CN101045749A - Preparation method of multivalence anti-venin yolk antibody - Google Patents
Preparation method of multivalence anti-venin yolk antibody Download PDFInfo
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- CN101045749A CN101045749A CN 200710027790 CN200710027790A CN101045749A CN 101045749 A CN101045749 A CN 101045749A CN 200710027790 CN200710027790 CN 200710027790 CN 200710027790 A CN200710027790 A CN 200710027790A CN 101045749 A CN101045749 A CN 101045749A
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Abstract
This invention relates to a preparation method of multivalent anti-snake venom chicken yolk antibody. It includes following steps:(1) collect different kinds of snake venom antigen; (2) preparation of chicken ovum of anti-snake venom chicken yolk antibody, and infuse snake venom to chicken for immunization, after 20 to 4 weeks continuously collect egg for 6 to 10 mouths.(3) extract multivalent anti-snake venom chicken yolk antibody. This product can replace horse serum for injection, and be able to developed as oral using drug which has extensive application prospects in prevention of snake bite.
Description
Technical field
The present invention relates to a kind of preparation method of snake venom antibody, relate in particular to a kind of anti-venin yolk antibody (Immunoglobulin-yolk, preparation method IgY).More particularly, be a kind of preparation method of multivalence anti-venin yolk antibody.
Background technology
The conventional production of present existing antivenin all is to prepare with the horse immunity, normally to use the snake venom immunity horses of detoxification earlier, blood plasma is further made with extra care and is purified behind gastric enzyme digestion, ammonium sulfate precipitation, and making to remove the segmental IgG of Fc is the polyclonal antiserum preparation of main component.
Summing up the antivenin of being produced in the prior art has the following disadvantages: the one, and goods mostly are liquid preparation, are difficult for preserving and transportation, and validity period is short; The 2nd, mostly be monovalent serum, need have before the medication clear and definite be diagnosed as which kind of snake and hinder and inject corresponding antivenin again, but the snakebite state of an illness is anxious, progress is fast, and the state of an illness is dangerous, mortality ratio is high, and makes a definite diagnosis the delay that will inevitably cause on treatment time before the medication; The 3rd, make with extra care, purifying process falls behind, so that this product specific activity is lower, and the anaphylaxis rate that causes is higher.The anaphylaxis of antivenin can appear at the different steps of conventional hypersensitive test of patient or intravenously administrable, and irritated back occurs uncomfortable in chest rapidly, palpitaition, and larynx blocks sense, vomiting, stomachache, heating, fash etc. seriously can cause death and die.And yield poorly, the cost height causes costing an arm and a leg, and is subjected to great restriction in clinical application.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of multivalence anti-venin yolk antibody, this method productive rate height, cost are low.The antibody of gained is safe, good stability, high specificity, supersensitivity are little, except being prepared into the injection formulations, also can be prepared into oral preparations, is widely used in snakebite prevention, the treatment.
The objective of the invention is to realize by following technical measures: a kind of preparation method of 3 valency anti-venin yolk antibodies, it may further comprise the steps:
(1) collects various venom antigens;
(2) prepare the anti-venin yolk antibody ovum gallinaceum: venom antigen is injected carry out immunity in the chicken body, 2～4 lasting collection in week back eggs 6～10 months;
(3) extract multivalence anti-venin yolk antibody (IgY).
Can obtain required snake venom with conventional collect means in the described step (1), also can utilize snake venom venon extractor (ZL200420046279.9) to gather snake venom, venom dilutes in 1: 4 ratio adding distil water, and is centrifugal, and low-temperature vacuum drying is a lyophilized powder.Snake poison lyophilized powder is dissolved in phosphate buffered saline buffer (PBS) solution, adds 0.25% glutaraldehyde after placing 2h under 30 ℃ the temperature, utilize SephadexG-25 chromatography desalination, measure snake venom LD
50, and fully emulsified and make antigen with freund adjuvant by 1: 1 (V/V).
