CN101899100B - Anti-HIV-I (Human Immunodeficiency Virus-I) polypeptides as well as coding sequences and applications thereof - Google Patents
Anti-HIV-I (Human Immunodeficiency Virus-I) polypeptides as well as coding sequences and applications thereof Download PDFInfo
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Abstract
The invention provides Anti-HIV-I (Human Immunodeficiency Virus-I) polypeptides and combined polypeptides as well as coding sequences, expression vectors, preparation methods and applications thereof. The polypeptides and the combined polypeptides are synthesized by the expression after gene recombination or a polypeptide synthesizer, simple in process, capable of improving the final yield and restricting the HIV-I incursion effect and is suitable for industrial production.
Description
This case is dividing an application of following patent application:
Application number: 200710037104.x;
The applying date: on February 2nd, 2007;
Denomination of invention: the polypeptide of HIV-I resisting, its encoding sequence and uses thereof.
Technical field
The present invention relates to the genetically engineered field, more specifically, the present invention relates to polypeptide, its encoding sequence of new HIV-I resisting and uses thereof.
Background technology
Human immunodeficiency virus (human immunodeficiency virus, HIV) causes acquired immune deficiency syndrome (AIDS) (acquired immunodeficiency syndrome, AIDS), i.e. acquired immune deficiency syndrome (AIDS).HIV belongs to the RNA Retroviridae, can be divided into I type (HIV-I) and II type (HIV-II), and wherein the HIV-I infection rate is high, hazardness is large.The infected carries virus throughout one's life, and in sequela in the latent period of several years, systemic immune system is subject to grievous injury, finally deathward.Because at present acquired immune deficiency syndrome (AIDS) still being lacked effective treatment means, from 1981 since first acquired immune deficiency syndrome (AIDS) case has been found in the USA New York, acquired immune deficiency syndrome (AIDS) has developed into a global prevailing disease, fast with its velocity of propagation, mortality ratio is high and can't effect a radical cure and had a strong impact on human health and social development, has been subject to the extensive attention of national governments and international community.HIV-I is the target spot of medicinal design from the whole process to invasion, time multiplexed cell system, assembling and the release etc. of cell, but still lacking now can the permanently effective medicine that does not have again toxic side effect, along with going deep into of research, virus more and more is subject to investigator's attention to adhesion and the fusion process of cell.
The grappling of HIV-I mainly is the process that film merges with invading, that processing by HIV-I adventitia precursor glycoprotein gp160 begins, gp160 is hydrolyzed into two fragments through the precursor processive enzyme: surface glycoprotein gp120 and transmembrane protein gp41, wherein gp120 does not contain transmembrane domains, it is connected with the gp41 non covalent bond, gp41 namely causes the infection of HIV-I after cell receptor is combined, the main combination with cell receptor CD4 of gp120 caused infection afterwards, thereby to the cracking of gp160, the binding site of gp41 and gp120 and be to understand the key that HIV-I infects mechanism and development anti-infectives with the interactional research of cell receptor, caused various countries scientists' very big concern, the research of each side is around this expansion.
1993, Jiang etc. find that the earliest the C-polypeptide SJ-2176 (630-659 residue) with fusion inhibition activity that is derived from the gp41CHR zone can suppress HIV-1 infection [Jiang S et al.HIV-1inhibition by a peptide[J] .Nature in the nmol/L level, 1993,365 (6442): 113.]。The Lu of Massachusetts Polytechnics etc. have carried out a series of hydrolysis research to gp41 with proteolytic enzyme, find that the C-polypeptide such as C43 (624～666), C34 (628～655), C28 (628～655) have active [the Weissenhorn W of the inhibition of fusion, Dessen A, Harrison SC, et al, Atomic structure of ectodomain from HIV-1gp41.Nature, 1997; 387:426.Chan D C, Fass D, Berger JM, et al, Core structure of gp41 from the HIV envelope glycoprotein.Cell, 1997,89 (2): 263-273].1994, Wild etc. find that again gp41C end polypeptide DP-178 (638-673 residue) suppresses HIV-1 and induces the efficient of Syncytium formation to increase substantially [Wild CT, Shugars DC, Greenwell TK, et al, Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1gp41are potent inhibitors of virus infection[J] .Proc.Natl.Acad.Sci.USA, 1994,91 (21): 9770-9774.]。