CN102516370A - Anti-HIV (human immunodeficiency virus) medicine target and use thereof in pharmacy - Google Patents

Abstract

The invention discloses an anti-HIV (human immunodeficiency virus) medicine target and a use thereof in pharmacy, wherein the medicine target aims at the mutual binding site between the Vif protein of a virus and the CBFbeta protein of a host cell; and the binding site comprises the Helix4 and Loop3 of the CBFbeta, as well as the former 98 amino acids, the 144th, 145th and 146th sites of (SLQ) amino acids at the N-terminal of the Vif. A potential novel HIV viral inhibitor is designed and selected by using the target. A monoclonal antibody and a polyclonal antibody using the medicine target and a preparation method for the same are provided. The medicine target is used for therapy for HIV, enables overcoming for the medicine resistance of therapy for HIV to be possible, provides new direction for therapy for HIV, and has great significance.

Description

The drug target of anti-HIV and the purposes in pharmacy thereof

Technical field

The invention discloses the drug target of a kind of anti-HIV, the purposes of this drug target in pharmacy also is provided simultaneously, belong to biological pharmacy technical field.

Background technology

The medicine of the present clinical listing of medicine of treatment AIDS can be divided into following several types according to drug target: absorption fusion, reversed transcriptive enzyme, intergrase, the proteolytic enzyme of virus has become screening and has prepared the main target spot of anti-AIDS medicine, and nearly development and clinical treatment during the last ten years mainly carries out to these several sites.Regrettably; Though these medicines can inhibitory phase be answered the activity of viral protein, have all in clinical treatment that all unfavorable factors are lasting like: medication cycle, occur resistance behind the life-time service, toxic side effect, sufferer are difficult to bear the pain, viral taskworkization (reactivation), medicine high price after the drug withdrawal are expensive, detection technique and equipment are limited etc.These problems make normally used anti-AIDS drug in therapeutic process, can receive restriction in various degree, and can't use widely.

For many defectives of HIV medicine, resistance is one of subject matter, and promptly virus amino acid mutation can take place repeatedly and produces crossing drug resistant and causes antiviral therapy failure.1996, the scientific research personnel proposed the strategy of " HAART " (HAART, efficient anti-reverse transcription therapy) to this, promptly adopt the mode of three kinds of drug combinations to treat AIDS.This method can suppress a plurality of links of HIV reproduction process simultaneously, reduces resistance to greatest extent, reduces the M & M that AIDS takes place HIV the infected, has improved patient's prognosis quality of life largely.Though find behind the clinical treatment of several years this method also come with some shortcomings as: the interaction between the medicine, toxic addition, produce the HIV cross-resistance, dosage is big and complicated, medicine is invalid etc. to the HIV that hides; But the most effective so far beyond doubt AIDS treatment means of HAART method, so Many researchers has taken up to solve the limitation that exists in its use.With long-range strategic eye; Best solution route is exactly thoroughly to understand HIV-1 virusology characteristic and immune mechanism; Quicken the development and the exploitation of newtype drug and target, increase the prescription of HAART, this also is the main direction and the means of following treating AIDS research.

APOBEC3G albumen is the natural anti-virus factor in the up-to-date found human body cell, but its antiviral function is but by HIV-1 Vif albumen institute antagonism.Vif can form the ECS mixture with ubiquitin protein ligase function with multiple protein in the host cell, causes the ubiquitinization of APOBEC3G also finally to be discerned, degrade by body endoproteinase body, thereby can't bring into play antiviral function.Inquire into the proteic mechanism of action of Vif and have important Research Significance for the design anti-AIDS drug.

Summary of the invention

The invention discloses the drug target of a kind of anti-HIV, utilize the medicine of the treatment HIV that this target spot designing and screening makes new advances.

The present invention also provides the purposes of this drug target in pharmacy, the monoclonal antibody of this drug target and polyclonal antibody and preparation method thereof and potential new virus suppressor factor.

Technical solution of the present invention is following:

Helix4 and the Loop3 of CBF β have been deleted through the molecular cloning means; And the N of Vif end; Or the amino acid sites of the 145th, 146 and 147 of the 21st of the Vif that suddenlyd change or Vif the 38th and Vif, detect CBF β and the Vif binding ability obviously descends through the co-immunoprecipitation method.