The mode difference of injection venom antigen immunity chicken in the described step (2), the preparation method of anti-venin yolk antibody ovum gallinaceum is following three kinds of modes:
1) will at least two kinds single venom antigens circulate successively and inject in single the chicken body, carry out antigen immune, immunity is 1 time weekly, injects a kind of venom antigen at every turn, a circulation immunity cycle enters next cycle after finishing immediately; Be the multivalence anti-venin yolk antibody ovum gallinaceum in first 2～4 week of circulation immunity cycle back collection egg;
2) be to mix venom antigen with at least two kinds of single venom antigen equal portions mixings, be injected into then in single the chicken body, carry out antigen immune; Initial immunity and 2 weeks of interval of immunity for the second time, follow-up immunization is 4 weeks at interval; 2～4 weeks back collection egg is the multivalence anti-venin yolk antibody ovum gallinaceum;
3) at least two kinds of single venom antigens are injected respectively in single the different chicken bodies, carry out antigen immune; Initial immunity and 2 weeks of interval of immunity for the second time, follow-up immunization is 4 weeks at interval; 2～4 weeks back collection egg is different unit price anti-venin yolk antibody ovum gallinaceums.
Described step (3) is extracted the mode difference of preparation multivalence anti-venin yolk antibody (IgY) according to preparation anti-venin yolk antibody ovum gallinaceum, and adopts following preparation method:
1. direct method: the egg that adopts water dilution method directly to give birth to from the hen after the immunity of injection venom antigen, from egg yolk liquid, extract multivalence anti-venin yolk antibody, wherein the ratio of deionized water and egg yolk liquid is 5～12: 1 (V/V), transfers pH to 4～8,4 ℃ placement 6～12h; 4 ℃ centrifugal, and rotating speed is 15min/19000g; Get the supernatant liquor ultrafiltration and concentration and can obtain multivalence anti-venin yolk antibody solution, strength of solution 15～25mg/ml.
2. hybrid system: the 1. described method of employing mode is extracted the unit price anti-venin yolk antibody respectively from different unit price anti-venin yolk antibody ovum gallinaceums, the antibody equal portions of the different snake venom that will obtain mix then, can obtain multivalence anti-venin yolk antibody.
The multivalence anti-venin yolk antibody (IgY) that above-mentioned steps (3) water dilution method extracts gained can directly be prepared into oral preparations, is used for the preventive usage of snakebite control.Also can be prepared into injection, use ammonium sulfate salting-out process purifying IgY, the steps include: in the IgY aqueous solution, to add saturated ammonium sulphate solution, in IgY solution, reach 40%～60% saturation ratio to ammonium sulfate through further purifying; Placed 2 hours for 4 ℃ then, centrifugal, abandon supernatant, precipitation is dissolved in phosphate buffered saline buffer (PBS), ultrafiltration desalination; The solution of gained is adopted DEAE Sepharos FF post, with 0.075,0.15 and the 0.3mol/L PBS gradient elution of pH 7.0, collect purpose solution respectively, ultrafiltration desalination, concentrated, freeze-drying, 4 ℃ of preservations.
The present invention has following beneficial effect compared with the prior art:
1, its structurally similar Mammals IgG of IgY, its molecular weight is about 180～210KD usually, comprises the heavy chain of 1 65-70KD and the light chain of 2 22-35KD.Compare with existing horse serum IgG, IgY has that productive rate height, cost are low, good stability, high specificity, and supersensitivity is little and significantly reduce owing to the antibody collection causes advantages such as injury to animal.
2, be a kind of high multivalence anti-venin IgY that tires, because of used hybrid antigen respectively from a plurality of subfamilies, the antibody that is produced has best cross reaction ratio to 11 kinds of snake venom of China, can under the true situation of acatalepsia, be used for the snakebite first aid immediately, to win effectively the valuable opportunity of treatment in time, generation and the reduction mortality ratio of avoiding serious pathology there is vital role.
