CN1056383C - Chinese trichosanthes protein, preparation method and use in pharmacy - Google Patents
Chinese trichosanthes protein, preparation method and use in pharmacy Download PDFInfo
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- CN1056383C CN1056383C CN97115693A CN97115693A CN1056383C CN 1056383 C CN1056383 C CN 1056383C CN 97115693 A CN97115693 A CN 97115693A CN 97115693 A CN97115693 A CN 97115693A CN 1056383 C CN1056383 C CN 1056383C
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Abstract
The present invention relates to new ribosome inactivating protein of Chinese trichosanthes protein for inhibiting HIV activity and simultaneously provides a preparation method thereof. Both the crude product and the pure product of the Chinese trichosanthes protein can be used for remarkably inhibiting the formation of HIV-1 induced C8166 plasmodium. For acute infection, the Chinese trichosanthes protein remarkably inhibits the expression of an HIV-IP24 antigen. When the concentration of a compound is 0.04 mug/ml, the inhibition ratio of 94P73CMI and the inhibition ratio of 94P75CMI to P24 antigen expression are respectively 54.8% and 52.6% (namely EC50 is less than 0.04 mug/ml). HIV-1 antigen positive cells are reduced by 47.0%. For chronic infection, the Chinese trichosanthes protein has no influence on the duplication of HIV-1IIIB. Even if under the condition of toxicity concentration, the level of the P24 antigen expression can not be changed a lot.
Description
The invention belongs to the anti-AIDS drug field, relate in particular to TRICHOBITACIN and control application in the medicine of acquired immune deficiency syndrome (AIDS) in preparation.
Acquired immune deficiency syndrome (AIDS) (AIDS) is the deadly disease of the destruction body immune system that caused by human immunodeficiency virus (HIV).Owing to still there is not preventible effective vaccine at present, anti-AIDS medicine becomes the main means of control AIDS.The anti-AIDS medicine of current clinical usefulness is chemical synthetic drug, and often toxic side effect is big, easily produces shortcomings such as resistance.Seeking the new anti-AIDS medicine or the research of lead compound from conventional medicament and natural resource, is important research aspect and very active field in the current domestic and international new drug development.At least found that from natural resource tens kinds of compounds have the activity of anti-HIV.In addition, to being that (Ribosomeinactivating proteins, the active research of anti-AIDS RIP) has become the emphasis of research at present both at home and abroad for the ribosome inactivating protein of representative with the Trichosanthin.RIP is that a class acts on the eukaryotic cell rrna, arrestin matter synthetic toxalbumin, and (the Trichosanthin of representative Trichosanthin wherein, TCS) be a kind of alkaline single chain protein of forming by 247 amino acid that from cucurbitaceous plant snakegourd (Trichosanthes kirilowii) napiform root, extracts, molecular weight is 27KDa, pI is 9.4, does not contain halfcystine and glycosyl [Chow et al, 1990; Collin et al, 1990].TCS has RNA N-glycosidase activity [Zhang and Liu, 1992].TCS is one of new drug few in number of China oneself initiative.
The objective of the invention is based on above-mentioned prior art basis, a kind of new active ribosome inactivating protein-TRICHOBITACIN (Trichobitacin) with inhibition HIV is provided, its preparation method is provided simultaneously.
In order to realize purpose of the present invention, the invention provides following technical scheme:
TRICHOBITACIN, pressed the residue behind the juice for a kind of piece root and to have separated the strand ribosome inactivating protein that obtains from the snakegourd plant, molecular weight 27,228Da, iso-electric point 9.6, sugar content 0.7-0.9%, have RNA N-glycosidase activity, N-end and C-terminal amino acid are Aspartic Acid and L-Ala, and N-holds 38 amino acid whose sequences, except that 31 be the Isoleucine, the same with Trichosanthin.