The another kind of optimal way of above-mentioned HIV virus drugs target spot does; Helix4 and the Loop3 of CBF β have been deleted through the molecular cloning means; And the N of Vif end; Or the amino acid sites of suddenlyd change the 145th, 146 and 147 of Vif the 21st amino acids or the 38th of Vif and Vif, the ability drop of HIV-1Vif degraded APOBEC3G.

HIV drug target provided by the invention is characterized in that:

This drug target is to the Vif albumen of virus and the site that mutually combines between the host cell CBF β albumen; This binding site comprises Helix4 and the Loop3 of CBF β, and the N of Vif end.

Described HIV virus drugs target spot, preferred sites are Vif the 21st amino acids or Vif the 38th amino acids.

The present invention provides the polyclonal antibody of a kind of anti-HIV-1 virus, it is characterized in that it being that to hold the polypeptide in preceding 98 amino acid scopes with Vif N be the polyclonal antibody of epitope, has the HIV-1 Vif of inhibition and CBF β bonded ability.

The Polyclonal Antibody Preparation method of said anti-HIV-1 virus, step comprises:

1) polypeptide or the sequence of sequence in Vif N holds preceding 98 amino acid scopes being provided is the polypeptide of epitope with the SLQ site;

2) with polypeptide and carrier protein couplet, and with the adjuvant immune animal;

3) get the antiserum(antisera) of animal,, promptly get said polyclonal antibody through antigen link coupled affinity chromatography column purification.

These above-mentioned Antibody Preparation steps are not unalterable, and those of ordinary skill in the art can reasonably revise according to practical situation and make it to meet application requiring.Such as whether adopting and carrier protein couplet, whether use affinity chromatography method purifying or the like.

The present invention provides the monoclonal antibody of a kind of anti-HIV-1 virus, it is characterized in that it being that to hold the polypeptide in preceding 97 amino acid scopes with Vif N be the monoclonal antibody of epitope, has the HIV-1 Vif of inhibition and CBF β bonded ability.

The MONOCLONAL ANTIBODIES SPECIFIC FOR method of said anti-HIV-1 virus, step comprises:

1) polypeptide or the sequence of sequence in Vif N holds preceding 98 amino acid scopes being provided is the polypeptide of epitope with the SLQ site;

2) with polypeptide and carrier protein couplet, and with the adjuvant immune mouse;

3) separating mouse boosting cell and SP2/0 myeloma cell strain merges;

4) add mouse peritoneal feeder cell and fused cell and cultivate altogether, and screen positive strain;

5) cloning screening can obtain cell strain of monoclonal antibody.

Equally, these steps are not unalterable yet, and those of ordinary skill in the art can reasonably revise according to practical situation and make it to meet application requiring.Such as whether using adjuvant, and select different myeloma cell strain or the like.

A kind of HIV viral inhibitors of the present invention, this viral inhibitors can effectively stop Vif-CBF β to mutually combine, thereby suppress the HIV virus replication.

The designing and screening method of a kind of HIV viral inhibitors of the present invention may further comprise the steps:

1) bonding interface through information biology means simulations Vif and CBF β is set up the model of bonding interface according to analog information, and with this model be restricted condition from MDDR, NCI screens the small molecules that obtains meeting model in the DBs such as ACD.Dock and manual screening through DOCK at last, according to binding pattern, reasonable structure, availability obtains final candidate molecules and carries out the biology screening.

2) the biology screening comprises following several respects:

A) co-immunoprecipitation detects the interference performance that candidate molecules mutually combines to Vif-CBF β;

B) Magi method validation medicine suppresses the ability of HIV;

C) mtt assay detection of drugs toxicity;

D) obtain the anti HIV-1 virus suppressor factor that can effectively stop Vif-CBF β to mutually combine.

According to drug mechanism is theoretical derivation, and HIV-1 Vif and CBF β binding site are that the inverase of target spot can be micromolecular compound, Chinese medicine, plant amedica extract, efficient part or effective constituent, nucleic acid class such as RNAi, polypeptide class or protein drug.