Enfuviritide (T-20) is anti-film fusion class new inhibitor [the Jason LaBonte et al.Enfuvirtide that first blocking-up HIV-1 invades target cell, Nature reviews drug discovery, May 2003, volume 2,345-346], form [Wild CT et al.Peptides corresponding to a predictive α-helical domain of human immunodeficiency virus type 1gp41are potent inhibitors of virus infection.Medical Sciences by 36 amino-acid residues, October 1994, Proc.Natl.Acad.Sci.USA, Vol.91, pp.9770-9774.], the HR2 end of its structural simulation HIV-1 transmembrane protein gp41 can suppress HIV-1 virus effectively.It suppresses mechanism and competes in conjunction with HR1[Isabel M et al.Di1ation of the Human Immunodeficiency Virus-1Envelope Glycoprotein Fusion Pore Revealed by the Inhibitory Action of a Synthetic Peptide from gp41.The Journal of Cell Biology virus and target cell for the synthetic T20 of manual simulation HR2, Volume 140, Number 2, January 26,1998,315-323.], thereby from ingress cut-out cell entry target cell.Clinical study shows polypeptide enfuviritide as oral inhibitor and infeasible, but can be used as subcutaneous injection.In the clinical observation of per 28 a days first phase that 78 routine HIV-1 virus carriers are carried out, find, traditional heavy dose is not very effective, but can carry out subcutaneous injection [Wheeler DA.et al.Safety for twice on the one by reducing dosage, tolerability, and plasma pharmacokinetics of high-strength formulations of enfuvirtide (T-20) in treatment experienced HIV-1-infected patients.Journal of clinical virology 2004.30 (2), 183-190].Pharmaceutical quantities is relevant with quantity of viruses, is hampered by technical problem but continue subcutaneous injection.Maximum viral reduction rate is enfuvirtide every day twice, each 100mg[Greenberg M et al.Enfuvirtide (T-20) and T-1249resistance:observations from phase II clinical trials of enfuvirtide in combination with oral antiretrovirals and a phase I/IIdose-ranging monotherapy trial of T-1249.Antiviral Ther 2002; 7:Suppl:S140.abstract.]。
T-1249 is another the anti-film fusion polypeptide inhibitor that develops on the basis of T-20, its binding zone partially overlaps with T-20 binding zone, but more go deep into drain tank zone [the Greenberg ML et al.In vitro antiviral activity of T-1249 of HR1 than T-20, a second generation fusion inhibitor.Antiviral Ther2002,7 (Suppl 1): S10.abstract.]。This six spirals hydrophobic region plays an important role in the process that film merges.T-1249 has carried out the clinical observation of per 14 days first phases in 115 HIV-1 virus carriers.The taking dose of T-1249 is 6.25-200mg, and once a day subcutaneous injection of some control group, some is twice, maximum viral reduction rate is subcutaneous injection every day T-1249 total amount 150-200mg[Eron J et al.A 14-day assessment of the safety, pharmacokinetics, and antiviral activity of T-1249, a peptide inhibitor of membrane fusion.In:Programs and abstracts of the Eighth Conference on Retroviruses and Opportunistic Infections, Chicago, February 4-8,2001.Alexandria, Va.:Foundation for Retrovirology and Human Health, 2001:47.abstract.]。
Take the HR1 of gp41 core and HR2 structure as the basis, through the synthetic HR1-HR2 series polypeptide of gene recombination, can form preferably Six helix bundle structure of a stability in addition, be similar to the nucleus that gp41 has the film fusion function.Add that at the C of HR1-HR2 end a HR1 forms the HR121 structure, perhaps add that at the N end HR2 forms the HR212 structure, these two fusion polypeptide all have preferably stability and solvability, show with pseudovirus and T cells in vitro fusion experiment, fusion polypeptide has potential inhibition [Ling Ni et al.Rational design of highly potent HIV-1fusion inhibitory proteins:Implication for developing antiviral therapeutics.Biochemical and Biophysical Research Communications 2005,332:831-836 to the film fusion of HIV-1.]。
The applicant had applied for that (publication number: CN1810830A), this application provided a kind of peptide inhibitor based on gp41C end peptide sequence to patent " anti-HIV-1 peptide C 22, its encoding sequence and preparation method thereof ".
At present the common problem that exists of hiv inhibitor medicine is, medicine enters human body and is easy to later on enzymolysis, and HIV easily develops immunity to drugs to medicine and because the drug molecule amount produces immunogenicity greatly, directly affected the result of use of medicine.This area is strong and be difficult for the novel polypeptide inhibitor of the small molecular weight of enzymolysis in the urgent need to developing a species specificity.
Summary of the invention
First purpose of the present invention provides the high anti-HIV-1 inhibitor of a series of activity, comprises polypeptide and combination polypeptide.
Second purpose of the present invention provides the encoding sequence of a series of HIV-I resisting inhibitor.
The 3rd purpose of the present invention provided the preparation method of this aspect polypeptide and combination polypeptide.
The 4th purpose of the present invention provides the purposes of polypeptide and the combination polypeptide of described HIV-I resisting inhibitor.