The present invention also provides the method for preparing above-mentioned TRICHOBITACIN, gets snakegourd piece root, pressed residue behind the juice to soak with physiological saline after, use acetone precipitation, after the collecting precipitation lyophilize,, get the peak I that column chromatography for separation goes out and get final product with Sephadex G-50 or G-75 and CM-Sephadex C-50 purifying.Use high-efficient liquid phase chromatogram HPLC then, methods such as SDS-gel electrophoresis SDS-PAGE and HPCE HPCE are identified its purity, with the electronics place revolve-mass spectrum ES-MS measures its molecular weight, isoelectric focusing electrophoresis is measured iso-electric point, measures partial amino-acid series with automatic sequencer behind the enzymolysis.
The present invention also provides TRICHOBITACIN to control the application of acquired immune deficiency syndrome (AIDS) medicine as preparation.
The pharmacological results with TRICHOBITACIN of the present invention illustrates drug action of the present invention and beneficial effect below:
1, TRICHOBITACIN induces the C8166 cell to form plasmodial restraining effect to HIV-1:
On the flat Tissue Culture Plate in 96 holes, testing compound is carried out 5 times of doubling dilutions with perfect medium, totally seven extent of dilution, every hole 100 μ l establish 3 repeating holes, and the normal cell contrast is set simultaneously, the contrast of HIV cells infected.Every hole drips 3 * 10
5The C8166 cell 100 μ l of/ml, every then hole adds the HIV-1 IIIB supernatant of 200 TCID50.Be that infection multiplicity is 0.007, the cumulative volume in every hole is 300 μ l.Put 37 ℃, 5%CO
2Cultivate in the incubator.Infect the back and observed the influence that testing compound forms synplasm on the 3rd day.Under 100 times inverted microscope, choose 5 nonoverlapping visuals field, counting HIV-1 IIIB induces the plastidogenetic synplasm number of C8166.EC50 reaches 50% o'clock drug level for suppressing synplasm formation.CC50 is the drug level when 50% host cell is produced cytotoxicity, and selectivity index (SI) is the ratio of CC50/EC50.Table 1 TRICHOBITACIN suppresses HIV-1 IIIB and induces C8166 to form synplasm and to the cytotoxic effect of C8166
| Compound | EC50 (μg/ml) | CC50 (μg/ml) | SI |
| Chinese trichosanthes protein crude product (93P62 salt precipitation) Chinese trichosanthes protein crude product (XBP94P23) Chinese trichosanthes protein (XBP94P27) Chinese trichosanthes protein (93P10CM-I) Chinese trichosanthes protein (93P18CM-I) Chinese trichosanthes protein (94P70CMI) Chinese trichosanthes protein (94P70CMII) Chinese trichosanthes protein (94P73CMI) Chinese trichosanthes protein (94P75CMI) | 0.01 0.0052 0.014 0.0118 0.0045 0.023 0.00273 0.005 0.001 | 1.24 0.55 2.1 3.39 4.23 1.71 0.84 2.0 0.6 | 124 105.8 150 287.3 940.0 74.4 307.7 400 600 |
As seen from Table 1, the purifying TRICHOBITACIN of TRICHOBITACIN crude product and different lot numbers all has remarkable vitro inhibition HIV-1 IIIB to induce the C8166 cell to form plasmodial effect.
2, TRICHOBITACIN is to the blocking effect of HIV cells infected fusion
Press (1987) such as Walker; The described method improvement of Johnson and Waker (1990) carries out.On 96 porocyte culture plates, with testing compound by 5 times of doubling dilutions, totally 4 extent of dilution, each extent of dilution is established 3 repeating holes, every hole 100 μ l.The control wells that does not contain testing compound is set simultaneously.Every hole drips 6 * 10
5The logarithmic phase C8166 cell 50 μ l and 2 * 10 of/ml
5The chronically infected H9 cell 50 μ l of the HIV-1 IIIB of/ml, promptly HIV-1 IIIB cells infected is 1: 3 with the ratio of the C8116 cell count that does not infect.Put 37 ℃, cultivate in the 5%CO2 incubator after 24 hours, under inverted microscope, merge the synplasm quantity that forms and infer the whether cohesive process of blocking virus and cell of compound by observation of cell."-" expression compound is not blocked the formation of cytogamy; "+" expression compound has been blocked the formation of cytogamy fully.
