CN103656635A - Preparation method of German cockroach allergen preparation - Google Patents
Preparation method of German cockroach allergen preparation Download PDFInfo
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- CN103656635A CN103656635A CN201310694219.1A CN201310694219A CN103656635A CN 103656635 A CN103656635 A CN 103656635A CN 201310694219 A CN201310694219 A CN 201310694219A CN 103656635 A CN103656635 A CN 103656635A
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Abstract
The invention provides a preparation method of a German cockroach allergen preparation. The method comprises steps as follows: (1), German cockroaches are inactivated; (2), the German cockroaches are washed and soaked with ethyl alcohol and preserved in a refrigerator with the temperature of subzero 80 DEG C for standby application after dried; (3), an appropriate quantity of the frozen German cockroaches are ground into powder in liquid nitrogen; (4), the German cockroaches are frozen after degreased by acetone and then freeze-dried into powder; (5), the German cockroach powder is weighed, an extracting solution is added, the mixture is sufficiently and uniformly mixed on a vortex mixer, and supernatant is absorbed; (6), the content and the proportion of allergens in a German cockroach allergen crude extract are detected by using a corresponding specific antibody resisting allergens; and (7), yeast or inset expressed recombinant protein is added, so that the consistency between the content and the proportion of the mixture and the content and the proportion of five main allergens of the German cockroaches is up to 65%-75%, and the allergen preparation is obtained. The German cockroach allergen crude extract extracted with the method can obtain main allergen protein of the German cockroaches to the greatest extent, the repeatability of the extract is good, and the error is smaller than 30%.
Description
Technical field
The present invention relates to biological pharmacy technical field, be specially a kind of preparation method of Groton bug anaphylactogen preparation.
Background technology
Anaphylactic disease is one of mankind's major disease.Its sickness rate is estimated to account for 22% of world population at present, and just to be greater than every year 1% speed, increases, and rises the most obvious with the sickness rate of child patient.Its hazardness can not be weighed with its mortality rate simply, and will consider the financial burden that the high outbreak frequency of disease causes to patient.Because anaphylactic disease mostly occurs young crowd, it is just more serious on human society and economic impact.China is in industrialization transformation process, its sickness rate significantly increases, and has had a strong impact on China get involved quality of life, the work efficiency of population, has increased the weight of patient's financial burden.The expense that only Europe is used for the treatment of autopath is every year over 50,000,000,000 Euros, and continuation is developing with 3 times of speedups to Other diseases.The cost of asthma and allergic rhinitis is often only respectively up to 64 and 6,000,000,000 dollars by the U.S..The whole nation still has hundreds of millions of people to have potential diagnosis and Treatment need.Therefore, active support China anaphylactogen in-vivo diagnostic and the research and development for the treatment of preparation, can effectively prevent that the medical expense infiltration of China being caused because of external product from going up, and reduces national medical expenditure and people burden.
Anaphylactogen standardization is a global problem, and China widens gradually in this field and American-European countries's gap.For example, the U.S. effectively manages anaphylactogen preparation, has set up specially FDA/CBER/LIB laboratory, is intended to set up the effectively rational quality clearance standard of biological value calibration method, setting of anaphylactogen reference material, autopath's reference serum pond, standard.1981, on American market, there is more than five thousand nonstandardized technique anaphylactogen product to sell, wherein the overwhelming majority does not all have safety and validation verification.And now, FDA is through anaphylactogen Standardization System standard for many years, only 19 kinds of the standardization anaphylactogens of official approval.Germany's institute of biological products-PEI institute (Paul Ehrlich Institute, PEI) has been carried out same strategy, forces to carry out standardization anaphylactogen in Germany, strengthens supervision, has closed tens of anaphylactogen manufacturing enterprises.European Union subsidizes by continuous three framework plans of science and technology, has set up the ELISA calibration method of the main allergic protein restructuring reference material rBet v1 of birch and standard, prepares to write European Pharmacopoeia.We utilize new extraction and recombinant technique in this project, prepare second filial generation standardization anaphylactogen preparation.
