CN101851274B - Polypeptides for suppressing invasion of hepatitis C virus - Google Patents
Polypeptides for suppressing invasion of hepatitis C virus Download PDFInfo
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Abstract
The invention discloses polypeptides for suppressing the invasion of hepatitis C virus (HCV). The invention provides the following ten polypeptides: the polypeptide 1 is as shown by the sequence I in a sequence table, the polypeptide II is as shown by the sequence 2, the polypeptide III is as shown by the sequence 3, the polypeptide IV is as shown by the sequence 4, the polypeptide V is as shown by the sequence 5, the polypeptide VI is as shown by the sequence 6, the polypeptide VII is as shown by the sequence 7, the polypeptide VIII is as shown by the sequence 8, the polypeptide IX is as shown by the sequence 9, and the polypeptide X is as shown by the sequence 10. The polypeptides have the following advantages that: the polypeptides consist of 16 to 24 amino acids so as to be short, synthesized chemically and prepared conveniently by recombinant expression; the sequences and the acting sites of the polypeptides are different from those of a conventional HCV invasion suppressor, the target is clear and the specificity suppresses the interaction between an HCV virus cyst membrane and a host acceptor CLDN1; drug resistance is not caused easily; and because the sequences of the polypeptides are originated from human protein CLDN1 rather than HCV virus cyst membrane protein, the polypeptides have a suppression effect on various gene-type HCVs.
Description
Technical field
The present invention relates to suppress the polypeptide that hepatitis C virus is invaded.
Background technology
Hepatitis C is that (Hepatitis C virus HCV) infects and to cause that quite a few patient can lapse to and be chronic infection (Chronic hepatitis C), liver cirrhosis (Cirrhosis) and hepatocellular carcinoma (Hepatocellular carcinoma after acute HIV infection by hepatitis C virus; HCC) (Kiyosawa K, Sodeyama T, Tanaka E; Gibo Y, Yoshizawa K, Nakano Y; Furuta S, Akahane Y, Nishioka K; Purcell RH; Et a1.Interrelationship of blood transfusion, non-A, non-B hepatitis andhepatocellular carcinoma:analysis by detection of antibody to hepatitis Cvirus.Hepatology.1990 Oct; 12 (4 Pt 1): 671-5.).The chronicity rate that HCV infects is very high, is 50%～85%, and very harmful to patient's health and lives, the related medical cost increases year by year, has become serious society and public health problem.According to the World Health Organization's investigation in 1999, global HCV the infected reaches 1.7 hundred million, and infection rate is 3.1% (Lauer GM, Walker BD.Hepatitis C virus infection.N EnglJ Med.2001 Jul 5; 345 (1): 41-52.).HCV the infected of China and Fa patient's sum all occupy first place in the world, and belong to one of third liver district occurred frequently.The general crowd's of China HCV infection rate is 3.2%, and the infected is about about forty-two million, and is fast rise trend in recent years (cma, " hepatitis C guideline of prevention and treatment ", 2005) always.More severe is the vaccine that does not also have to prevent the HCV infection at present in the world, and third liver that can't block New Development infects.And the present medicine that does not also have directly to be directed against HCV virus target spot.The combination therapy of polyoxyethylene glycol Interferon, rabbit (PEG-IFN) and ribavirin (Ribavirin) is as efficient not high (about 50%) of present unique treatment means for China and global popular strain (1b and 1a genotype); Treatment cycle is long, expensive and the intensive spinoff is arranged.Therefore, researching and developing novel anti-HCV medicament and regimen will be extremely important to the control of hepatitis C.
HCV has unique genome structure and life cycle.HCV belongs to flaviviridae (Flaviviridae) sub-thread positive chain RNA virus, and the main human pathogen of this section also comprises: Yellow fever virus, Dengue virus, West Nile virus etc.The HCV genome is about 9.6kb, comprises a big ORFs, encodes one by about 3000 precursor proteins that amino acid is formed; The ORFs both sides are non-translational region (Untranslatedregions, UTRs) (Choo QL, Kuo G; Weiner AJ, Overby LR, Bradley DW; Houghton M.Isolation of a cDNA clone derived from a blood-borne non-A, non-B viralhepatitis genome.Science.1989 Apr 21; 244 (4902): 359-62.).Under the combined action of HCV precursor protein via the proteolytic enzyme of host and encoding viral, the processing maturation is four structural protein: Core, E1, E2 and p7, and six Nonstructural Protein: NS2, NS3, NS4A, NS4B, NS5A and NS5B.Wherein E1 and E2 are membrane glycoprotein.One of important hereditary feature of HCV is its genomic height variability, and wherein E1 and E2 zone degree of variation is the highest, and 3 ' and 5 '-UTR is comparatively conservative.This characteristic has been brought the difficulty of certain degree for design and exploitation HCV vaccine.HCV can be divided into 6 oligogene types and nearly hundred kinds of different subtypes at present, according to the method for the current international practice, representes the HCV genotype with Arabic numeral, representes gene hypotype (like 1b, 2a, 3c etc.) with the English alphabet of small letter.Gene 1 type is global distribution, accounts for more than 70% of all HCV infection.HCV and many other RNA viruses are similar, and behind the infection host, through the certain period, in the infected's body, forming with a dominant strain is master's relevant mutant virus crowd, is called quasispecies (Quasispecies).HCV is complete, and life cycle is divided into usually: virus absorption and intrusion host cell; The translation of precursor protein, processing and ripe; Viral genome is duplicated; Links such as virus assembling.