3, multivalence Ovum Gallus domesticus Flavus snake venom Antibody Preparation cost is low, output is big, safe, except that alternative horse serum for the injection, also can be developed as oral preparations, the latter is having broad application prospects aspect the snakebite prevention, society and economic benefit are more considerable.
Embodiment
Now in conjunction with the embodiments the present invention is carried out concrete elaboration, but protection scope of the present invention is not limited thereto, those skilled in the art also can realize purpose of the present invention by above content.
The preparation of one: 3 valency (Naja+adder+agkistrodon acutus) of embodiment antisnake venom IgY
(1) 3 kind of venom antigen preparation:
3 kinds of single venom antigen preparations: with the homemade snake venom venon extractor (patent No.: ZL200420046279.9) extract grow up Chinese cobra, homemade round spot adder, ahylysantinfarctase respectively, venom was by (V/V) adding distil water dilution in 1: 4, centrifugal, low-temperature vacuum drying is a lyophilized powder.Get the snake poison lyophilized powder of 50mg and be dissolved in 5ml 0.1M phosphate buffered saline buffer (PBS) solution, add 0.5ml 0.25% glutaraldehyde after placing 2h under 30 ℃ the temperature, utilize SephadexG-25 chromatography desalination, measure snake venom LD
50, and fully emulsified and make antigen with freund adjuvant by 1: 1 (V/V).
(2) with 3 kinds of snake venom attenuation antigen immune hens:
Get 22 ages in week Lay Hangzhoupro hen (body weight: 1.0 ± 0.1kg), with 3 kinds of venom antigens respectively through chicken double-vane, chest, belly and back subcutaneous and muscle multidigit point inject.Immunity is 1 time weekly, injects a kind of single venom antigen at every turn, circulates successively to inject in the chicken body by NNAV antigen, homemade round spot viper venom antigen, ahylysantinfarctase antigen and carries out the immunity of chicken body.The 2nd week in all after dates of first circulation immunity is collected egg, continues to collect 6～10 months.The egg of collecting gained is 3 valency antisnake venom IgY ovum gallinaceums.
After numbering,, leave and take the preceding egg of immunity as negative control in 4 ℃ of preservations.
(3) extraction, purifying lgY:
Adopt water dilution method directly to extract from 3 valency anti-venin yolk antibody ovum gallinaceums, the egg that the hen after the 3 kinds of venom antigen immunity of injection of promptly learning from else's experience gives birth to extracts 3 valency anti-venin yolk antibody IgY from egg yolk liquid.The ratio of deionized water and egg yolk liquid 9: 1 (V/V) is transferred pH to 5.1, places 8h for 4 ℃, and 4 ℃ of centrifugal 15min/19000g, supernatant are through ultrafiltration and concentration, strength of solution 20mg/ml.
With ammonium sulfate salting-out process purifying IgY, add saturated ammonium sulphate to ammonium sulfate saturation ratio in IgY solution and be 50%, 4 ℃ of placement 2 hours, centrifugal, abandon supernatant, precipitation is dissolved in PBS, ultrafiltration desalination; With the solution of gained, adopt DEAESepharos FF post then, with 0.075,0.15 and 0.3mol/L PB (pH 7.0) gradient elution, collect purpose solution respectively, the ultrafiltration desalination, concentrate freeze-drying, 4 ℃ of preservations.
Embodiment two:
Different with embodiment one is: in the described step (2) with 3 kinds (Chinese cobra, homemade round spot adder, agkistrodon acutus) through the single venom antigen of attenuation in 1: 1: 1 ratio mixing.Get 22 ages in week Lay Hangzhoupro hen (body weight: 1.0 ± 0.1kg), will mix in the body of venom antigen and muscle multidigit point injection chicken subcutaneous through chicken double-vane, chest, belly and back.Initial immunity and 2 weeks of interval of immunity for the second time, follow-up immunization was spaced apart for 4 weeks.The 2nd week behind initial immunity is collected egg, and the collected egg in 2 week backs is 3 valency antisnake venom IgY ovum gallinaceums.