As far back as the sixties in last century, just there is Blatta seu periplaneta to cause the report of asthma.But until in recent years,, the effect of Blatta seu periplaneta in asthma just receives people's concern gradually.Data shows, in U.S.'s asthmatic patient, the incidence rate of Blatta seu periplaneta allergy reaches 27.5～42%, and Europe is 3.9～24.5%, and Africa is 30～44.6%, in China, in the big cities such as Beijing, Shanghai, the irritated rate of the Blatta seu periplaneta of children asthma is also up to 27.8～43.8%.The continuous rising of asthma prevalence, relevant with Blatta seu periplaneta pollution aggravation to a great extent, Blatta seu periplaneta has become the second largest indoor anaphylactogen that is only second to dirt demodicid mite.At present common Groton bug anaphylactogen is out identified, makes the foundation that has been prepared with of second filial generation standardization Groton bug anaphylactogen preparation.But, up to the present, there is no the Groton bug preparation technology who publishes, and Groton bug anaphylactogen preparation there is larger clinical demand, Yi Ci our company develops second filial generation standardization Groton bug anaphylactogen preparation.
Summary of the invention
Technical problem solved by the invention is to provide a kind of preparation method of Groton bug anaphylactogen preparation, to solve the problem in above-mentioned background technology.
Technical problem solved by the invention realizes by the following technical solutions: a kind of preparation method of Groton bug anaphylactogen preparation, comprises the steps:
(1) by Groton bug deactivation in-80 ℃ of refrigerators;
(2) with 75% ethanol, clean, soak 15 minutes, air-dry being placed on-80 ℃ Refrigerator store is standby;
(3) get appropriate freezing Groton bug grind into powder in liquid nitrogen;
(4) use after acetone defat 2 times, be placed in-80 ℃ of refrigerator and cooled and freeze, in vacuum freeze drier, be lyophilized into subsequently powder;
(5) take Groton bug powder, ratio according to Groton bug powder 1g extracting solution 1L adds corresponding extracting solution, on vortex agitator, fully mix, put extracting on 4 ℃ of shaking tables, at rotating speed, be 12000g afterwards, centrifugal 30min under 4 ℃ of conditions, draws supernatant, is Groton bug anaphylactogen crude extract;
(6) by the specific antibody of anti-Blag2 or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin and a kind of less important allergen Bla g 5, identify content and the ratio of main allergen Blag2 in Groton bug anaphylactogen crude extract or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin and a kind of less important allergen Bla g 5;
(7) recombinant protein that adds yeast or insecticide to express according to qualification result in step (6), it is reached and five kinds of main allergen Blag2 of Groton bug self or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin, 1 kind of less important allergen Bla g 5 content and ratio are basically identical, obtain anaphylactogen preparation of the present invention.
It is 0.5～4h that described Groton bug powder adds extraction time on the rearmounted 4 ℃ of shaking tables of extracting solution.
Described extracting solution is high phosphatizing phthalate buffer (PBS), and filling a prescription is: NaCl:35.1g～70.2g, KCl:0.2g, Na
2hPO
4: 1.42g, KH
2pO
4: 0.27g, adds 800mlddH
2o regulates pH value 5.0～9.0, is settled to 1L.
Five kinds of main allergen content of described Groton bug self and the concordance of ratio are 65%～75%.
Described Groton bug, for pure lines, is multiplied into by a pair of parents.
Compared with prior art, the present invention has following beneficial effect: the Groton bug anaphylactogen crude extract that the inventive method is extracted can farthest obtain Groton bug main allergen albumen, extract favorable repeatability, and error is less than 30%.
Accompanying drawing explanation
Fig. 1 is 1 hour result of different N aCl concentration PBS solution extracting Groton bug in the present invention.