First link of HCV host cells infected is to utilize the surface of cell membrane acceptor to accomplish the invasion procedure of virion.Over the past two years, active about the operation irregularity of discovery and evaluation HCV acceptor, and Tremendous achievements.The specificity HCV acceptor that has been found that at present mainly comprises: CD81, SR-BI, Claudin-1 (CLDN1) and Occludin (OCLN).The CD81 molecule belongs to one of tetratransmembrane albumen family member, mainly interacts through its extracellular region and HCV envelope protein E2, accomplishes mediation virus and invades host cell (Pileri P, Uematsu Y; Campagnoli S, Galli G, Falugi F; Petracca R, Weiner AJ, Houghton M; Rosa D, Grandi G, Abrignani S.Binding of hepatitis C virus to CD81.Science.1998 Oct 30; 282 (5390): 938-41.).E1 and E2 have the E2 of enhancing and CD81 bonded ability after being combined into heterodimer.SR-BI (Scavengerreceptor class B type I) is the secondary transmembrane protein of being made up of 509 amino acid, has a big extracellular region, and its extracellular region contains 9 glycosylation sites.SR-BI mainly combines (Scarselli, E., Ansuini, H., Cerino with the E2 of HCV; R., Roccasecca, R.M., Acali, S.; Filocamo, G., Traboni, C., Nicosia; A., Cortese, R.and Vitelli, A. (2002) The human scavenger receptorclass B type I is a novel candidate receptor for the hepatitis C virus.EMBOJ.21,5017-5025.).Evidence suggests in addition: SR-BI also can be used as the low-density lipoprotein (LDL) of acetylize and oxidation and the acceptor of RHDL (HDLs), and lipoprotein such as HDLs can promote the infection of HCV through SR-BI.
What need emphatically point out is: tetratransmembrane albumen CLDN1 closely connects one of the component of (Tight junction) as cell; Can be used as new accessory receptor (the Evans MJ of HCV in 2007 by doctor Rice of U.S. Rockefeller University and colleague's report thereof; Von Hahn T, Tscherne DM, Syder AJ; Panis M;
B, Hatziioannou T, McKeating JA; Bieniasz PD, Rice CM.Claudin-l is a hepatitisC virus co-receptor required for a late step in entry.Nature.2007 Apr12; 446 (7137): 801-5.).Can be although cross expression CLDN1 so that HCV gets into non-sensitive clone 293T; Can but still have a series of associated problem awaiting acknowledgements: the envelope protein of HCV interact with CLDN1? Why can't some non-sensitive cells (like HeLa) HCV after crossing expression CLDN1 still get into? In addition, the molecule mechanism people that get into about CLDN1 mediation HCV also know little about it.The suppressor factor of invading to HCV at present, mainly concentrates on anti-HCV envelope protein and previous acceptor CD81 that finds and the neutralizing antibody of SR-BI.In view of HCV novel receptor CLDN1, OCLN and cell Tight junction structure get into the vital role that possibly bring into play in the host cell process at HCV; Simultaneously; In order to continue some innovative researches, press for the polypeptide viroid entry inhibitors that obtains to the HCV novel receptor than short amino acid sequence to the HCV acceptor.This type peptide inhibitor should have be prone to synthetic, target spot is clear and definite, spinoff is little, to the effective characteristics of several genes C-type virus C.
Summary of the invention
The purpose of this invention is to provide and suppress the polypeptide that hepatitis C virus is invaded.
The polypeptide that inhibition hepatitis C virus provided by the invention is invaded is in following ten peptide species any one:
Polypeptide I: aminoacid sequence is shown in the sequence 1 of sequence table; NH
2-MANAGLQLLGFILAFLGW-COOH;
Polypeptide II: aminoacid sequence is shown in the sequence 2 of sequence table; NH
2-LLGFILAFLGWIGAIVST-COOH;
Polypeptide III: aminoacid sequence is shown in the sequence 3 of sequence table; NH
2-FILAFLGWIGAIVSTALP-COOH;
Polypeptide IV: aminoacid sequence is shown in the sequence 4 of sequence table; NH
2-AFLGWIGAIVSTALPQWR-COOH;
Polypeptide V: aminoacid sequence is shown in the sequence 5 of sequence table; NH
2-GWIGAIVSTALPQWRIYS-COOH;
Polypeptide VI: aminoacid sequence is shown in the sequence 6 of sequence table; NH
2-GAIVSTALPQWRIYSYAG-COOH;
Polypeptide VII: aminoacid sequence is shown in the sequence 7 of sequence table; NH
2-MANAGLQLLGFILAFL-COOH;
Polypeptide VIII: aminoacid sequence is shown in the sequence 8 of sequence table; NH
2-MANAGLQLLGFILAFLGWIG-COOH;
Polypeptide IX: aminoacid sequence is shown in the sequence 9 of sequence table; NH
2-MANAGLQLLGFILAFLGWIGAI-COOH;
Polypeptide X: aminoacid sequence is shown in the sequence 10 of sequence table; NH
2-MANAGLQLLGFILAFLGWIGAIVS-COOH.
The present invention also protects the verivate of said polypeptide, for (1) or (2) as follows:
(1) said polypeptide is carried out one or more amino acid whose insertions and/or replacement and/or disappearance, the verivate that obtains;
(2) said polypeptide or (1) said verivate are connected the verivate that obtains with carrier.
Use conformation as the amino acid of D-type, the rare amino acid of nature existence or manually modified amino acid replacement the one or more amino-acid residues in the said polypeptide, can increase bioavailability and improve antiviral activity.Said carrier is including, but not limited to polypeptide concatermer, protein, polyoxyethylene glycol and lipid material etc.The verivate of said polypeptide or said polypeptide can directly or with other molecule coupling be used to prepare the medicine that suppresses multiple infection of coated virus, or be developed as the novel antiviral medicine as leading peptide.
The application in preparation hepatitis C virus entry inhibitors of said polypeptide or said verivate also belongs to protection scope of the present invention.
The present invention also protects a kind of hepatitis C virus entry inhibitors, and its activeconstituents is said polypeptide or said verivate.
Said hepatitis C virus can be the hepatitis C virus of H77 genotype (1a), the hepatitis C virus of Con1 genotype (1b) or the hepatitis C virus of JFH1 genotype (2a).The hepatitis C virus of said H77 genotype (1a) specifically can be the hepatitis C virus of DNA shown in the sequence 11 that has sequence table in the genomic dna; Said Con1 genotype (1b) hepatitis C virus specifically can be the hepatitis C virus of DNA shown in the sequence 12 that has sequence table in the genomic dna; The hepatitis C virus of said JFH1 genotype (2a) specifically can be the hepatitis C virus of DNA shown in the sequence 13 that has sequence table in the genomic dna.
