CN105106953A - Antibody-based drug for treating psoriasis - Google Patents
Antibody-based drug for treating psoriasis Download PDFInfo
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- CN105106953A CN105106953A CN201510519923.2A CN201510519923A CN105106953A CN 105106953 A CN105106953 A CN 105106953A CN 201510519923 A CN201510519923 A CN 201510519923A CN 105106953 A CN105106953 A CN 105106953A
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- 229940079593 drug Drugs 0.000 title abstract description 7
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention provides an antibody-based drug for treating psoriasis. A pathogenic bacterium is directly used as an antigen for immunization, and remarkable antibody effect is acquired; the pathogenic bacterium for use belongs to an existing bacterial strain, a dander sample can also be acquired from an affected part of a patient with pathogenic bacterium, the sample is cultured on a Sabouraud's dextrose agar medium containing chloramphenicol to obtain fungi, culturing by streaking is performed to obtain a single colony, a culture is verified, the single colony is selected and inoculated to a liquid medium and subjected to amplified cultivation, centrifuging is performed to collect bacteria, formaldehyde inactivating is then performed to prepare an antigen, the antigen is used to directly immunize a new Zealand white rabbit to obtain an antibody, and the drug prepared with the antibody has remarkable alleviating effect for treating patients with psoriasis.
Description
Technical field
The present invention relates to curing psoriasis medicine field, especially one is used for the treatment of psoriasic antibody drug.
Background technology
Psoriasis is that the common chronic inflammatory skin that people suffer from is sick, and the cause of disease there is no definite final conclusion.Psoriasis is divided into four types, and wherein psoriasis vulgaris is the most common.Typical lesions is boundary clearly erythema, pimple, the multilamellar squama of surface coverage drying.This course of disease is long, and Relapse rate shows effect, and clinical treatment is comparatively thorny.Current psoriatic medicine is a lot, but due to its cause of disease intricate, the curative effect of many medicines can not be determined, only to improve patients symptomatic.Traditional medicine has more untoward reaction, and by contrast, antipsoriatic biological preparation is strong targetedly, determined curative effect, the advantage that untoward reaction is few.Have many have developed for psoriasis immunopathogenesis much to have the specific biological preparation of target position in recent years, majority demonstrates good application prospect.But have no research from affected part this culture of isolated of label taking of patient fungus that goes out to cure the disease to the antibody doing antigen and reentry.
Summary of the invention
Technical problem to be solved by this invention is to provide one to be used for the treatment of psoriasic antibody drug.
For solving the problems of the technologies described above, technical scheme of the present invention is:
One is used for the treatment of psoriasic antibody drug, and be the polyclonal antibody that psoriasis fungus (pathogenic bacterium) is prepared as antigen, described polyclonal antibody is obtained by following method:
(1) prepare antigen, operated in accordance with the following methods respectively by pathogenic bacterium, wherein, described pathogenic bacterium are Aspergillus fumigatus, Oidium tropicale, Candida parapsilosis and/or aspergillus niger:
In A pathogenic bacterium inoculation potato dextrose medium, 28 DEG C, 160rpm shakes cultivation, and bacterium liquid is centrifugal, collecting precipitation, in precipitation, add 3.7% formaldehyde, 4 DEG C of deactivations of spending the night;
Thalline after the deactivation of b collected by centrifugation, resuspended with normal saline, cell Ultrasonic Pulverization instrument is pulverized, and BCA method is prepared into the solution of total protein concentration 0.5mg/ml after measuring;
C gets protein solution and isopyknic Freund's complete adjuvant is mixed with first immunisation antigen, and to be prepared into booster immunization antigen for subsequent use with equal-volume incomplete Freund's adjuvant;
(2) antibody preparation: first immunisation, after 1ml first immunisation antigen subcutaneous injection new zealand white rabbit 15,30,45 days respectively with adding the further immune strengthening of antigen, immune strengthening inject for the last time after one week in gather antiserum, obtain the antibody serum that various isolation identification goes out fungus.
Preferably, be above-mentionedly used for the treatment of psoriasic antibody drug, in described step (1), pathogenic bacterium are Oidium tropicale and/or Candida parapsilosis.
Preferably, be above-mentionedly used for the treatment of psoriasic antibody drug, also comprise the acceptable adjuvant of pharmacy, excipient.
Preferably, being above-mentionedly used for the treatment of psoriasic antibody drug, is unguentum.