The specific embodiment
In order to make technological means of the present invention, creation characteristic, workflow, using method reach object and effect is easy to understand, below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, rather than whole embodiment.Embodiment based in the present invention, those of ordinary skills, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
A preparation method for Groton bug anaphylactogen preparation, comprises the steps:
(1) by Groton bug deactivation in-80 ℃ of refrigerators;
(2) with 75% ethanol, clean, soak 15 minutes, air-dry being placed on-80 ℃ Refrigerator store is standby;
(3) get appropriate freezing Groton bug grind into powder in liquid nitrogen;
(4) use after acetone defat 2 times, be placed in-80 ℃ of refrigerator and cooled and freeze, in vacuum freeze drier, be lyophilized into subsequently powder;
(5) take Groton bug powder, ratio according to Groton bug powder 1g extracting solution 1L adds corresponding extracting solution, on vortex agitator, fully mix, put extracting 1h on 4 ℃ of shaking tables, at rotating speed, be 12000g afterwards, centrifugal 30min under 4 ℃ of conditions, draws supernatant, is Groton bug anaphylactogen crude extract;
(6) by the specific antibody of anti-Blag2 or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin and a kind of less important allergen Bla g 5 anaphylactogen, identify content and the ratio of main allergen Blag2 in Groton bug anaphylactogen crude extract or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin and a kind of less important allergen Bla g 5;
(7) recombinant protein that adds yeast or insecticide to express according to qualification result in step (6), make main allergen Blag2 or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin, the concordance of a kind of less important allergen Bla g 5 content and ratio is 68%, obtains anaphylactogen preparation of the present invention.
A preparation method for Groton bug anaphylactogen preparation, comprises the steps:
(1) by Groton bug deactivation in-80 ℃ of refrigerators;
(2) with 75% ethanol, clean, soak 15 minutes, air-dry being placed on-80 ℃ Refrigerator store is standby;
(3) get appropriate freezing Groton bug grind into powder in liquid nitrogen;
(4) use after acetone defat 2 times, be placed in-80 ℃ of refrigerator and cooled and freeze, in vacuum freeze drier, be lyophilized into subsequently powder;
(5) take Groton bug powder, ratio according to Groton bug powder 1g extracting solution 1L adds corresponding extracting solution, on vortex agitator, fully mix, put extracting 1.5h on 4 ℃ of shaking tables, at rotating speed, be 12000g afterwards, centrifugal 30min under 4 ℃ of conditions, draws supernatant, is Groton bug anaphylactogen crude extract;
(6) by the specific antibody of anti-Blag2 or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin and a kind of less important allergen Bla g 5 anaphylactogen, identify content and the ratio of main allergen Blag2 in Groton bug anaphylactogen crude extract or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin and a kind of less important allergen Bla g 5;
(7) recombinant protein that adds yeast or insecticide to express according to qualification result in step (6), make main allergen Blag2 or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin, the concordance of a kind of less important allergen Bla g 5 content and ratio is 70%, obtains anaphylactogen preparation of the present invention.
A preparation method for Groton bug anaphylactogen preparation, comprises the steps:
(1) by Groton bug deactivation in-80 ℃ of refrigerators;
(2) with 75% ethanol, clean, soak 15 minutes, air-dry being placed on-80 ℃ Refrigerator store is standby;
(3) get appropriate freezing Groton bug grind into powder in liquid nitrogen;
(4) use after acetone defat 2 times, be placed in-80 ℃ of refrigerator and cooled and freeze, in vacuum freeze drier, be lyophilized into subsequently powder;
(5) take Groton bug powder, ratio according to Groton bug powder 1g extracting solution 1L adds corresponding extracting solution, on vortex agitator, fully mix, put extracting 2h on 4 ℃ of shaking tables, at rotating speed, be 12000g afterwards, centrifugal 30min under 4 ℃ of conditions, draws supernatant, is Groton bug anaphylactogen crude extract;