The application in the medicine of preparation treatment or prevention of hepatitis c of said polypeptide or said verivate also belongs to protection scope of the present invention.
The present invention also protects the medicine of a kind of treatment or prevention of hepatitis c, and its activeconstituents is said polypeptide or said verivate.
Polypeptide of the present invention can directly be used for the treatment that HCV infects separately, also can unite use with known one or more anti-HCV medicaments, and these medicines include but not limited to Interferon, rabbit, ribavirin etc.
The present invention has designed the inhibitory polypeptide library with the acceptor that HCV virus gets into host cell for target spot, and the candidate that screening has obtained to have stronger antiviral activity suppresses polypeptide.After candidate's peptide sequence and structure carried out the accent design and optimize, obtain a series of polypeptide with anti-HCV activity.These polypeptide can suppress the HCV pseudovirus can suppress infectious HCV virus again, and its main action target spot is a blocking virus and the early stage entering that suppresses viral of combining of its acceptor.
Polypeptide provided by the present invention has typical αLuo Xuanjiegou in organic phase or when touching membrane structures such as lipid bilayer.
Polypeptide provided by the present invention has following advantage:
(1) preparation is convenient: form by 16-24 amino-acid residue, and shorter relatively, both can also be convenient to prepare through the method preparation of chemosynthesis through recombinant expressed mode.
(2) peptide sequence is different with existing HCV entry inhibitors with action site: polypeptide action target spot provided by the present invention is clear and definite, for suppressing the interaction of HCV virus envelope and host receptor CLDN1 specifically.
(3) be difficult for producing resistance: because peptide sequence provided by the invention derives from human protein CLDN1, but not derive from HCV virus envelope albumen, so its HCV to several genes type (hypotype) is inhibited.
Description of drawings
Fig. 1 is the influence of the polypeptide libraries in various acceptors source to HCV pseudovirus invasion efficiency.
Fig. 2 is the circular dichroism spectrogram of polypeptide 1.
Fig. 3 is that the intrusion of the HCVpp pseudovirus of 1 pair of H77 genotype of polypeptide (1a) suppresses ability.
Fig. 4 is that the intrusion of the HCVpp pseudovirus of 1 pair of Con1 genotype of polypeptide (1b) suppresses ability.
Fig. 5 is that the intrusion of the HCVpp pseudovirus of 1 pair of JFH1 genotype of polypeptide (2a) suppresses ability.
Fig. 6 is that immunofluorescence dyeing detected the Subcellular Localization of HCV acceptor Claudin-1 after polypeptide 1 was handled the Huh7 cell; The result shows that polypeptide 1 processing does not influence expression level and the location thereof of cell surface receptor Claudin-1.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
293T cell: available from U.S. classical collection cell bank ATCC, catalog number (Cat.No.): CRL-11268.The Huh7 cell: (SMMC-7721) is available from U.S. Apath company.Plasmid pNL4.3-Luc-R
-E
-Available from NIH's AIDS research and reference reagent plan (NIH AIDS Research and Reference Reagent Program).HepG2: available from U.S. classical collection cell bank ATCC.Foetal calf serum and DMEM substratum are available from Invitrogen company, and transfection reagent is available from Giantagen company.Consumptive materials such as disposable Tissue Culture Plate and transfer pipet are available from Corning company.
The design of embodiment 1, antiviral polypeptide
The principle of design of antiviral polypeptide is following.
The primary link of HCV infection host is that the E1 and the E2 envelope protein of virus interacts with host cell surface acceptor SR-BI, CD81, CLDN1 and OCLN successively, accomplishes the first step of virus infection through cell endocytic and film fusion.In theory, the specific region of cell surface receptor has the ability that combines the HCV envelope protein, through the polypeptide libraries method for screening, can obtain corresponding competitive polypeptide.
In view of the reasonableness of above experimental design, be the basic design polypeptide libraries with the aminoacid sequence of HCV acceptor SR-BI, CD81, CLDN1 and OCLN, every long 18 amino acid of polypeptide, two adjacent polypeptide have 5 amino acid whose repeat regions (overlap).
Polypeptide libraries comprises 113 polypeptide altogether.With pseudovirus (HCVpp) and Huh7 SMMC-7721 is screening model; 113 polypeptide (primary dcreening operation concentration is 50 μ M) are carried out antiviral screening; Obtain the polypeptide (polypeptide 1 and polypeptide 2) of 2 antiviral activity＞90%, lay respectively at the N end (see figure 1) of acceptor CLDN1.Fig. 1 is polypeptide libraries screening synoptic diagram; Acceptor 1-4 among the figure represents HCV acceptor CD81, SR-BI, CLDN1 and OCLN respectively; Wherein No. 58 and No. 59 two polypeptide demonstrate good antiviral activity.
Polypeptide 1 (CL58): MANAGLQLLGFILAFLGW;
Polypeptide 4 (CL58.4): AFLGWIGAIVSTALPQWR.
Wherein, inhibition efficient＞99% of polypeptide 1 under the concentration of treatment of 50 μ M, and the inhibition effect of polypeptide 4 is poor slightly than CL58, inhibition efficient＞90% under the final concentration of 50 μ M.
According to the sequence of above two polypeptide, the aminoacid sequence of polypeptide 1 and polypeptide 4 is carried out the accent design and optimizes, obtain 10 polypeptide, see table 1.
Table 110 amino acid sequence of polypeptide
| Peptide sequence (NH 2-polypeptide-COOH) | |
| CL58 (polypeptide 1) | MANAGLQLLGFILAFLGW |
| CL58.2 (polypeptide 2) | LLGFILAFLGWIGAIVST |
| CL58.3 (polypeptide 3) | FILAFLGWIGAIVSTALP |
| CL58.4 (polypeptide 4) | AFLGWIGAIVSTALPQWR |
| CL58.5 (polypeptide 5) | GWIGAIVSTALPQWRIYS |
| CL58.6 (polypeptide 6) | GAIVSTALPQWRIYSYAG |
| CL58-2 (polypeptide 7) | MANAGLQLLGFILAFL |
| CL58+2 (polypeptide 8) | MANAGLQLLGFILAFLGWIG |
| CL58+4 (polypeptide 9) | MANAGLQLLGFILAFLGWIGAI |
| CL58+6 (polypeptide 10) | MANAGLQLLGFILAFLGWIGAIVS |
Polypeptide 1, polypeptide 2, polypeptide 3, polypeptide 4, polypeptide 5, polypeptide 6, polypeptide 7, polypeptide 8, polypeptide 9 and polypeptide 10 among each embodiment all refers to each the bar polypeptide in the table 1 later on.