The above-mentioned preparation method being used for the treatment of psoriasic antibody drug, concrete preparation process is as follows:
(1) prepare antigen, operated in accordance with the following methods respectively by pathogenic bacterium, wherein, described pathogenic bacterium are Aspergillus fumigatus, Oidium tropicale, Candida parapsilosis and/or aspergillus niger:
In A pathogenic bacterium inoculation potato dextrose medium, 28 DEG C, 160rpm shakes cultivation, and bacterium liquid is centrifugal, collecting precipitation, in precipitation, add 3.7% formaldehyde, 4 DEG C of deactivations of spending the night;
Thalline after the deactivation of b collected by centrifugation, resuspended with normal saline, cell Ultrasonic Pulverization instrument is pulverized, and BCA method is prepared into the solution of total protein concentration 0.5mg/ml after measuring;
C gets protein solution and isopyknic Freund's complete adjuvant is mixed with first immunisation antigen, and to be prepared into booster immunization antigen for subsequent use with equal-volume incomplete Freund's adjuvant;
(2) antibody preparation: first immunisation, after 1ml first immunisation antigen subcutaneous injection new zealand white rabbit 15,30,45 days respectively with adding the further immune strengthening of antigen, immune strengthening inject for the last time after one week in gather antiserum, obtain the antibody serum that various isolation identification goes out fungus.
Preferably, the above-mentioned preparation method being used for the treatment of psoriasic antibody drug, in described step (1), pathogenic bacterium are Oidium tropicale and/or Candida parapsilosis.
Preferably, the above-mentioned preparation method being used for the treatment of psoriasic antibody drug, in described step (1), pathogenic bacterium are obtained by following method:
(1) label taking originally: after conventional 75% alcohol disinfecting, epidermis cell (scurf) and Skin Deep cell are got in the affected part of scraping psoriasis people, are inoculated on the Sabouraud's dextrose agar inclined-plane containing 0.05% chloromycetin, is placed in 28 DEG C and cultivates 10-15 days;
(2) fungus is separated: adopt line partition method to inoculate on Sabouraud's dextrose agar flat board, cultivate under being placed in 28 DEG C of temperature, wait to turn out single bacterium colony, qualification fungus, preserve by Freezing Glycerine method and identify strain.
Preferably, the above-mentioned preparation method being used for the treatment of psoriasic antibody drug, by the serum of the various antibody of described step (2) gained, carry out titration, after suitably diluting with physiological saline solution, be mixed in proportion, add 3% water-solubleazone and emulsifiable paste matrix makes unguentum.
The invention has the beneficial effects as follows:
Above-mentionedly be used for the treatment of psoriasic antibody drug, pathogenic bacterium are adopted directly to carry out immunity as antigen and obtain significant antibody effects, the pathogenic bacterium used are existing bacterial strain, also scurf specimen can be obtained from psoriasis people affected part, specimen is turned out fungus on the weak glucose agar medium in husky fort containing chloromycetin, line separation and Culture goes out single bacterium colony, through identifying strain, picking list colony inoculation is to fluid medium amplification culture, collected by centrifugation thalline obtains, formalin-inactivated is adopted to prepare antigen again, antibody is obtained with this antigen direct immunization new zealand white rabbit, with this antibody for main medicine of preparing has significant remission effect for the treatment of psoriasis people, its preparation method is simple, has no side effect, can prepare medicine targetedly carry out autotelic targeted therapy according to the pathogenic bacterium of single patient.
Detailed description of the invention
Below in conjunction with specific embodiment, technical scheme of the present invention is further described.
Embodiment 1
Pathogenic bacterium are separated
1, after conventional 75% alcohol disinfecting in psoriasis people sufferer place, epidermis cell (scurf) and Skin Deep cell (patient specimen pacifies outpatient service by generation and Hai Xin out-patient department provides) are got in scraping affected part, obtain 11 kinds of samples altogether from 11 patient's sufferers.11 kinds of samples are inoculated on the Sabouraud's dextrose agar inclined-plane containing 0.05% chloromycetin respectively, are placed in 28 DEG C and cultivate 2 weeks.Sabouraud's dextrose agar presses Chinese Pharmacopoeia preparation.
2, the fungus turned out of picking, adopts line partition method to inoculate on Sabouraud's dextrose agar flat board, cultivates, wait to turn out single bacterium colony under being placed in 28 DEG C of temperature.Carry out perusal record and often plant bacterium colony state, determine yeast-like fungus and mycete, then mycete is made smear light Microscopic observation spore shape with after cotton blue dyeing, in conjunction with colonial morphology, determined the type of mycete by the contrast of fungus storehouse; Yeast-like fungus adopts Candida chromogenic medium qualification, chooses yeast streak inoculation on Candida chromogenic medium, and be placed in 35 DEG C and cultivate 48 hours, observed and recorded color changes, according to different colours qualification strain.The strain identified is preserved by Freezing Glycerine method.It is Aspergillus fumigatus, Oidium tropicale, Candida parapsilosis and aspergillus niger respectively that Dual culture goes out 4 kinds of pathogenic bacterium.Wherein Oidium tropicale, Candida parapsilosis is strain common in 11 kinds of samples.