Table 2 TRICHOBITACIN is to HIV-1 IIIB and the influence of cell bonded
| Compound | Concentration (μ g/ml) | Merge and suppress |
| TRICHOBITACIN (93P10CMI) TRICHOBITACIN (93P18CMI) TRICHOBITACIN (94P70CMI) TRICHOBITACIN (94P73CMI) TRICHOBITACIN (94P75CNI) | 13.2 13.8 6.0 4.35 5.85 | - - - - - |
After the CD4 receptors bind on the HIV-1 on cells infected surface and non-infected cells surface, can cause cytogamy to form synplasm.The medicine that acts on this target spot will be blocked the generation of merging.As seen from Table 2, TRICHOBITACIN all can not be blocked the fusion of cells infected.
3, TRICHOBITACIN is to the influence of virus replication in the HIV-1 acute infection cell
Improve slightly with reference to (1987) methods such as (1990) such as Lee-Huang and Walker.
Testing compound is diluted with perfect medium, establish 4 extent of dilution, each extent of dilution three hole, every hole 100 μ l.The control wells that does not contain compound is set simultaneously.Collect logarithmic phase C8166 cell suspension 50 μ l, promptly every hole contains 5 * 10
4Individual cell.Put 37 ℃, 5%CO
2Cultivate in the incubator, make compound and cell preact 60 minutes.Every hole adds the HIV-1 IIIB 50 μ l that contain 200 TCID50.Be that MOI is 0.004.Put 37 ℃, 5%CO
2Cultivated 72 hours in the incubator.The blue dye exclusion method counting of platform phenol with 0.8%, the calculating survivaling cell number.The collecting cell suspension, TGL-16C desk centrifuge 3000rpm, centrifugal 5 minutes.Collect culture supernatant and be used for ELISA mensuration P24 antigen levels.Reset and add 5%Triton X-100 solution 50 μ l deactivation and lytic viruses on per 450 μ l.Put then～20 ℃ of preservations, standby.With 50 μ l substratum re-suspended cell throw outs, cell suspension is added drop-wise in the hole of 8 hole slides, after the drying, fix 15 minutes with cold acetone, put-20 ℃ of preservations then.Be used for indirect IF staining and detect the HIV positive cell.
The effect that table 3 TRICHOBITACIN is duplicated acute infection HIV
| Compound | Concentration (μ g/ml) | Cell survivaling number (per-cent of experimental and control) | HIV antigen-positive cell decrement (%) | P24 antigen presentation inhibiting rate (%) |
| TRICHOBITACIN (94P73CMI) (lot number 1) | 5.0 1.0 0.2 0.04 | 41.9 81.4 102.3 99.7 | 73.8 64.9 53.0 47.0 | 86.3 81.3 58.2 54.8 |
| TRICHOBITACIN (94P75CMI) | 5.0 1.0 0.2 0.04 | 51.2 74.4 76.7 79.1 | 92.6 58.9 59.4 47.0 | 84.1 80.1 61.7 52.6 |
Infect in the acute infection system of C8166 at HIV-1 IIIB, at first make compound pre-treatment C8166 cell 90 minutes, infect with HIV-1 IIIB then.Judge the influence that ribosome inactivating protein duplicates HIV by the variation that detects HIV antigen-positive cell percentage and HIV P24 antigenic expression.As seen from Table 3, TRICHOBITACIN all can suppress HIV-1 IIIB duplicating in acute infection C8166 cell significantly.The antigenic expression of HIV antigen-positive cell and P24 has certain dependency.4, TRICHOBITACIN to HIV-1 chronic infection cell expressing P24 antigenic influence carry out with reference to (1992) methods such as Kinchington.
Testing compound is carried out doubling dilution with perfect medium on 96 hole microtest plates, each compound is established 4 extent of dilution, and each extent of dilution is established three holes, every hole 100 μ l.The control wells that does not contain compound is set simultaneously.Collect the 4th day the chronically infected H9 cell of HIV-1 IIIB in recovery back, transfer cell concn to 4 * 10
5/ ml, every hole adds cells infected suspension 100 μ l.Be that every hole contains 4 * 10
4Cell count.Put 37 ℃, cultivated 96 hours in the 5%CO2 incubator.The careful 150 μ l supernatants of drawing in every hole are used for ELISA and measure HIV P24 antigen levels after the 5%Triton X-100 deactivation.Measure the toxic action of compound with the MTT method to H9/HIV-1 IIIB cell.