Synthetic and the chemical property determination of embodiment 2, polypeptide
One, polypeptide 1 and contrast polypeptide is synthetic
1, polypeptide 1 is synthetic
Synthesize polypeptide 1 by U.S. Genscript company through chemical process.With DMSO dissolving polypeptide 1, be mixed with the storing solution of 10mM ,-20 ℃ of freezing preservations.
2, the contrast polypeptide is synthetic
Contrast polypeptide (NH
2-polypeptide-COOH): SWLRDIWDWICEVLSDFK.
The contrast polypeptide is similarly the sequence that derives from CLDN1, and the sequence number in original polypeptide libraries is No. 60, and position next-door neighbour No. 58 and No. 59 polypeptide are at the C of No. 58 and No. 59 polypeptide end; Through screening, the contrast polypeptide does not have any influence to the intrusion of HCV.
Synthesize the contrast polypeptide by U.S. Genscript company through chemical process.With DMSO dissolving contrast polypeptide, be mixed with the storing solution of 10mM ,-20 ℃ of freezing preservations.
Two, the physicochemical property of polypeptide 1 (circular dichroism spectrum analysis)
1, experiment purpose
Through measuring the helicity or the helical content of polypeptide 1, confirm the secondary structure that polypeptide 1 is possible.
2, laboratory apparatus, reagent and method
Utilize Jasco J-720 circular dichroism spectrum (CD) appearance (Jasco, Easton, MD), (50mMKH under 25 ℃ of mild conditionss
2PO
4/ K
2HPO
4/ 100mM KCl, pH7) and contain 50% alpha-helix and induce (50mM KH under the condition of reagent 2,2,2 tfifluoroethyl alcohol (TFE)
2PO
4/ K
2HPO
4/ 100mM KCl, pH7 damping fluid/50%TFE), measure the average residue molar ellipticity coefficient of polypeptide 1 respectively.The polypeptide storage mother liquor of 500 μ M is joined later in the quartzy testing tube of 0.02cm through 10 times of dilutions, and the scanning through 190 to 250nm obtains the ellipse coefficient of product polypeptide.Under the 222nm wavelength, measure the alpha-helix property that the polypeptide molar ellipticity coefficient that obtains is used to assess polypeptide.
3, experimental result
Typical alpha-helix on CD figure, is shown as 208nm and there are two negative peaks at the 222nm place.As can be seen from Figure 2, polypeptide 1 is containing under the 50%TFE condition, two negative peaks occur at 208nm and 222nm place.Show that polypeptide 1 has helical conformation at thin aqueous phase (structure that for example contains film), (Buffer) then is unordered conformation in the aqueous solution.
Polypeptide with embodiment 2 preparations carries out the detection of subsequent implementation example.
The inhibition ability that embodiment 3,1 pair of virus of polypeptide are invaded
One, the preparation of the HCVpp pseudovirus of range gene type
1, the preparation of the HCVpp pseudovirus of H77 genotype (1a)
(1) preparation of recombinant plasmid
According to the sequence in the HCVdb DB (http://www.hcvdb.org), by the H77 gene shown in the sequence 11 of Genscript company composition sequence table.Cut recognition site through HindIII and EcoRI enzyme,, obtain recombinant plasmid pcDNA3-H77 (the H77 gene clone is in CMV promotor downstream) the H77 gene insertion vector plasmid pcDNA3 (Invitrogen) shown in the sequence 11.
(2) preparation of the genotypic HCVpp pseudovirus of H77
The 293T cell is inoculated in the 10cm Tissue Culture Dish in transfection previous day, and cell density is 50% during transfection.With pNL4.3-Luc-R
-E
-Plasmid and pcDNA3-H77 cotransfection 293T cell, wherein pNL4.3-Luc-R
-E
-: pcDNA3-H77: the amount of transfection reagent is 15 μ g: 15 μ g: 60 μ L.Collected viral supernatant (HCVpp) in 48 hours after the transfection, filter packing, be stored in-80 ℃ with 0.45 μ M filter.
2, the preparation of the HCVpp pseudovirus of Con1 genotype (1b)
(1) preparation of recombinant plasmid
According to the sequence in the HCVdb DB (http://www.hcvdb.org), by the Con1 gene shown in the sequence 12 of Genscript company composition sequence table.Cut recognition site through HindIII and EcoRI enzyme,, obtain recombinant plasmid pcDNA3-Con1 (the Con1 gene clone is in CMV promotor downstream) the Con1 gene insertion vector plasmid pcDNA3 shown in the sequence 12.
(2) preparation of the genotypic HCVpp pseudovirus of Con1
(2) with step 1.
3, the preparation of the HCVpp pseudovirus of JFH1 genotype (2a)
(1) preparation of recombinant plasmid
According to the sequence in the HCVdb DB (http://www.hcvdb.org), by the JFH1 gene shown in the sequence 13 of Genscript company composition sequence table.Cut recognition site through HindIII and EcoRI enzyme,, obtain recombinant plasmid pcDNA3-JFH1 (the JFH1 gene clone is in CMV promotor downstream) the JFH1 gene insertion vector plasmid pcDNA3 shown in the sequence 13.。
(2) preparation of the genotypic HCVpp pseudovirus of JFH1
(2) with step 1.
PcDNA3-H77, pcDNA3-Con1 and pcDNA3-JFH1 are referred to as HCV envelope protein expression plasmid pcDNA3-E1E2.