A first aspect of the present invention provides a kind of polypeptide of the HIV of inhibition virus, and described polypeptide contains the aminoacid sequence shown in the SEQ ID NO:l (C22:ELDKWASLWNWF NITNWLWYIK).In one embodiment, described polypeptide is the fusion rotein of the polypeptide shown in the SEQID NO:1 and TrpLE.Preferably, described polypeptide has the aminoacid sequence shown in the SEQ ID NO:1.
A second aspect of the present invention provides a kind of polypeptide of the HIV of inhibition virus, and described polypeptide contains the aminoacid sequence (M3:EPSCKASRCKSIPPQCHCAN) shown in the SEQ ID NO:2.In one embodiment, described polypeptide is the polypeptide shown in the SEQ ID NO:2 and the fusion rotein of TrpLE.Preferably, described polypeptide has the aminoacid sequence shown in the SEQ ID NO:2.
A third aspect of the present invention provides a kind of combination polypeptide of the HIV of inhibition virus, it is characterized in that, this combination polypeptide comprises any two or three aminoacid sequences shown in SEQ ID NO:l, SEQ ID NO:2 or the SEQ ID NO:3 (CD4M9).Preferably, described combination polypeptide contains the sequence of sequence, SEQ ID NO:2 and SEQ ID NO:3 of sequence, SEQID NO:1 and SEQ ID NO:3 of SEQ ID NO:1 and SEQ ID NO:2 or the sequence of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and its mode of connection can have the various arrangement combination.Better, described mode of connection is shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.
A fourth aspect of the present invention provides a kind of encoding sequence of aforementioned polypeptides.Preferably, described encoding sequence is selected from a kind of in lower group:
(1) nucleotide sequence shown in the SEQ ID NO:4;
(2) nucleotide sequence shown in the SEQ ID NO:7.
A fifth aspect of the present invention provides a kind of encoding sequence of the aforesaid combination polypeptide of encoding.
A sixth aspect of the present invention provides a kind of application of aforementioned polypeptides, i.e. the application of aforementioned polypeptides in the medicine of preparation prevention or treatment HIV virus infection.Better, aforementioned polypeptides is for the preparation of the vaccine of prevention HIV virus infection.
A seventh aspect of the present invention provides a kind of application of aforesaid combination polypeptide, i.e. the application of aforesaid combination polypeptide in the medicine of preparation prevention or treatment HIV virus infection.Preferably, the aforesaid combination polypeptide is for the preparation of the vaccine of prevention HIV virus infection.
A eighth aspect of the present invention provides a kind of carrier, and described carrier contains the sequence of coding aforementioned polypeptides or combination polypeptide.Preferably, described carrier is selected from a kind of among the pTMHa30-51.
A ninth aspect of the present invention provides a kind of genetically engineered host cell, it is characterized in that the host cell that it is transformed or transduce by above-mentioned carrier.
A tenth aspect of the present invention provides a kind of preparation method who prepares the polypeptide that suppresses HIV virus, it is characterized in that, the method comprises:
(a) under the condition of the polypeptide that is fit to expression inhibiting HIV virus, cultivate above-mentioned host cell;
(b) from culture, isolate the polypeptide that suppresses HIV virus.
A eleventh aspect of the present invention provides a kind of preparation method who prepares the combination polypeptide that suppresses HIV virus, it is characterized in that, the method comprises:
(a) under the condition of the combination polypeptide that is fit to expression inhibiting HIV virus, cultivate above-mentioned host cell;
(b) from culture, isolate the combination polypeptide that suppresses HIV virus.
A twelveth aspect of the present invention, provide a kind of can with the antibody of aforementioned polypeptides specific binding.Preferably, described antibody is monoclonal antibody.
A thirteenth aspect of the present invention provides a kind of antibody that can be combined with the aforesaid combination polypeptid specificity.Preferably, described antibody is monoclonal antibody.
A fourteenth aspect of the present invention provides a kind of HIV vaccine, the carrier that described vaccine comprises above-mentioned any one or the polypeptide more than two kinds and pharmaceutically allows.Better, described vaccine contains the aminoacid sequence shown in the aminoacid sequence shown in the aminoacid sequence shown in the aminoacid sequence shown in SEQ ID NO:1 and the SEQ ID NO:2, SEQID NO:1 and the SEQ ID NO:3, SEQID NO:2 and the SEQ ID NO:3 or SEQ ID NO:1, SEQ ID NO:2 or the SEQ ID NO:3.
A fifteenth aspect of the present invention provides a kind of HIV vaccine, the carrier that described vaccine comprises above-mentioned any one or the combination polypeptide more than two kinds and pharmaceutically allows.The mode of connection of described combination polypeptide is a lot, does not enumerate one by one at this.Better, described vaccine contains the aminoacid sequence shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or the SEQ ID NO:16, and perhaps described vaccine contains among the SEQ ID NO:13,14,15 or 16 any two, three kinds even four kinds of combination polypeptide.