Embodiment 2
Prepare antigen
A bacterium is cultivated, picking 4 kinds of fungal inoculum contain in 5ml potato dextrose medium test tube respectively, be placed in 28 DEG C, 160rpm shakes incubator and cultivates 36h (24-48h all can realize), and then in the triangular flask being transferred to containing 100ml potato dextrose medium, be placed in 28 DEG C, 160rpm shakes incubator and cultivates 60h (48-72h all can realize).Collect liquid culture, 6000rpm, centrifugal 10min, supernatant discarded.
B formalin-inactivated, in the precipitation after centrifugal, add 3.7% formaldehyde, 4 DEG C are spent the night, same to pelleted by centrifugation, with normal saline flushing 3-4 time.
C deactivation is verified, the thalline respectively after picking deactivation, and line is coated on the weak glucose agar medium of husky guarantor, cultivates 60h (48-72h all can realize) for 28 DEG C, asepsis growth deactivation success.
Prepared by d antigen, the thalline normal saline after sterilizing is resuspended, and cell Ultrasonic Pulverization instrument is pulverized, and BCA method is prepared into the solution of total antigen amount 0.5mg/ml after measuring.
Embodiment 3
The preparation of antibody
Immune rabbit, first immunisation, get protein solution 1ml (containing total protein 0.5mg) and mix with isopyknic Freund's complete adjuvant, mortar grinder to emulsion enters water and does not scatter, and illustrates that emulsifying is complete.The preparation method of booster immunization antigen is the same, as long as use incomplete Freund's adjuvant instead to replace Freund's complete adjuvant.15, within 30,45 days, add the further immune strengthening of antigen respectively, immune strengthening inject for the last time after one week in gather antiserum.
5, measure tiring of antibody, adopt indirect elisa method mensuration polyclonal antiserum and tire.Before immunity inoculation, rabbit ear edge venous blood sampling 1ml is as negative control, and before the 4th immunity, every rabbit is got blood 1ml and is used for surveying antibody titer, and the antibody titer that the antigen of 4 kinds of inoculations obtains is all 1:6400.
6, get blood and obtain antibody, after the 4th immunity about 1 week, by rabbit fasting 24h, to prevent Hyperlipemia; Be fixed on by rabbit on rabbit plate, etherization artery gets blood.To the centrifuge tube of blood be collected, 37 DEG C of tilting 1-2h, then after 4 DEG C of tilting 3-4h, the serum of precipitation is moved in new centrifuge tube, sludged blood is with 3000rpm, and 4 DEG C of centrifugal 10min, carefully draw supernatant, merge twice serum, 3000rpm, 4 DEG C of centrifugal 10min, in a small amount subpackage, prevent multigelation, be stored in-80 DEG C.
Embodiment 4
Treat the preparation of psoriatic antibody drug:
Adopt physiological saline solution that antibody serum is for subsequent use according to 1:20 dilute serum (dilute serum all can realize in 1:10-1:100 (v/v) scope), first in the water-bath of 40 DEG C, the emulsifiable paste matrix (Guangzhou Bo Xin Fine Chemical Co., Ltd) of certain volume (the present embodiment selects 160ml) is melted the four kinds of each 10ml of antibody serum and cumulative volume 3% (g/ml) water-solubleazone that add dilution more respectively, mixing, makes unguentum.Prepare rear 4 DEG C of preservation medicines.
| Reagent name | Producer |
| Chloromycetin | Solarbio |
| Glucose | Tianjin Chemical Reagents Factory No.1 |
| Peptone | Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd |
| Agar | Solarbio |
| Formaldehyde | The luxuriant chemical reagent factory of the Tianjin mayor |
| Cotton blue dye liquor | Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd |
| Potato culture | Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd |
| Candida culture medium | Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd |
| Buddhist formula adjuvant | SiGMA |
Embodiment 5
Compliance test result
Smear use (wherein 5 is the patient extracting sample) to 13 patients, smear 2 every day, gently rub a few minutes.6 weeks courses for the treatment of, observing effect.Wherein have 8 patients (comprising the patient that 5 were extracted sample) medication after one month symptom obviously alleviate, the area of skin lesion reduces, 3 conditions of patients are had to be controlled, the area of skin lesion does not increase, prove that this medicament is effective to psoriatic treatment, especially specific aim Dispersal risk medication effect is remarkable.