Table 4 TRICHOBITACIN is to the influence of chronic infection cell expressing P24 antigen levels
| Compound | Concentration (μ g/ml) | Cell survivaling number (per-cent of experimental and control) | P24 antigenic expression (per-cent of experimental and control) |
| TRICHOBITACIN (94P73CMI) | 5.0 1.0 0.2 0 04 | -0.5 13.5 43.3 88.2 | 66.0 83.4 118.2 116.2 |
| TRICHOBITACIN (94P75CMI) | 5.0 1.0 0.2 0.04 | 0.2 9.0 26.5 68.8 | 57.7 52.2 98.4 94.5 |
The ideal medicine should suppress acute infection and can suppress chronically infected HIV again and duplicate.The clinical of the overwhelming majority all can not reach this purpose with inverase now.As seen from Table 4, ribosome inactivating protein does not influence HIV-1 IIIB and duplicates in that chronic infection H9 is intracellular.
The beneficial effect that can draw TRICHOBITACIN of the present invention from above-mentioned experimental result is:
TRICHOBITACIN crude product and pure product all suppress HIV-1 significantly and induce the plasmodial formation of C8166; In acute infection, TRICHOBITACIN has suppressed the antigenic expression of HIV-1 P24 significantly, when compound concentration was 0.04 μ g/ml, 94P73CMI and 94P75CMI were respectively 54.8% and 52.6% (being that EC50 all is lower than 0.04 μ g/ml) to P24 antigen presentation inhibiting rate.The HIV-1 antigen-positive cell has reduced 47.0%; In chronic infection, TRICHOBITACIN does not have influence to duplicating of HIV-1IIIB, even under toxic concentration, the P24 antigenic expression also changes not quite.Therefore, TRICHOBITACIN of the present invention is a kind of new ribosome inactivating protein with HIV (human immunodeficiency virus)-resistant activity.
Further specify essentiality content of the present invention below in conjunction with accompanying drawing with embodiment, but content of the present invention is not limited thereto.
Fig. 1 is the Sephadex G-75 column chromatography figure of TRICHOBITACIN of the present invention
Fig. 2 is the ion exchange column CM-Sephadex C-50 column chromatography figure of TRICHOBITACIN of the present invention
Embodiment one:
1. preparation TRICHOBITACIN: get snakegourd (Trichosanthes kirilowii Maxim (Cucurbitaceae) piece root, after having pressed residue behind the juice to soak with physiological saline, use acetone precipitation, after the collecting precipitation lyophilize, with molecular sieve Sephadex G-50 or G-75 and ion exchange column CM-Sephadex C-50 purifying, when ion-exchange chromatography, two peaks appear, and peak I is TRICHOBITACIN (Trichobitacin).
Use HPLC then, methods such as SDS-PAGE and HPCE are identified its purity, measure its molecular weight with ES-MS, and isoelectric focusing electrophoresis is measured iso-electric point, measures partial amino-acid series with automatic sequencer behind the enzymolysis.
2, the trypsin digestion of TRICHOBITACIN: get the 19mg TRICHOBITACIN, add 0.6ml 8mol/l urea, heating is 2 minutes in 100 ℃ of boiling water, adds 0.6ml distilled water and 0.6ml 0.4mol/l NH
4HCO
3, under agitation add the trypsinase aqueous solution 0.6ml that 0.3mg handled through TPCK, vibrate 37 ℃ of enzymolysis 24 hours.
3. the mensuration of the mixed peptide quality after the trypsin digestion TRICHOBITACIN: with the solution lyophilize behind the above-mentioned enzymolysis, measure the mixed peptide quality with MALDI-TOF-MS and FAB-MS respectively, MALDI-TOF-MS selects 2 respectively for use, 5-resorcylic acid (2,5-Dihydroxybenzoic acid, DHB) and alpha-cyano-4-hydroxycinnamic acid (α-Cyano-4-hydroxycinnamic acid CHCA) makes matrix, and FAB-MS makes matrix with glycerine.