Two, the inhibition ability of 1 pair of virus of polypeptide
1, the HCV pseudovirus is invaded experiment
1. pseudovirus is packed
The 293T cell is inoculated in the 10cm Tissue Culture Dish in transfection previous day, and cell density is 50% during transfection.With pNL4.3-Luc-R
-E
-Plasmid and pcDNA3-E1E2 plasmid (pcDNA3-H77, pcDNA3-Con1 or pcDNA3-JFH1) cotransfection 293T cell, wherein pNL4.3-Luc-R
-E
-: pcDNA3-E1E2: the amount of transfection reagent is 15 μ g: 15 μ g: 60 μ L.Collected viral supernatant (HCVpp) in 48 hours after the transfection, filter packing, be stored in-80 ℃ with 0.45 μ M filter.
2. pseudovirus is invaded experiment
The Huh7 cell inoculation in 96 orifice plates, is cultured to 90% and converges density; Getting the viral supernatant that 1. 100 μ L steps obtain mixes with the polypeptide 1 of each concentration (0,5,10,15,20 μ M); Adding the Huh7 cell then infects; Lysing cell carries out luciferase reporter gene detection (detection reagent is available from Promega company, and instrument is the Modulus Microplate Luminometer of Turner BioSystems company) after 48 hours.
Result such as Fig. 3, Fig. 4 and shown in Figure 5, polypeptide 1 (CL58) all has the obvious suppression effect for the virus in the main epidemic strain 1a of the HCV of China, 1b and 2a hypotype.
The median effective dose of embodiment 4, antiviral polypeptide
1, the preparation of the genotypic HCVpp pseudovirus of H77
With 1 of the step 1 of embodiment 3.
2, median effective dose is measured
The viral supernatant of getting 100 μ L steps 1 preparations mixes with each polypeptide respectively that (every peptide species is provided with some concentration respectively; The polypeptide final concentration is respectively 20 μ M, 10 μ M, 5 μ M, 2.5 μ M, 1.25 μ M, 0.625 μ M, 0.3125 μ M and 0 μ M); Mixture added among the SMMC-7721 Huh7 infected 3 hours; Change the fresh DMEM substratum (purchase of Invitrogen company) that contains 10% foetal calf serum and continue to cultivate 48 hours, utilize luciferase detection system (available from Promega company) to carry out the mensuration of virus infection efficient.The result sees table 2.
The IC50 value of table 210 polypeptide
| IC50，μM | IC50，μM | ||
| CL58 (polypeptide 1) | 2.1±0.5 | CL58.6 (polypeptide 6) | 23.8±2.1 |
| CL58.2 (polypeptide 2) | 4.3±0.3 | CL58-2 (polypeptide 7) | 7.6±0.9 |
| CL58.3 (polypeptide 3) | 8.9±1.0 | CL58+2 (polypeptide 8) | 17.8±1.1 |
| CL58.4 (polypeptide 4) | 12.5±1.5 | CL58+4 (polypeptide 9) | 4.0±0.3 |
| CL58.5 (polypeptide 5) | 21.5±1.9 | CL58+6 (polypeptide 10) | 5.1±0.6 |
The cytotoxicity of embodiment 5,1 couple of SMMC-7721 Huh7 of polypeptide and HepG2,293T cell etc.
Utilize MTT detection kit (available from Promega company) to analyze the isocellular cytotoxicity analysis of cytotoxicity of 1 pair of various cell of polypeptide (SMMC-7721 Huh7, HepG2 and 293T) respectively.Analytical procedure to every kind of cell is following: cell inoculation is in 96 orifice plates; Be incubated in the DMEM substratum that contains 10% foetal calf serum to cell density and reach 50%; Get the polypeptide 1 (2 μ M, 4 μ M, 8 μ M, 16 μ M, 32 μ M, 64 μ M and 128 μ M) of different final concentrations and handled above-mentioned cell 48 hours; Operate according to Promega company test kit specification sheets, measure cytotoxicity.
The result: polypeptide 1 for the isocellular toxicity of SMMC-7721 Huh7, HepG2 and 293T (mld) all greater than 100 μ M.
The antiviral-mechanism research of embodiment 6, polypeptide 1
Because the sequence of polypeptide 1 derives from the acceptor CLDN1 of HCV; For further investigation polypeptide 1 suppresses the mechanism that HCV invades; Inquire into whether the antiviral activity of polypeptide 1 cause owing to the Subcellular Localization that has changed acceptor CLDN1; Use the polypeptide 1 of 20 μ M and the solvent DMSO of equal volume after 24 hours, to utilize the cellular localization situation of the methods analyst CLDN1 of immunofluorescence dyeing respectively as control treatment Huh7 cell.
Cell inoculation carries out handled in 96 orifice plates that contain slide.After processing finishes, with PBS washed cell 3 times, adopt 2% Paraformaldehyde 96 fixed cell after, handle penetrating cell 3 times with Triton-X100, each 10 minutes.Carry out cell dyeing so that the antibody of anti-Claudin-1 and FITC fluorescent mark two are anti-respectively, nucleus is with the DAPI mark.Gather image through Leica TCS SP5.
The result is as shown in Figure 6, and polypeptide 1 is handled expression amount and the Subcellular Localization that the Huh7 liver cancer cell does not change HCV acceptor CLDN1.Wherein DMSO handles as negative control.CLDN1 albumen still is positioned the tight junction of cell (Tight Junction) after two kinds of processing.The result shows the action target spot of polypeptide 1 not the host, so its cytotoxicity maybe be extremely low.