Embodiment
This research is mainly started with from the three aspects: that stops HIV-I gp41 to wear film, obtains each crucial polypeptide, by drug cocktail therapy (treatment) the active centre of three or two polypeptide is combined into a combination polypeptide, inquires into effect and the mechanism of action of its treatment HIV-I.This laboratory was take trypsin inhibitor MBI structure as the basis in recent years, designed a kind of peptide inhibitor M3 (SEQ IDNO:2:EPSCKASRCKSIPPQCHCAN), furin enzyme (processing proteases) has been suppressed active Ki value reach 3.26 * 10
-9M, the cell experiment effect that suppresses HIV-I is also better.C end polypeptide with the gp41 core texture compares with the power that the core protein of class gp41 is combined respectively, and the C22 polypeptide for preparing with nearest this laboratory has extraordinary inhibition to HIV-I, through computer simulation, designs serial class C22 polypeptide; For the structural analysis of CD4 and gp120 binding, from CD4M3～CD4M9 series peptide, IC50 becomes 0.1～1.0 μ M of CD4M9 from the 40 μ M of CD4M3, and inhibition is greatly improved.This research is according to the principle of drug cocktail therapy (treatment), utilizing single polypeptide to come the block film fusion is that certain difficulty is arranged, the polypeptide that must use multi-medicament or have a plurality of active centre can use, and perhaps uses the combination of polypeptide of the present invention and other drug, can improve the effect for the treatment of acquired immune deficiency syndrome (AIDS).The above-mentioned three polypeptide active centers of analysis-by-synthesis with these three or two synthetic new active polypeptide of polypeptide active central. set, filter out the polypeptide or the combination polypeptide drugs that can be used for the HIV-I treatment at last.
The inventor finds can greatly simplify the means of production of HIV-I resisting polypeptide making up polypeptide with fusion rotein pTMHa30-51 system expression HIV-I resisting through extensive and deep research, improves the yield of polypeptide.
Polypeptide of the present invention can be recombinant polypeptide or synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the synthetic product of Peptide synthesizer, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).
The sequence of combination polypeptide of the present invention is can be by Peptide synthesizer synthetic or link together by the dna sequence dna of round pcr with single polypeptide.The present invention relates to prepare the method that the present invention makes up polypeptide a series of containing, it comprises step:
A. the structure of expression vector;
B. cultivate host cell, described host cell contains above-mentioned combination expression of polypeptides precursor;
C. separation and purification goes out described combination polypeptide amalgamation protein;
D. obtain described combination polypeptide with chemical cracking;
E. isolate the combination polypeptide.
Among the present invention, the nucleotide sequence of HIV-I resisting combination polypeptide can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell is viral or other carriers.In a word, as long as can copy in host and stablize, any plasmid and carrier can be used.A key character of expression vector is usually to contain replication orgin, promotor, marker gene and translation controlling elements.
Method well-known to those having ordinary skill in the art can be used for make up and contain HIV-I resisting combination peptide coding dna sequence dna and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can be effectively connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor, but eukaryotic promoter comprises CMV early promoter, HSV thymus gland kinase promoter, early stage and late period SV40 promotor and the promotor expressed of some other known controlling gene in protokaryon and eukaryotic cell or its virus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected marker groups, phenotypic character with the host cell that is provided for selecting transforming, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness such as eukaryotic cell, or be used for colibacillary tsiklomitsin or kalamycin resistance.
Comprise above-mentioned suitable dna sequence dna and the suitable carrier of promotor or control sequence, can be used for transforming or transduction to suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, such as bacterial cell; Or the eukaryotic cell such as low, such as yeast cell; Or higher eucaryotic cells, such as mammalian cell.Representative example has: intestinal bacteria, streptomyces; Eukaryotic cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblasts such as CHO, COS cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be processed with the CaCl2 method in exponential growth after date results, and used step is well-known in this area.Another kind method is to use MgCl
2If necessary, transforming also the method for available electroporation carries out.When the host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the coded polypeptide of encoding sequence of the present invention or combination polypeptide.According to used host cell, substratum used in the cultivation can be selected from various conventional mediums.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (such as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular be expressed or be secreted into to recombinant polypeptide in the above methods can in cell or at cytolemma.If necessary, can utilize its physics, chemical separating and purifying protein by various separation methods with other characteristics.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processes, process the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, ultrafiltration, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods with protein precipitant.
In a preference, HIV-I resisting combination polypeptide of the present invention is the formal representation with fusion rotein, and then processes through chemical cracking.With method separated products such as sieve chromatographies, can obtain combination polypeptide of the present invention at last.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, such as Fab ' or (Fab) 2 fragments; Heavy chain of antibody; Light chain of antibody; Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the polypeptide of the present invention of purifying or combination polypeptide can be applied to animal to induce the generation of polyclonal antibody.Similarly, the cell of expression polypeptide of the present invention or combination polypeptide can be used to immune animal and produces antibody.