4. TRICHOBITACIN is separated with HPLC through the peptide section of trypsin digestion: the TRICHOBITACIN enzymolysis is after 24 hours, and is centrifugal, and supernatant liquor is directly used high-efficient liquid phase chromatogram HPLC reversed-phase column (μ Bondapak C18,10mm * 30cm) separate; The precipitation part is washed several times through the N-methylmorpholine of pH8.1, and with the 65%TFA dissolving, centrifugal, clear liquid separates with HPLC, gets final product by following condition wash-out then.
Elutriant: A:0.1%TFA-H
2O B:0.08%TFA+80%CH
3CN
Gradient: time 0-43-50-55-58min
B％ 0-65-100-100-0
Flow velocity: 2mL/min detects wavelength: 230nm
5. the sequential analysis of partial peptide section: the peptide section after the HPLC separation and purification checks order by hand with automatic sequence instrument, DABITC/PITC double coupling method, and compares the identical peptide section with Trichosanthin of TRICHOBITACIN with mass spectroscopy, thereby determines the sequence of corresponding peptides.
TRICHOBITACIN by method for preparing, it is a kind of strand ribosome inactivating protein, molecular weight 27,228Da, iso-electric point 9.6, sugar content 0.7-0.9%, have RNA N-glycosidase activity, N-end and C-terminal amino acid are Aspartic Acid and L-Ala, and N-holds 38 amino acid whose sequences, except that 31 be the Isoleucine, the same with Trichosanthin.
Claims (3)
1, TRICHOBITACIN, it is characterized in that separating the residue after the piece root of snakegourd plant has been pressed juice a kind of strand ribosome inactivating protein that obtains, molecular weight 27,228Da, iso-electric point 9.6, sugar content 0.7-0.9%, have RNA N-glycosidase activity, N-end and C-terminal amino acid are Aspartic Acid and L-Ala, and N-holds 38 amino acid whose sequences, except that 31 be the Isoleucine, the same with Trichosanthin.
2, the method for preparing claim 1 TRICHOBITACIN, it is characterized in that getting snakegourd piece root, after having pressed residue behind the juice to soak with physiological saline, use acetone precipitation, after the collecting precipitation lyophilize, with Sephadex G-50 or G-75 and CM-Sephadex C-50 purifying, get the peak I that column chromatography for separation goes out and get final product.
3, claim 1 TRICHOBITACIN is controlled the application of acquired immune deficiency syndrome (AIDS) medicine as preparation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97115693A CN1056383C (en) | 1997-11-29 | 1997-11-29 | Chinese trichosanthes protein, preparation method and use in pharmacy |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97115693A CN1056383C (en) | 1997-11-29 | 1997-11-29 | Chinese trichosanthes protein, preparation method and use in pharmacy |
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| Publication Number | Publication Date |
|---|---|
| CN1209435A CN1209435A (en) | 1999-03-03 |
| CN1056383C true CN1056383C (en) | 2000-09-13 |
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| CN97115693A Expired - Fee Related CN1056383C (en) | 1997-11-29 | 1997-11-29 | Chinese trichosanthes protein, preparation method and use in pharmacy |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0256907A1 (en) * | 1986-07-15 | 1988-02-24 | Sanofi S.A. | Protein synthesis inhibitor, process for its isolation, use, and pharmaceutial compositions containing it |
| WO1988009123A1 (en) * | 1987-05-29 | 1988-12-01 | Genelabs Incorporated | Method of selectively inhibiting hiv |
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1997
- 1997-11-29 CN CN97115693A patent/CN1056383C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0256907A1 (en) * | 1986-07-15 | 1988-02-24 | Sanofi S.A. | Protein synthesis inhibitor, process for its isolation, use, and pharmaceutial compositions containing it |
| WO1988009123A1 (en) * | 1987-05-29 | 1988-12-01 | Genelabs Incorporated | Method of selectively inhibiting hiv |
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| Publication number | Publication date |
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| CN1209435A (en) | 1999-03-03 |
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