Sequence table
< 110>Institute of Pathogen Biology, Chinese Academy of Medical Sciences
< 120>suppress the polypeptide that hepatitis C virus is invaded
<130>CCGNARY102180
<160>13
<210>1
<211>18
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>1
Met?Ala?Asn?Ala?Gly?Leu?Gln?Leu?Leu?Gly?Phe?Ile?Leu?Ala?Phe?Leu
1 5 10 15
Gly?Trp
<210>2
<211>18
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>2
Leu?Leu?Gly?Phe?Ile?Leu?Ala?Phe?Leu?Gly?Trp?Ile?Gly?Ala?Ile?Val
1 5 10 15
Ser?Thr
<210>3
<211>18
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>3
Phe?Ile?Leu?Ala?Phe?Leu?Gly?Trp?Ile?Gly?Ala?Ile?Val?Ser?Thr?Ala
1 5 10 15
Leu?Pro
<210>4
<211>18
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>4
Ala?Phe?Leu?Gly?Trp?Ile?Gly?Ala?Ile?Val?Ser?Thr?Ala?Leu?Pro?Gln
1 5 10 15
Trp?Arg
<210>5
<211>18
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>5
Gly?Trp?Ile?Gly?Ala?Ile?Val?Ser?Thr?Ala?Leu?Pro?Gln?Trp?Arg?Ile
1 5 10 15
Tyr?Ser
<210>6
<211>18
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>6
Gly?Ala?Ile?Val?Ser?Thr?Ala?Leu?Pro?Gln?Trp?Arg?Ile?Tyr?Ser?Tyr
1 5 10 15
Ala?Gly
<210>7
<211>16
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>7
Met?Ala?Asn?Ala?Gly?Leu?Gln?Leu?Leu?Gly?Phe?Ile?Leu?Ala?Phe?Leu
1 5 10 15
<210>8
<211>20
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>8
Met?Ala?Asn?Ala?Gly?Leu?Gln?Leu?Leu?Gly?Phe?Ile?Leu?Ala?Phe?Leu
1 5 10 15
Gly?Trp?Ile?Gly
20
<210>9
<211>22
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>9
Met?Ala?Asn?Ala?Gly?Leu?Gln?Leu?Leu?Gly?Phe?Ile?Leu?Ala?Phe?Leu
1 5 10 15
Gly?Trp?Ile?Gly?Ala?Ile
20
<210>10
<211>24
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>10
Met?Ala?Asn?Ala?Gly?Leu?Gln?Leu?Leu?Gly?Phe?Ile?Leu?Ala?Phe?Leu
1 5 10 15
Gly?Trp?Ile?Gly?Ala?Ile?Val?Ser
20
<210>11
<211>1677
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>11
taccaagtgc?gcaattcctc?ggggctttac?catgtcacca?atgattgccc?taactcgagt 60
attgtgtacg?aggcggccga?tgccatcctg?cacactccgg?ggtgtgtccc?ttgcgttcgc 120
gagggtaacg?cctcgaggtg?ttgggtggcg?gtgaccccca?cggtggccac?cagggacggc 180
aaactcccca?caacgcagct?tcgacgtcat?atcgatctgc?ttgtcgggag?cgccaccctc 240
tgctcggccc?tctacgtggg?ggacctgtgc?gggtctgtct?ttcttgttgg?tcaactgttt 300
accttctctc?ccaggcgcca?ctggacgacg?caagactgca?attgttctat?ctatcccggc 360
catataacgg?gtcatcgcat?ggcatgggat?atgatgatga?actggtcccc?tacggcagcg 420
ttggtggtag?ctcagctgct?ccggatccca?caagccatca?tggacatgat?cgctggtgct 480
cactggggag?tcctggcggg?catagcgtat?ttctccatgg?tggggaactg?ggcgaaggtc 540
ctggtagtgc?tgctgctatt?tgccggcgtc?gacgcggaaa?cccacgtcac?cgggggaagt 600
gccggccgca?ccacggctgg?gcttgttggt?ctccttacac?caggcgccaa?gcagaacatc 660
caactgatca?acaccaacgg?cagttggcac?atcaatagca?cggccttgaa?ctgcaatgaa 720
agccttaaca?ccggctggtt?agcagggctc?ttctatcagc?acaaattcaa?ctcttcaggc 780
tgtcctgaga?ggttggccag?ctgccgacgc?cttaccgatt?ttgcccaggg?ctggggtcct 840
atcagttatg?ccaacggaag?cggcctcgac?gaacgcccct?actgctggca?ctaccctcca 900
agaccttgtg?gcattgtgcc?cgcaaagagc?gtgtgtggcc?cggtatattg?cttcactccc 960
agccccgtgg?tggtgggaac?gaccgacagg?tcgggcgcgc?ctacctacag?ctggggtgca 1020
aatgatacgg?atgtcttcgt?ccttaacaac?accaggccac?cgctgggcaa?ttggttcggt 1080
tgtacctgga?tgaactcaac?tggattcacc?aaagtgtgcg?gagcgccccc?ttgtgtcatc 1140
ggaggggtgg?gcaacaacac?cttgctctgc?cccactgatt?gtttccgcaa?gcatccggaa 1200
gccacatact?ctcggtgcgg?ctccggtccc?tggattacac?ccaggtgcat?ggtcgactac 1260
ccgtataggc?tttggcacta?tccttgtacc?atcaattaca?ccatattcaa?agtcaggatg 1320
tacgtgggag?gggtcgagca?caggctggaa?gcggcctgca?actggacgcg?gggcgaacgc 1380
tgtgatctgg?aagacaggga?caggtccgag?ctcagcccat?tgctgctgtc?caccacacag 1440
tggcaggtcc?ttccgtgttc?tttcacgacc?ctgccagcct?tgtccaccgg?cctcatccac 1500
ctccaccaga?acattgtgga?cgtgcagtac?ttgtacgggg?tagggtcaag?catcgcgtcc 1560
tgggccatta?agtgggagta?cgtcgttctc?ctgttcctcc?tgcttgcaga?cgcgcgcgtc 1620
tgctcctgct?tgtggatgat?gttactcata?tcccaagcgg?aggcggcttt?ggagaac 1677
<210>12
<211>1677
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>12
tatgaagtgc?gcaacgtatc?cggagtgtac?catgtcacga?acgactgctc?caacgcaagc 60
attgtgtatg?aggcagcgga?catgatcatg?catacccccg?ggtgcgtgcc?ctgcgttcgg 120
gagaacaact?cctcccgctg?ctgggtagcg?ctcactccca?cgctcgcggc?caggaacgct 180
agcgtcccca?ctacgacgat?acgacgccat?gtcgatttgc?tcgttggggc?ggctgctctc 240
tgctccgcta?tgtacgtggg?agatctctgc?ggatctgttt?tcctcgtcgc?ccagctgttc 300
accttctcgc?ctcgccggca?cgagacagta?caggactgca?attgctcaat?atatcccggc 360
cacgtgacag?gtcaccgtat?ggcttgggat?atgatgatga?actggtcacc?tacagcagcc 420