Monoclonal antibody of the present invention can utilize hybridoma technology to prepare.Each antibody-like of the present invention can utilize polypeptide of the present invention or combination polypeptide, obtains by the routine immunization technology.These polypeptide or combination polypeptide can utilize the recombination method preparation or utilize Peptide synthesizer synthetic.Can or make up polypeptide and come immune animal and produce with the polypeptide of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form antibody of being combined of polypeptide of the present invention or combination polypeptide; The antibody of being combined with the posttranslational modification form (such as the polypeptide of glycosylation or phosphorylation) can come immune animal and obtains with the polypeptide that produces in the eukaryotic cell (for example yeast or insect cell) or combination polypeptide.
The available polypeptide of the present invention of the production of polyclonal antibody or combination polypeptide immune animal, such as rabbit, sheep etc.Multiple adjuvant can be used for strengthening immune response, includes but not limited to freund's adjuvant etc.
Polypeptide of the present invention, combination polypeptide or encoding sequence of the present invention can be used for the medicine for preparing detection, prevent and/or treat the HIV virus infection.Prevent and/or treat the vaccine of HIV virus infection especially for preparation.
The present invention also provides a kind of pharmaceutical composition, and it contains polypeptide of the present invention or combination polypeptide and pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as Tablet and Capsula can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, Tablet and Capsula should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, and every day is 5-90 microgram/kg body weight approximately, and is better, and every day is 24 micrograms/kg body weight approximately.In addition, polypeptide of the present invention also can use with the other treatment agent.Certainly, concrete dosage also should be considered the factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The invention has the advantages that:
(1) the combination polypeptide of the present invention's announcement is the principle according to cocktail therapy HIV-I, start with from three importances of process that the HIV-I film melts, with the synthetic polypeptide in the active centre of these three key points, make this polypeptide exercise the function of drug cocktail therapy (treatment), thereby can be used as effectively HIV-I resisting membrane fusion inhibitor.
(2) polypeptide of the present invention or combination polypeptide can cracking can obtain once the step by the fusion rotein of expressing, or obtains by Peptide synthesizer is synthetic, and the preparation method is easy to operate.
(3) polypeptide of the present invention or combination polypeptide have tiring of higher inhibition HIV virus through appropriate design.
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, used reagent all can buy in market.
The gene of embodiment 1 combination polypeptide is synthetic
With reference to the intestinal bacteria preference codon, chemosynthesis combination peptide C 22 strand oligonucleotide templates (SEQ ID NO:4) (5 ' GAA TTG GAT AAA TGG GCG TCG CTG TGG AAT TGG TTT AAT ATT ACC AAT TGG CTG TGG TAT ATT AAA3 ') and forward primer (SEQ ID NO:5) (5 ' GCG AAG CTT ATG GAA TTG GAT AAA TGG GCG 3 ') and reverse primer (SEQ ID NO:6): 5 ' GCG GGA TCC TTA TTT AAT ATA CCA CAG CCA3 ' obtains the C22 gene with the PCR reaction.C22-M3 (SEQ ID NO:13), C22-CD4M9 (SEQ ID NO:14), M3-CD4M9 (SEQ ID NO:15), C22-CD4M9-M3 (SEQ ID NO:16) all carry out gene with reference to this principle and synthesize, forward primer at each combination polypeptide is introduced restriction enzyme site HindIII, design limit restriction enzyme site Ecol I and terminator codon in reverse primer.M3 strand oligonucleotide template (SEQ ID NO:7) (CTT GGA GCA CTA CGA TCT TCA CTT ACG TTC TCT TCA TAT CGA GGA TTG ACG GTA CGA CGA TTG), forward primer (SEQ ID NO:8) (5 ' GCG AAG CTT ATG CTT GGA GCA CTA CGA TCT TCA CTT 3 ') and reverse primer (SEQ ID NO:9): (5 ' GCG GGA TCC TTA CAA TCG TCG TAC CGT CTT TCC 3 '); CD4M9 strand oligonucleotide template (SEQ ID NO:10) (ACG TTG AAT CGA TCA ACG TTC AAT TCT ACG CTA TCA AAT CCG AAT AAT CAG TTC ACG CGA CCG TCA CTT ACG CGA ACG CCG GGA), forward primer (SEQ ID NO:11) (5 ' GCG AAG CTT ATG ACG TTG AAT CGA TCA ACG TTC AAT 3 ') and reverse primer (SEQ ID NO:12) (5 ' GCG GGA TCC TTA TCC CGG CGT TCG CGT AAG TGA 3 ').