Above-mentioned reference embodiment is to this kind of detailed description being used for the treatment of psoriasic antibody drug and carrying out; illustrative instead of determinate; several embodiments can be listed according to institute's limited range; therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention.
Claims (5)
1. be used for the treatment of a psoriasic antibody drug, it is characterized in that: the polyclonal antibody prepared as antigen for psoriasis fungus, described polyclonal antibody is obtained by following method:
(1) prepare antigen, operated in accordance with the following methods respectively by pathogenic bacterium, wherein, described pathogenic bacterium are Aspergillus fumigatus, Oidium tropicale, Candida parapsilosis and/or aspergillus niger:
In A pathogenic bacterium inoculation potato dextrose medium, 28 DEG C, 160rpm shakes cultivation, and bacterium liquid is centrifugal, collecting precipitation, in precipitation, add 3.7% formaldehyde, 4 DEG C of deactivations of spending the night;
Thalline after the deactivation of b collected by centrifugation, resuspended with normal saline, cell Ultrasonic Pulverization instrument is pulverized, and BCA method is prepared into the solution of total protein concentration 0.5mg/ml after measuring;
C gets protein solution and isopyknic Freund's complete adjuvant is mixed with first immunisation antigen, and to be prepared into booster immunization antigen for subsequent use with equal-volume incomplete Freund's adjuvant;
(2) antibody preparation: first immunisation, after 1ml first immunisation antigen subcutaneous injection new zealand white rabbit 15,30,45 days respectively with adding the further immune strengthening of antigen, immune strengthening inject for the last time after one week in gather antiserum, obtain the antibody serum that various isolation identification goes out fungus.
2. be according to claim 1ly used for the treatment of psoriasic antibody drug, it is characterized in that: in described step (1), pathogenic bacterium are Oidium tropicale and/or Candida parapsilosis.
3. be according to claim 1ly used for the treatment of psoriasic antibody drug, it is characterized in that: in described step (1), pathogenic bacterium are obtained by following method:
(1) label taking originally: after conventional 75% alcohol disinfecting, epidermis cell and Skin Deep cell are got in the affected part of scraping psoriasis people, are inoculated on the Sabouraud's dextrose agar inclined-plane containing 0.05% chloromycetin, is placed in 28 DEG C and cultivates 10-15 days;
(2) fungus is separated: adopt line partition method to inoculate on Sabouraud's dextrose agar flat board, cultivate under being placed in 28 DEG C of temperature, wait to turn out single bacterium colony, qualification fungus, preserve by Freezing Glycerine method and identify strain.
4. be according to claim 1ly used for the treatment of psoriasic antibody drug, it is characterized in that: be unguentum.
5. be according to claim 1ly used for the treatment of psoriasic antibody drug, it is characterized in that: by the serum of the various antibody of described step (2) gained, carry out titration, after suitably diluting with physiological saline solution, be mixed in proportion, add 3% water-solubleazone and emulsifiable paste matrix makes unguentum.
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| CN201510519923.2A CN105106953A (en) | 2015-08-21 | 2015-08-21 | Antibody-based drug for treating psoriasis |
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Cited By (2)
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| CN106279349A (en) * | 2016-08-31 | 2017-01-04 | 广东工业大学 | For preparing extracting method and the preparation method of yolk antibody thereof of the dermatophytosis suppressor proteins antigen of yolk antibody |
| CN106397586A (en) * | 2016-08-31 | 2017-02-15 | 广东工业大学 | Anti-dermatophyte specific yolk antibody, preparation method and application thereof |
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| CN104844709A (en) * | 2014-05-27 | 2015-08-19 | 青岛大学附属医院 | Preparation for specific aspergillus fumigatus infection resisting chicken egg yolk antibody and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106279349A (en) * | 2016-08-31 | 2017-01-04 | 广东工业大学 | For preparing extracting method and the preparation method of yolk antibody thereof of the dermatophytosis suppressor proteins antigen of yolk antibody |
| CN106397586A (en) * | 2016-08-31 | 2017-02-15 | 广东工业大学 | Anti-dermatophyte specific yolk antibody, preparation method and application thereof |
| CN106397586B (en) * | 2016-08-31 | 2019-12-06 | 广东工业大学 | specific yolk antibody for resisting dermatophytes and preparation method and application thereof |
| CN106279349B (en) * | 2016-08-31 | 2019-12-10 | 广东工业大学 | extraction method of dermatophyte cytoplasm protein antigen for preparing yolk antibody and preparation method of yolk antibody |
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