ctagtggtat?cgcagttact?ccggatccca?caagctgtcg?tggatatggt?ggcgggggcc 480
cattggggag?tcctagcggg?ccttgcctac?tattccatgg?tggggaactg?ggctaaggtt 540
ctgattgtga?tgctactctt?tgccggcgtt?gacgggggaa?cctatgtgac?aggggggacg 600
atggccaaaa?acaccctcgg?gattacgtcc?ctcttttcac?ccgggtcatc?ccagaaaatc 660
cagcttgtaa?acaccaacgg?cagctggcac?atcaacagga?ctgccctgaa?ctgcaatgac 720
tccctcaaca?ctgggttcct?tgctgcgctg?ttctacgtgc?acaagttcaa?ctcatctgga 780
tgcccagagc?gcatggccag?ctgcagcccc?atcgacgcgt?tcgctcaggg?gtgggggccc 840
atcacttaca?atgagtcaca?cagctcggac?cagaggcctt?attgttggca?ctacgcaccc 900
cggccgtgcg?gtatcgtacc?cgcggcgcag?gtgtgtggtc?cagtgtactg?cttcacccca 960
agccctgtcg?tggtggggac?gaccgaccgg?ttcggcgtcc?ctacgtacag?ttggggggag 1020
aatgagacgg?acgtgctgct?tcttaacaac?acgcggccgc?cgcaaggcaa?ctggtttggc 1080
tgtacatgga?tgaatagcac?tgggttcacc?aagacgtgcg?ggggcccccc?gtgtaacatc 1140
ggggggatcg?gcaataaaac?cttgacctgc?cccacggact?gcttccggaa?gcaccccgag 1200
gccacttaca?ccaagtgtgg?ttcggggcct?tggttgacac?ccagatgctt?ggtccactac 1260
ccatacaggc?tttggcacta?cccctgcact?gtcaacttta?ccatcttcaa?ggttaggatg 1320
tacgtggggg?gagtggagca?caggctcgaa?gccgcatgca?attggactcg?aggagagcgt 1380
tgtaacctgg?aggacaggga?cagatcagag?cttagcccgc?tgctgctgtc?tacaacggag 1440
tggcaggtat?tgccctgttc?cttcaccacc?ctaccggctc?tgtccactgg?tttgatccat 1500
ctccatcaga?acgtcgtgga?cgtacaatac?ctgtacggta?tagggtcggc?ggttgtctcc 1560
tttgcaatca?aatgggagta?tgtcctgttg?ctcttccttc?ttctggcgga?cgcgcgcgtc 1620
tgtgcctgct?tgtggatgat?gctgctgata?gctcaagctg?aggccgccct?agagaac 1677
<210>13
<211>1677
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>13
gcccaggtga?agaataccag?tagcagctac?atggtgacca?atgactgctc?caatgacagc 60
atcacttggc?agctcgaggc?tgcggttctc?cacgtccccg?ggtgcgtccc?gtgcgagaga 120
gtggggaata?cgtcacggtg?ttgggtgcca?gtctcgccaa?acatggctgt?gcggcagccc 180
ggtgccctca?cgcagggtct?gcggacgcac?atcgatatgg?ttgtgatgtc?cgccaccttc 240
tgctctgctc?tctacgtggg?ggacctctgt?ggcggggtga?tgctcgcggc?ccaggtgttc 300
atcgtctcgc?cgcagtacca?ctggtttgtg?caagaatgca?attgctccat?ctaccctggc 360
accatcactg?gacaccgcat?ggcatgggac?atgatgatga?actggtcgcc?cacggccacc 420
atgatcctgg?cgtacgtgat?gcgcgtcccc?gaggtcatca?tagacatcgt?tagcggggct 480
cactggggcg?tcatgttcgg?cttggcctac?ttctctatgc?agggagcgtg?ggcgaaggtc 540
attgtcatcc?ttctgctggc?cgctggggtg?gacgcgggca?ccaccaccgt?tggaggcgct 600
gttgcacgtt?ccaccaacgt?gattgccggc?gtgttcagcc?atggccctca?gcagaacatt 660
cagctcatta?acaccaacgg?cagttggcac?atcaaccgta?ctgccttgaa?ttgcaatgac 720
tccttgaaca?ccggctttct?cgcggccttg?ttctacacca?accgctttaa?ctcgtcaggg 780
tgtccagggc?gcctgtccgc?ctgccgcaac?atcgaggctt?tccggatagg?gtggggcacc 840
ctacagtacg?aggataatgt?caccaatcca?gaggatatga?ggccgtactg?ctggcactac 900
cccccaaagc?cgtgtggcgt?agtccccgcg?aggtctgtgt?gtggcccagt?gtactgtttc 960
acccccagcc?cggtagtagt?gggcacgacc?gacagacgtg?gagtgcccac?ctacacatgg 1020
ggagagaatg?agacagatgt?cttcctactg?aacagcaccc?gaccgccgca?gggctcatgg 1080
ttcggctgca?cgtggatgaa?ctccactggt?ttcaccaaga?cttgtggcgc?gccaccttgc 1140
cgcaccagag?ctgacttcaa?cgccagcacg?gacttgttgt?gccctacgga?ttgttttagg 1200
aagcatcctg?atgccactta?tattaagtgt?ggttctgggc?cctggctcac?accaaagtgc 1260
ctggtccact?acccttacag?actctggcat?tacccctgca?cagtcaattt?taccatcttc 1320
aagataagaa?tgtatgtagg?gggggttgag?cacaggctca?cggccgcatg?caacttcact 1380
cgtggggatc?gctgcgactt?ggaggacagg?gacaggagtc?agctgtctcc?tctgttgcac 1440
tctaccacgg?aatgggccat?cctgccctgc?acctactcag?acttacccgc?tttgtcaact 1500
ggtcttctcc?accttcacca?gaacatcgtg?gacgtacaat?acatgtatgg?cctctcacct 1560
gctatcacaa?aatacgtcgt?tcgatgggag?tgggtggtac?tcttattcct?gctcttagcg 1620
gacgccagag?tctgcgcctg?cttgtggatg?ctcatcttgt?tgggccaggc?cgaagca 1677
Claims (13)
1. polypeptide is in following ten peptide species any one:
Polypeptide I: aminoacid sequence is shown in the sequence 1 of sequence table;
Polypeptide II: aminoacid sequence is shown in the sequence 2 of sequence table;
Polypeptide III: aminoacid sequence is shown in the sequence 3 of sequence table;
Polypeptide IV: aminoacid sequence is shown in the sequence 4 of sequence table;
Polypeptide V: aminoacid sequence is shown in the sequence 5 of sequence table;
Polypeptide VI: aminoacid sequence is shown in the sequence 6 of sequence table;
Polypeptide VII: aminoacid sequence is shown in the sequence 7 of sequence table;
Polypeptide VIII: aminoacid sequence is shown in the sequence 8 of sequence table;
Polypeptide IX: aminoacid sequence is shown in the sequence 9 of sequence table;
Polypeptide X: aminoacid sequence is shown in the sequence 10 of sequence table.