The PCR reaction conditions: (50mM KCl, 10mM Tris-HCl, pH8.3,2.0mM MgCl2,200 μ M dNTPs) add each 50pmol of amplimer in 50 μ l reaction systems.Fully beginning PCR circulation behind the mixing: 95 ℃ of sex change 1 minute, 94 ℃ of sex change 5 minutes, 53 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, and carried out altogether 30 circulations.At last 72 ℃ of insulations 10 minutes.The PCR product is identified through 2% agarose gel electrophoresis, is used for the clone after reclaiming.
Embodiment 2 combination polypeptide plasmid constructions
The PCR fragment that obtains among the embodiment 1 is used respectively restriction enzyme HindIII and BamHI double digestion, be connected with the carrier pTMHa30 (available from Amersham company) of same double digestion, the carrier that connects (50 μ l competent cells need 25ngDNA) transforms in competence e. coli bl21 (DE3), volume should not surpass 5% of competent cell, rotates gently several times mixing content.Ice bath 30 minutes; Pipe is put into 42 ℃ of water-baths, regularly 90 seconds heat-shockeds; Fast pipe is transferred to ice bath 90 seconds, made the cell cooling; Every pipe adds 800 μ lLB substratum, and 37 ℃ of slow shaking 45 minutes make the antibiotics resistance marker gene of bacteria resuscitation and expression plasmid coding; Low-speed centrifugal 2 minutes removes supernatant, stays approximately 100 μ l substratum in Eppendorf tube, resuspended thalline; Spread the bacterium device with glass bacterium liquid is even on the agar plate upper berth; Flat-plate inverted is placed 37 ℃ of constant incubators, bacterium colony can occur after 12-16 hour.The picking positive colony is identified behind the coated plate, and the result shows that plasmid construction is correct.
Embodiment 3: the present invention makes up the expression of polypeptide
The recombinant plasmid that obtains among the embodiment 2 is converted into respectively among the E.coli BL21 (DE3), choose single bacterium colony in 37 ℃ of overnight incubation, transfer in LB (contain kana 10 μ g/mls) in by 1% next day, 37 ℃ are cultured to OD600 and are about at 0.6～0.8 o'clock, adding final concentration is 0.5mmol/L IPTG, continue to cultivate 3～4h, induce target protein to express, centrifugal, collect thalline, after the carrying out ultrasonic bacteria breaking, centrifugal again, cleer and peaceful precipitation in the collection is respectively through SDS-PAGE (separation gel 15%, spacer gel 4.5%) analysis purposes protein expression position.The result shows that expression product is in precipitation.
Embodiment 4: the present invention makes up the purifying of polypeptide
With the thalline 10mM Tris-HCl (pH=8.0) that collects among the embodiment 3,1mM EDTA, twice of 10%sucrose supersound washing, use again 10mMTris-HCl (pH=8.0), 1mMEDTA, the 1%TritonX-100 supersound washing once, inclusion body buffer A (6MGuHCl, 50mM phosphate buffer (pH=8.0), DTT) the Ni post of upper pre-equilibration after the dissolving, be washed till baseline, Buffer C wash-out (6MGuHCl with bufferB (8M Urea, 50mM phosphate buffer (pH=6.3)), Glacial acetic acid, pH=2.0), collect elutriant, the water dialysed overnight.Get albumen, after the freeze-drying, with the dissolving of 70% formic acid, use the CNBr cracking after 3 hours, the water dialysed overnight.Again dissolve with buffer A after the freeze-drying, the Ni post of upper pre-equilibration, and be washed till baseline with buffer A, collect all effluent liquid, with the oxidation of oxidized form Triptide, after the water dialysis, with reversed-phase HPLC separate targets albumen (Agilent of Agilent company 1100 type HPLC), obtain respectively C22-M3, C22-CD4M9, M3-CD4M9 or C22-CD4M9-M3 combination polypeptide.
Embodiment 5: cytotoxicity and HIV-1 that the present invention makes up polypeptide enter the active detection of inhibition
The antiviral activity test
Get 10
5The MT-2 cell envelope is seeded in the 96-porocyte culture plate, and the experiment polypeptide (5,1.67,0.56,0.19,0.021,0mg/ml, every sample do 2 hole parallel tests) that adds continuous 3 times of dilutions was hatched 2 hours.HTLV-IIIB (biomedicine section of University of Maryland provides) with 100 half cytopathogenic effect titres (TCID50) infects every porocyte, collects cell conditioned medium liquid after 5 days, measures the HIV-1P24 Yield of Antigen.
Cell toxicity test
10
5The MT-2 cell is seeded in the 96-porocyte culture plate, adds different dilution experiment polypeptide (1,0.5,0.25,0.125,0.064,0.032,0.016,0mg/ml, every sample do 2 hole parallel tests), cultivates after 5 days, adds MTT reagent, measures the OD value.