The ratio 5: 1 of deionized water and egg yolk liquid is transferred pH to 4 in the described step (3), and 4 ℃ of placement 6h, 4 ℃ of centrifugal 15min/19000g, supernatant are through ultrafiltration and concentration, strength of solution 15mg/ml; Use ammonium sulfate salting-out process purifying IgY then, add saturated ammonium sulphate to ammonium sulfate saturation ratio in IgY solution and be 40%, 4 ℃ of placement 2 hours, centrifugal, abandon supernatant, precipitation is dissolved in PBS, ultrafiltration desalination; With the solution of gained, adopt DEAE Sepharos FF post then, with 0.075,0.15 and the 0.3mol/L PB gradient elution of pH 7.0, collect purpose solution respectively, ultrafiltration desalination, concentrated, freeze-drying, 4 ℃ of preservations.
Embodiment three:
Different with embodiment one, two is: in the described step (2) respectively with Chinese cobra, homemade round spot adder, agkistrodon acutus subcutaneous and muscle multidigit point injects the Lay Hangzhoupro hen (body weight: in 1.0 ± 0.1kg) bodies in 3 22 ages in week respectively through chicken double-vane, chest, belly and back through the single venom antigen of attenuation.Initial immunity and 2 weeks of interval of immunity for the second time, follow-up immunization was spaced apart for 4 weeks.The 2nd week behind initial immunity is collected egg, and the collected egg in 2 week backs is 3 kinds of different unit price antisnake venom IgY ovum gallinaceums.
Adopt water dilution method directly to extract 3 kinds of unit price antisnake venom IgY in the described step (3), then that these 3 kinds of unit price antisnake venom IgY are even by 1: 1: 1 mixed.
The ratio 12: 1 of deionized water and egg yolk liquid is transferred pH to 8 in the described step (3), and 4 ℃ of placement 12h, 4 ℃ of centrifugal 15min/19000g, supernatant are through ultrafiltration and concentration, strength of solution 25mg/ml; Use ammonium sulfate salting-out process purifying IgY then, add saturated ammonium sulphate to ammonium sulfate saturation ratio in IgY solution and be 60%, 4 ℃ of placement 2 hours, centrifugal, abandon supernatant, precipitation is dissolved in PBS, ultrafiltration desalination; With the solution of gained, adopt DEAE Sepharos FF post then, with 0.075,0.15 and the 0.3mol/L PB gradient elution of pH7.0, collect purpose solution respectively, ultrafiltration desalination, concentrated, freeze-drying, 4 ℃ of preservations.
Test example one: 3 valency antisnake venom lgY tires and purity detecting and to the neutralization and the protectiveness experiment of 11 kinds of snake venom
The foregoing description one～three 3 prepared valency antisnake venom lgY are tired and purity detecting and to the neutralization of 11 kinds of snake venom and protectiveness experiment:
(1) purity and molecular weight determination: (concrete steps are with reference to the patent of having applied for to adopt the SDS-PAGE method, application number: 200610035761.6), the result shows: 3 valency antisnake venom lgY aqueous extracts contain 7 dense colored zones, and wherein 6 is impurity band, and molecular weight is respectively between 25KD and the 50～66KD; Removed 4 impurity bands between 56～66KD through 50% ammonium sulfate precipitation, further removed near 2 impurity bands the 25KD through the DEAE column chromatography; 3 valency antisnake venom lgY chromatography things only show a dense purpose band that dyes on non-reduced type SDS-PAGE, reach electrophoresis purity.After adding DTT, the lgY disulfide linkage is opened, and shows that the heavy chain of lgY is about 65KD, and light chain is about 35KD, calculates the IgY molecular weight in view of the above and is about 200KD.