Positively effect of the present invention is: the drug target that discloses a kind of anti-HIV.Utilize this target spot designing and screening novel HIV viral inhibitors of potential that made new advances.The monoclonal antibody of utilizing this drug target and polyclonal antibody and preparation method thereof are provided.Be used for the treatment of HIV, make the resistance that overcomes HIV treatment become possibility,, be significant for the treatment of HIV provides new direction.

Description of drawings

Fig. 1 proves that for the FRET experiment Vif and CBF β are interactional through direct combination;

Fig. 2 needs CBF β for the degraded of Vif mediation APOBECEG;

Fig. 3 is the discriminating on the CBF β and the important site of Vif bonded;

Fig. 4 is the discriminating on the Vif and the important site of CBF β bonded;

Fig. 5 is drug target potential new small molecule viral inhibitors screening figure;

Fig. 6 is that the binding ability of Vif L145A two mutants and CBF β detects figure.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are used to explain that youngster of the present invention is not used in restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these surnames of equal value fall within the application's appended claims institute restricted portion equally.

The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

Confirm with the important site of Vif bonded on the embodiment 1:CBF β

FRET experiment proof Vif and CBF β marriage relation

Make up YFP-Vif and CFP-CBF β expression vector, above-mentioned expression vector cotransfection is gone into the 293T cell with Lipofectamine2000.Transfectional cell is observed through Zeiss LSM510-Meta confocal imaging system after 37 degree are cultivated 24-36h, and uses Zen2009 software that imaging is analyzed and revised.Utilizing acceptor optical quenching technology to carry out fluorescence resonance energy migration (FRET) analyzes: the YFP-Vif fusion rotein is carried out optical quenching 5s, subsequently the cancellation level of donor is measured, and with cancellation before compare.FRET is efficient through using grand calculating of FRET in the LSM imaging software.The result shows that YFP-Vif and CFP-CBF β are interactional through direct bonded mode.(Fig. 1)

According to CBF β crystalline structure, make up 6 two mutants of CBF β, be respectively: Δ 1 (2-14), Δ 2 (2-30), Δ 3 (2-50), Δ 4 (59-68), Δ 5 (69-90), Δ 6 (129-140).

In the 293T cell respectively with the above-mentioned 6 kinds of CBF β mutation expression carriers of the common transfection of Vif-HA.Collect cell behind the 48h, carry out cracking.Adopt the co-immunoprecipitation method, detect the binding ability of different mutants and Vif-HA.Experimental result shows that Δ 5 (69-90) has lost binding ability with Vif basically, explains that this zone is extremely important to the two combination.

Binding site comprises Helix4 and the Loop3 (referring to Fig. 3) of CBF β, among the figure, and (a) CBF β two mutants collection of illustrative plates; (b), (c) the co-immunoprecipitation method detects the binding ability of Vif albumen and CBF β two mutants; (d) CBF beta structure synoptic diagram; To the Vif structure, make up the N end deletion mutant of Vif, preferred sites is the 21st of Vif or Vif the 38th amino acids site (referring to Fig. 4), and the sudden change (referring to Fig. 6) of Vif the 145th amino acids (among the SLQ a bit).Detect through co-immunoprecipitation among Fig. 4, (a) the important site of Vif Δ N collection of illustrative plates and Vif indicates; (b) Vif N end deletion mutant (Vif Δ N) obviously descends with the binding ability of CBF β; (c) binding ability of VifW21A or VifW38A two mutants and CBF β obviously descends.The binding ability of Vif L145A two mutants and CBF β obviously descends among Fig. 6.

Embodiment 2:The degraded of the APOBEC3G of Vif mediation needs CBF β

Utilize the RNA perturbation technique to knock out the endogenous CBF β in the 293T cell, and set up stable expression cell line through screening.Cotransfection Vif and APOBEC3G expression plasmid in the 293T cell of disappearance CBF β, and be the experiment contrast group with APOBEC3G corotation VR1012.After the transfection 48 hours, collect cell, cracking is carried out western and is detected.The experiment proof has or not the not influence of amount of APOBEC3G in the Vif pair cell under the situation that does not have CBF β to exist.Referring to Fig. 2: a) in the cell CBF β to the influence of the APOBEC3G degraded of Vif mediation; B) CBF β gets into the influence of virus to the APOBEC3G packing.