The result shows: the LD50 that the combination polypeptide such as C22-M3 (SEQ ID NO:13), C22-CD4M9 (SEQ ID NO:14), M3-CD4M9 (SEQ ID NO:15), C22-CD4M9-M3 (SEQ ID NO:16) suppress HIV-1 all reaches and is better than the 0.11mM level.
Each makes up polypeptide and does not show any toxicity at working concentration.
The preparation of embodiment 6C22-M3 combination polypeptide rabbit source property polyclonal antibody
According to molecular cloning guide (Cold spring harbor laboratory edition, 1989), the C22-M3 combination polypeptide that purifying among the embodiment 4 is obtained mixes rear immunizing rabbit with freund adjuvant, get blood after strengthening three times, make the rabbit source polyclonal antibody antiserum(antisera) of anti-C22-M3 combination polypeptide.
The preparation of embodiment 7 pharmaceutical compositions
Prepare the medicine injection take physiological saline as solvent, the medicine dissolution amount of every injection is 0.6 milligram.Body weight is 50 kilograms patient, injects twice every day, each potion.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Sequence table
＜110〉Tongji University
＜120〉polypeptide of HIV-I resisting, its encoding sequence and uses thereof
<130>061055
<160>16
<170>PatentIn version 3.1
<210>1
<211>22
<212>PRT
<213>C22
<400>1
Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asn Ile Thr Asn
1 5 10 15
Trp Leu Trp Tyr Ile Lys
20
<210>2
<211>21
<212>PRT
<213>M3
<400>2
Glu Pro Ser Asp Ala Arg Ser Glu Cys Lys Arg Ser Ile Ala Pro Asn
1 5 10 15
Cys His Ala Ala Asn
20
<210>3
<211>28
<212>PRT
<213>CD4M9
<400>3
Cys Asn Leu Ala Ser Cys Asn Leu Arg Cys Asp Ser Leu Gly Leu Leu
1 5 10 15
Val Lys Cys Ala Gly Ser Glu Cys Ala Cys Gly Pro
20 25
<210>4
<211>66
<212>DNA
＜213〉C22 strand Nucleotide
<400>4
gaattggata aatgggcgtc gctgtggaat tggtttaata ttaccaattg gctgtggtat 60
attaaa 66
<210>5
<211>30
<212>DNA
＜213〉forward primer
<220>
<221>misc_feature
<222>(1)..(31)
＜223〉artificial sequence
<400>5
gcgaagctta tggaattgga taaatgggcg 30
<210>6
<211>30
<212>DNA
＜213〉reverse primer
<220>
<221>misc_feature
<222>(1)..(30)
＜223〉artificial sequence
<400>6
gcgggatcct tatttaatat accacagcca 30
<210>7
<211>63
<212>DNA
＜213〉M3 strand Nucleotide
<400>7
cttggagcac tacgatcttc acttacgttc tcttcatatc gaggattgac ggtacgacga 60
ttg 63
<210>8
<211>36
<212>DNA
＜213〉forward primer
<400>8
gcgaagctta tgcttggagc actacgatct tcactt 36
<210>9
<211>33
<212>DNA
＜213〉reverse primer
<400>9
gcgggatcct tacaatcgtc gtaccgtctt tcc 33
<210>10
<211>84
<212>DNA
＜213〉CD4M9 strand Nucleotide
<400>10
acgttgaatc gatcaacgtt caattctacg ctatcaaatc cgaataatca gttcacgcga 60
ccgtcactta cgcgaacgcc ggga 84
<210>11
<211>36
<212>DNA
＜213〉forward primer
<220>
<221>misc_feature
<222>(1)..(36)
＜223〉artificial sequence
<400>11
gcgaagctta tgacgttgaa tcgatcaacg ttcaat 36
<210>12
<211>33
<212>DNA
＜213〉reverse primer
<220>
<221>misc_feature
<222>(1)..(33)
＜223〉artificial sequence
<400>12
gcgggatcct tatcccggcg ttcgcgtaag tga 33
<210>13
<211>43
<212>PRT
<213>C22-M3
<400>13
Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asn Ile Thr Asn
1 5 10 15
Trp Leu Trp Tyr Ile Lys Glu Pro Ser Asp Ala Arg Ser Glu Cys Lys
20 25 30
Arg Ser Ile Ala Pro Asn Cys His Ala Ala Asn
35 40
<210>14
<211>50
<212>PRT
<213>C22-CD4M9
<400>14
Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asn Ile Thr Asn
1 5 10 15
Trp Leu Trp Tyr Ile Lys Cys Asn Leu Ala Ser Cys Asn Leu Arg Cys
20 25 30
Asp Ser Leu Gly Leu Leu Val Lys Cys Ala Gly Ser Glu Cys Ala Cys
35 40 45
Gly Pro
50
<210>15
<211>49
<212>PRT
<213>M3-CD4M9
<400>15
Glu Pro Ser Asp Ala Arg Ser Glu Cys Lys Arg Ser Ile Ala Pro Asn
1 5 10 15
Cys His Ala Ala Asn Cys Asn Leu Ala Ser Cys Asn Leu Arg Cys Asp
20 25 30
Ser Leu Gly Leu Leu Val Lys Cys Ala Gly Ser Glu Cys Ala Cys Gly
35 40 45
Pro
<210>16
<211>71
<212>PRT
<213>C22-CD4M9-M3
<400>16
Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asn Ile Thr Asn
1 5 10 15
Trp Leu Trp Tyr Ile Lys Cys Asn Leu Ala Ser Cys Asn Leu Arg Cys
20 25 30
Asp Ser Leu Gly Leu Leu Val Lys Cys Ala Gly Ser Glu Cys Ala Cys
35 40 45
Gly Pro Glu Pro Ser Asp Ala Arg Ser Glu Cys Lys Arg Ser Ile Ala
50 55 60
Pro Asn Cys His Ala Ala Asn
65 70
Claims (8)
1. a combination polypeptide is characterized in that, the aminoacid sequence of this combination polypeptide is SEQ ID NO:15.
