of the polypeptide is respectively such as SEQ ID NO:13 to SEQ ID NO:In 23 shown in any sequence.

   6. polypeptide, its derivative or its officinal salt according to claim 4 or 5, wherein, N ends connection acetyl group, oligopeptide sequence or the lipophilic group of the polypeptide, and/or

   C-terminal connection amide groups, oligopeptide sequence or lipophilic group；Preferably,

   The N-terminal acetylation of the polypeptide, and/or C-terminal amidatioon.

   7. a kind of pharmaceutical composition, it includes polypeptide, its derivative or its officinal salt any one of claim 1 to 6；Alternatively, described pharmaceutical composition is also included and can pharmaceutically connect The carrier or auxiliary material received.

   8. the purposes of polypeptide, its derivative or its officinal salt in the medicine for preparing treatment and/or prevention and/or the infection of auxiliary treatment coating viroid any one of claim 1 to 6；Specifically, the coating viroid infection is HIV associated diseases or AIDS.

   9. the polypeptide, its derivative or its officinal salt any one of claim 1 to 6 are preparing or are being used as the purposes in HIV fusion inhibitors or inverase.

   10. a kind of method for suppressing HIV in vivo or in vitro, the step of including the use of the polypeptide any one of the claim 1 to 6 of effective dose, its derivative or its officinal salt.

   11. a kind of method for the cell fusion for suppressing HIV-1 Env mediation in vivo or in vitro, the step of including the use of the polypeptide any one of the claim 1 to 6 of effective dose, its derivative or its officinal salt.

   12. it is a kind of treat and/or prevention and/or the infection of auxiliary treatment coating viroid method, including the step of give polypeptide any one of the claim 1 to 6 of subject's effective dose, its derivative or its officinal salt；Specifically, the coating viroid infection is HIV associated diseases or AIDS.

   13. purposes of the polypeptide in screening treatment and/or prevention and/or auxiliary treatment coating viroid infection medicine any one of claim 1 to 6；Specifically, the coating viroid infection is HIV associated diseases or AIDS.

   14. purposes of the polypeptide in screening HIV fusion inhibitors or inverase any one of claim 1 to 6.

   15. encode the nucleotide sequence of polypeptide any one of claim 1 to 6. 16. a kind of nucleic acid construct, it includes the nucleotide sequence described in claim 15；Specifically, the nucleic acid construct is recombinant vector；More specifically, the recombinant vector is recombinant expression carrier.

   17. a kind of recombinant host cell, it includes the nucleic acid construct described in claim 16.