Embodiment 3

The monoclonal antibody of anti-HIV-1 virus is characterized in that it being that to hold the polypeptide in preceding 98 amino acid scopes with Vif N be the monoclonal antibody of epitope, has the HIV-1 Vif of inhibition and CBF β bonded ability.

The MONOCLONAL ANTIBODIES SPECIFIC FOR method of said anti-HIV-1 virus, step comprises:

1) antigenic selection and acquisition: the polypeptide of sequence in Vif N holds preceding 98 amino acid scopes is provided; Carry out the comparison of antigen immune originality, select the strong and water-soluble small peptide sequence preferably of immunogenicity as antigen.The corresponding sequence of adjusting of the little peptide of selecting is added the His label, be built among the prokaryotic expression carrier pET28a.The expression vector of this little peptide is transformed in the e. coli bl21 expresses, the soluble proteins supernatant is carried out purifying with the nickel post.Measure the little peptide concentration that obtains behind the purifying, and with His antibody little peptide is carried out western and detect.Western detects positive, prepares immune mouse.

2) immunity of mouse: purebred BALB/C mice, mouse 6～8 weeks of age.Initial immunity, subcutaneous multi-point injection, immunogen dosage 50ug/, injection volume is 800ul, Fu Shi Freund's complete adjuvant and immunogen solution volume ratio are 1:1.After three weeks, inject the antigen of same amount, select freund 's incomplete adjuvant for use with the abdominal injection mode.After 2～3 weeks, booster immunization adopts muscle injection mode, and the injections of antigens amount is 200ug/, selects freund 's incomplete adjuvant for use.Mouse in the immunologic process is cut tail get blood, detect antibody expression situation in the mice serum, thereby judge the immune effect of mouse with western.

3) merge with the SP2/0 myeloma cell strain: the mouse that immunity is good is put to death, extracting spleen cell.Myeloma cell SP2/0 and mouse boosting cell are mixed with 1:10, adopt PEG4000 to merge as fusogen.

4) cell after the fusion is inoculated in 96 orifice plates with the mode of limiting dilution, in the HAT substratum, screens.

5) collect cell conditioned medium, western detects the ability of different monoclonal cell crowd secretory antibodies.

Embodiment 4:A kind of polyclonal antibody of anti-HIV-1 virus

Holding the polypeptide in preceding 98 amino acid scopes with Vif N is the polyclonal antibody of epitope, has the HIV-1 Vif of inhibition and CBF β bonded ability.

The Polyclonal Antibody Preparation method of said anti-HIV-1 virus:

1) antigenic selection and procurement process are consistent with embodiment 3.

2) immune animal: select two of new zealand rabbits, each immune 200ug immunogen, the initial stage is used the Fu Shi Freund's complete adjuvant, and the later stage is used Freund.Respectively the 0th, 2,4,5,6,7,8,9 weeks were carried out immunity, and since the 5th week, vein is got blood and is followed the tracks of and detect immune effect.

3) get the antiserum(antisera) of animal,, promptly get said polyclonal antibody through antigen link coupled affinity chromatography column purification.

Embodiment 5:The potential new virus inhibitor screening of this drug target.

HIV-1 expression vector transient transfection is gone in the 293T cell, after 24 hours, cell is got off with trysinization, divide equally in 12 orifice plates, 1ml substratum about 1.0 * 10 is contained in every hole ⁵Individual cell adds medicine, and making drug effect concentration is 10uM/ml.Behind the drug effect 24h, collect viral supernatant and be used to do the Magi infection experiment; Collect the cytotoxicity of cell western detection intracellular virus protein expression situation and medicine and (, can judge protein expression influence in the medicine pair cell according to p55 among the figure referring to Fig. 5 b; Actin tentatively judges the toxicity of medicine pair cell).