(6) by the specific antibody of anti-Blag2 or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin and a kind of less important allergen Bla g 5 anaphylactogen, identify content and the ratio of main allergen Blag2 in Groton bug anaphylactogen crude extract or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin and a kind of less important allergen Bla g 5;
(7) recombinant protein that adds yeast or insecticide to express according to qualification result in step (6), make main allergen Blag2 or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin, the concordance of a kind of less important allergen Bla g 5 content and ratio is 72%, obtains anaphylactogen preparation of the present invention.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technical staff of the industry should understand; the present invention is not restricted to the described embodiments; that in above-described embodiment and description, describes just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (5)
1. a preparation method for Groton bug anaphylactogen preparation, is characterized in that: comprise the steps:
(1) by Groton bug deactivation in-80 ℃ of refrigerators;
(2) with 75% ethanol, clean, soak 15 minutes, air-dry being placed on-80 ℃ Refrigerator store is standby;
(3) get appropriate freezing Groton bug grind into powder in liquid nitrogen;
(4) use after acetone defat 2 times, be placed in-80 ℃ of refrigerator and cooled and freeze, in vacuum freeze drier, be lyophilized into subsequently powder;
(5) take Groton bug powder, ratio according to Groton bug powder 1g extracting solution 1L adds corresponding extracting solution, on vortex agitator, fully mix, put extracting on 4 ℃ of shaking tables, at rotating speed, be 12000g afterwards, centrifugal 30min under 4 ℃ of conditions, draws supernatant, is Groton bug anaphylactogen crude extract;
(6) by the specific antibody of anti-Blag2 or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin and a kind of less important allergen Bla g 5 anaphylactogen, identify content and the ratio of main allergen Blag2 in Groton bug anaphylactogen crude extract or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin and a kind of less important allergen Bla g 5;
(7) recombinant protein that adds yeast or insecticide to express according to qualification result in step (6), it is reached and five kinds of main allergen Blag2 of Groton bug self or Blag9, Blag6.01, Blag7, BlagGSTD1, Blagtrypsin, the concordance of a kind of less important allergen Bla g 5 content and ratio is 65%～75%, obtains anaphylactogen preparation of the present invention.
2. the preparation method of a kind of Groton bug anaphylactogen preparation according to claim 1, is characterized in that: it is 0.5～4h that described Groton bug powder adds extraction time on the rearmounted 4 ℃ of shaking tables of extracting solution.
3. the preparation method of a kind of Groton bug anaphylactogen preparation according to claim 1, is characterized in that: described extracting solution is high phosphatizing phthalate buffer (PBS), and filling a prescription is: NaCl:35.1g～70.2g, KCl:0.2g, Na
2hPO
4: 1.42g, KH
2pO
4: 0.27g, adds 800mlddH
2o regulates pH value 5.0～9.0, is settled to 1L.
4. the preparation method of a kind of Groton bug anaphylactogen preparation according to claim 1, is characterized in that: five kinds of main allergen content of described Groton bug self and the concordance of ratio are 65%～75%.
5. the preparation method of a kind of Groton bug anaphylactogen preparation according to claim 1, is characterized in that: described Groton bug, for pure lines, is multiplied into by a pair of parents.
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| CN102227214A (en) * | 2008-11-28 | 2011-10-26 | 切尔卡西亚有限公司 | Compositions with reduced dimer formation |
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|---|---|---|---|---|
| CN102227214A (en) * | 2008-11-28 | 2011-10-26 | 切尔卡西亚有限公司 | Compositions with reduced dimer formation |
Non-Patent Citations (2)
| Title |
|---|
| 姚湧等: "安徽省3种常见蜚蝙的可溶性蛋自质组成分析", 《安徽医科人学学报》, vol. 36, no. 2, 30 April 2001 (2001-04-30), pages 114 - 115 * |
| 黄艳红等: "5种德国小镰全虫蛋自提取方法的效果比较", 《中国媒介生物学及控制杂质》, vol. 23, no. 3, 30 June 2012 (2012-06-30), pages 209 - 211 * |
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Effective date of registration: 20160622 Address after: 121000 Jinzhou city of Liaoning Province Taihe District South West Ling in a beautiful day. D 35-97 Patentee after: He Shaoheng Address before: 232000 No. ten, No. seven, weft Road, Huainan hi tech Industrial Development Zone, Anhui, China Patentee before: ANHUI RAIN MIX BIOTECHNOLOGY CO., LTD. |