2. nucleic acid molecule, its combination polypeptide claimed in claim 1 of encoding.
3. the application of combination polypeptide claimed in claim 1 in the medicine of preparation prevention or treatment HIV virus infection.
4. a carrier is characterized in that, it contains nucleic acid molecule claimed in claim 2.
5. a genetically engineered host cell is characterized in that, the host cell that it is transformed or transduce by carrier claimed in claim 4.
6. a preparation method who prepares the combination polypeptide that suppresses HIV virus is characterized in that, the method comprises:
(a) under the condition of the polypeptide that is fit to expression inhibiting HIV virus or combination polypeptide, cultivate host cell claimed in claim 5;
(b) from culture, isolate the combination polypeptide that suppresses HIV virus.
7. antibody that energy is combined with combination polypeptid specificity claimed in claim 1.
8. a HIV vaccine is characterized in that, the carrier that described vaccine comprises combination polypeptide claimed in claim 1 and pharmaceutically allows.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010142581 CN101899100B (en) | 2007-02-02 | 2007-02-02 | Anti-HIV-I (Human Immunodeficiency Virus-I) polypeptides as well as coding sequences and applications thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201010142581 CN101899100B (en) | 2007-02-02 | 2007-02-02 | Anti-HIV-I (Human Immunodeficiency Virus-I) polypeptides as well as coding sequences and applications thereof |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200710037104XA Division CN101235083B (en) | 2007-02-02 | 2007-02-02 | HIV-I resisting polypeptide, coded sequence and use thereof |
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| CN101899100B true CN101899100B (en) | 2013-04-03 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1561229A (en) * | 2001-08-21 | 2005-01-05 | 马里兰大学生物技术研究所 | Virus coat protein/receptor chimeras and methods of use |
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2007
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1561229A (en) * | 2001-08-21 | 2005-01-05 | 马里兰大学生物技术研究所 | Virus coat protein/receptor chimeras and methods of use |
Non-Patent Citations (10)
| Title |
|---|
| Claudio Vita等.Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein.《PNAS》.1999,第96卷(第23期),13091-13096. |
| HIV-1进入细胞机制及进入抑制剂的研究进展;吴钦梅;《中国病原生物学杂志》;20060630;第1卷(第3期);229-231 * |
| HIV融合抑制剂的研究进展;史卫国等;《中国新药杂志》;20061231;第15卷(第17期);1429-1435 * |
| Hu Tao等.Template-assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor.《FEBS Journal》.2006,第273卷3907-3914. |
| Michael J. Root等.Protein Design of an HIV-1 Entry Inhibitor.《SCIENCE》.2001,第291卷884-888. |
| Protein Design of an HIV-1 Entry Inhibitor;Michael J. Root等;《SCIENCE》;20010202;第291卷;884-888 * |
| Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein;Claudio Vita等;《PNAS》;19991109;第96卷(第23期);13091-13096 * |
| Template-assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor;Hu Tao等;《FEBS Journal》;20061231;第273卷;3907-3914 * |
| 史卫国等.HIV融合抑制剂的研究进展.《中国新药杂志》.2006,第15卷(第17期),1429-1435. |
| 吴钦梅.HIV-1进入细胞机制及进入抑制剂的研究进展.《中国病原生物学杂志》.2006,第1卷(第3期),229-231. |
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| Publication number | Publication date |
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| CN101899100A (en) | 2010-12-01 |
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