The Magi infection experiment: 24h before infection, the MAGI-CCR5 cell suspension in the D-10 substratum, is cultivated on 6 porocyte culture plates, make cell density when infecting, reach 30-40%.Before the infection; Take out the nutrient solution in the hole, the dilution virus of difference is added 500 μ l contain in the DMEM substratum of final concentration 20 μ g/ml DEAE-dextran, add in the hole after the mixing; At 37 ℃; Cultivate after two hours in the 5% CO2 incubator, add the normal D-10 substratum of 2ml, similarity condition is cultivated 48h down.The sucking-off culture supernatant, the every hole of culture plate adds the stationary liquid of 1ml, and room temperature held 5 minutes discards stationary liquid; With PBS washing 2 times, add 0.6ml colour developing liquid, be placed on 16-20h in 37 ℃ of no CO2 incubators; Microscopically is observed blue spot, if high-visible, the color development stopping reaction; Outwell colour developing liquid,, finally add distilled water with the thorough rinsing of PBS 2 times; In microscopically numeration, calculate medicine to the inhibiting rate of virus (referring to Fig. 5 a, column diagram representes to add the relative infection ability of virus behind the different pharmaceutical).

Claims (9)

1. a HIV virus drugs target spot is characterized in that, this drug target is to the Vif albumen of virus and the site that mutually combines between the host cell CBF β albumen; This binding site comprises Helix4 and the Loop3 of CBF β, and preceding 98 amino acid and the 144th, 145,146 (SLQ) amino acid of the N of Vif end.

2. HIV virus drugs target spot according to claim 1 is characterized in that: preferred sites is Vif the 21st amino acids or the 38th amino acids.

3. the polyclonal antibody of anti-HIV-1 virus is characterized in that it being to hold the polypeptide in preceding 98 amino acid scopes or be the polyclonal antibody of epitope to the SLQ site with Vif N, has the HIV-1 Vif of inhibition and CBF β bonded ability.

4. the Polyclonal Antibody Preparation method of the said anti-HIV-1 of claim 3 virus, step comprises:

1) polypeptide or the sequence of sequence in Vif N holds preceding 98 amino acid scopes being provided is the polypeptide of epitope with the SLQ site;

2) with polypeptide and carrier protein couplet, and with the adjuvant immune animal;

3) get the antiserum(antisera) of animal,, promptly get said polyclonal antibody through antigen link coupled affinity chromatography column purification.

5. the monoclonal antibody of anti-HIV-1 virus is characterized in that it being to hold the polypeptide in preceding 98 amino acid scopes or be the monoclonal antibody of epitope to the SLQ site with Vif N, has the HIV-1 Vif of inhibition and CBF β bonded ability.

6. the MONOCLONAL ANTIBODIES SPECIFIC FOR method of the said anti-HIV-1 of claim 5 virus, step comprises:

1) polypeptide or the sequence of sequence in Vif N holds preceding 98 amino acid scopes being provided is the polypeptide of epitope with the SLQ site;

2) with polypeptide and carrier protein couplet, and with the adjuvant immune mouse;

3) separating mouse boosting cell and SP2/0 myeloma cell strain merges;

4) add mouse peritoneal feeder cell and fused cell and cultivate altogether, and screen positive strain;

5) cloning screening can obtain cell strain of monoclonal antibody.

7. HIV viral inhibitors, this viral inhibitors can effectively stop Vif-CBF β to mutually combine, thereby suppresses the HIV virus replication.

8. the screening method of the said HIV viral inhibitors of claim 7 may further comprise the steps:

1) bonding interface through information biology means simulations Vif and CBF β is set up the model of bonding interface according to analog information, and with this model be restricted condition from MDDR, NCI screens the small molecules that obtains meeting model in the DBs such as ACD; Dock and manual screening through DOCK at last, according to binding pattern, reasonable structure, availability obtains final candidate molecules and carries out the biology screening;

2) the biology screening comprises following several respects:

A) co-immunoprecipitation detects the interference performance that candidate molecules mutually combines to Vif-CBF β;

B) Magi method validation medicine suppresses the ability of HIV;

C) mtt assay detection of drugs toxicity;

D) obtain the anti HIV-1 virus suppressor factor that can effectively stop Vif-CBF β to mutually combine.

9. claim 1 or 2 described HIV-1 Vif and CBF β binding site are that the inverase of target spot can be micromolecular compound, Chinese medicine, plant amedica extract, efficient part or effective constituent, nucleic acid class such as RNAi, polypeptide class or protein drug.

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