2. the application of the said polypeptide of claim 1 in preparation hepatitis C virus entry inhibitors; Said polypeptide is polypeptide I, and said hepatitis C virus is the genotypic hepatitis C virus of H77, the genotypic hepatitis C virus of Con1 or the genotypic hepatitis C virus of JFH1.
3. application as claimed in claim 2 is characterized in that: the genotypic hepatitis C virus of said H77 is the hepatitis C virus that has DNA shown in the sequence 11 of sequence table in the genomic dna; The genotypic hepatitis C virus of said Con1 is the hepatitis C virus that has DNA shown in the sequence 12 of sequence table in the genomic dna; The genotypic hepatitis C virus of said JFH1 is the hepatitis C virus that has DNA shown in the sequence 13 of sequence table in the genomic dna.
4. the application of the said polypeptide of claim 1 in preparation hepatitis C virus entry inhibitors, said polypeptide are polypeptide II to the polypeptide X any one, said hepatitis C virus is the genotypic hepatitis C virus of H77.
5. application as claimed in claim 4 is characterized in that: the genotypic hepatitis C virus of said H77 is the hepatitis C virus that has DNA shown in the sequence 11 of sequence table in the genomic dna.
6. hepatitis C virus entry inhibitors, its activeconstituents is the said polypeptide of claim 1; Said polypeptide is polypeptide I; Said hepatitis C virus is the genotypic hepatitis C virus of H77, the genotypic hepatitis C virus of Con1 or the genotypic hepatitis C virus of JFH1.
7. suppressor factor as claimed in claim 6 is characterized in that: the genotypic hepatitis C virus of said H77 is the hepatitis C virus that has DNA shown in the sequence 11 of sequence table in the genomic dna; The genotypic hepatitis C virus of said Con1 is the hepatitis C virus that has DNA shown in the sequence 12 of sequence table in the genomic dna; The genotypic hepatitis C virus of said JFH1 is the hepatitis C virus that has DNA shown in the sequence 13 of sequence table in the genomic dna.
8. hepatitis C virus entry inhibitors, its activeconstituents is the said polypeptide of claim 1; Said polypeptide is polypeptide II to the polypeptide X any one; Said hepatitis C virus is the genotypic hepatitis C virus of H77.
9. suppressor factor as claimed in claim 8 is characterized in that: the genotypic hepatitis C virus of said H77 is the hepatitis C virus that has DNA shown in the sequence 11 of sequence table in the genomic dna.
10. the application of the said polypeptide of claim 1 in the medicine of preparation treatment or prevention of hepatitis c; Said polypeptide is polypeptide I, and said hepatitis C virus is the genotypic hepatitis C virus of H77, the genotypic hepatitis C virus of Con1 or the genotypic hepatitis C virus of JFH1.
11. the application of the said polypeptide of claim 1 in the medicine of preparation treatment or prevention of hepatitis c, said polypeptide are polypeptide II to the polypeptide X any one, said hepatitis C virus is the genotypic hepatitis C virus of H77.
12. the treatment or the medicine of prevention of hepatitis c, its activeconstituents is the said polypeptide of claim 1; Said polypeptide is polypeptide I; Said hepatitis C virus is the genotypic hepatitis C virus of H77, the genotypic hepatitis C virus of Con1 or the genotypic hepatitis C virus of JFH1.
13. the treatment or the medicine of prevention of hepatitis c, its activeconstituents is the said polypeptide of claim 1; Said polypeptide is polypeptide II to the polypeptide X any one; Said hepatitis C virus is the genotypic hepatitis C virus of H77.
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| CN2010101363682A CN101851274B (en) | 2010-03-29 | 2010-03-29 | Polypeptides for suppressing invasion of hepatitis C virus |
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| CN2010101363682A CN101851274B (en) | 2010-03-29 | 2010-03-29 | Polypeptides for suppressing invasion of hepatitis C virus |
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| JP6158171B2 (en) * | 2011-05-02 | 2017-07-05 | ザ・ジョンズ・ホプキンス・ユニバーシティ | Synthetic hepatitis C genome, production method thereof and use thereof |
| CN102382171B (en) * | 2011-09-27 | 2013-04-17 | 中国人民解放军第四军医大学 | Polypeptide for inhibiting hepatitis C virus infected cell and application thereof |
| CN103965292B (en) * | 2013-02-01 | 2020-04-03 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Structure and application of Japanese encephalitis virus envelope protein binding peptide |
| CN103548775B (en) * | 2013-10-23 | 2015-03-18 | 中国科学院武汉病毒研究所 | Construction method of CD81 (cluster of differentiation 81) and OCLN (occludin) dual-transgenosis murine, and application of the |
| CN107304224B (en) * | 2016-04-18 | 2020-07-07 | 中国医学科学院病原生物学研究所 | Polypeptide for inhibiting hepatitis C virus infection and application thereof |
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