(2) tire and cross-immunity is measured: (concrete steps are with reference to the patent of having applied for to adopt double immunodiffusion and indirect Elisa method, application number: 200610035761.6), it is active and tire to measure cross immunity between 11 kinds of hypertoxic snake venom of 3 valency antisnake venom IgY and China.(anti-cobra venom IgY, anti-echidnotoxin IgY, anti-ahylysantinfarctase IgY) compares with the unit price positive control, the cross immunity rate of 3 valency antisnake venom IgY and Naja, adder and agkistrodon acutus snake venom is greater than 45%, to the cross immunity rate of 8 kinds of snake venom such as Gold-banded Krait, coral snake, Bangladesh Naja, Ophiophagus hannan (Cantor), pallas pit viper, mountain Trimeresurus mucrosquamatus, Trimeresurus stejnegeri, Burma's viper greater than 30%.
(3) Western Blot detects
Get 11 kinds of snake venom through the SDS-PAGE electrophoresis, change pvdf membrane over to, slowly swayed in the confining liquid 1 hour.One～three prepared 3 valency antisnake venom IgY are hatched through the foregoing description, and 4 ℃ leave standstill 12h, use the PBST rinsing, and the anti-chicken IgY-HRP of rabbit is hatched 1h, rinsing, DAB colour developing.The result shows: all snake venom all have positive reaction on pvdf membrane, visible 7～13 tawny bands, molecular weight illustrate that from 100～0.7KDa 3 valency antisnake venom IgY all have combination specifically to the major antigen of 11 kinds of snake venom.
(4) protection of animal experiment: selecting NIH for use is healthy mice, is divided into 22 groups of (test group: 11 groups at random; Control group: 11 groups), 10 every group.
Test group: with 3 valency antisnake venom IgY chromatography things (10mg/kg) respectively with 11 kinds of snake venom (2 times of LD
50Dosage) in sterile test tube, mix, put in 25 ℃ of constant water bath box and hatch 60min, abdominal injection (0.2ml/ only).
Control group: with 11 kinds of snake venom (2 times of LD
50Dosage) directly carry out intraperitoneal administration (0.2ml/ only), respectively organize the survival rate of mouse after the record administration in the 24h.The result shows: 10 mouse of each test group all survive, and control group is all dead in 4～10h.
Above-mentioned test shows, but in the 3 valency antisnake venom IgY specificitys and 11 kinds of snake venom, can protect mouse to avoid the lethal hit of 11 kinds of snake venom to body.
Claims (9)
1, a kind of preparation method of multivalence anti-venin yolk antibody, it is characterized in that: it may further comprise the steps:
(1) collects various venom antigens;
(2) prepare the anti-venin yolk antibody ovum gallinaceum: venom antigen is injected carry out immunity in the chicken body, 2～4 lasting collection in week back eggs 6～10 months;
(3) extract multivalence anti-venin yolk antibody.
2, the preparation method of multivalence anti-venin yolk antibody according to claim 1, it is characterized in that: the mode of injection venom antigen immunity chicken is in the described step (2): at least two kinds of single venom antigens are circulated successively inject in the body of single chicken, carry out antigen immune, immunity is 1 time weekly, injects a kind of venom antigen at every turn.
3, the preparation method of multivalence anti-venin yolk antibody according to claim 1, it is characterized in that: the mode of injection venom antigen immunity chicken is in the described step (2): with at least two kinds of single venom antigen equal portions mixings is to mix venom antigen, be injected into then in the body of single chicken, carry out antigen immune; Initial immunity and 2 weeks of interval of immunity for the second time, follow-up immunization is 4 weeks at interval.
4, the preparation method of multivalence anti-venin yolk antibody according to claim 1, it is characterized in that: the mode of injection venom antigen immunity chicken is in the described step (2): at least two kinds of single venom antigens are injected respectively in the body of single different chickens, carry out antigen immune; Initial immunity and 2 weeks of interval of immunity for the second time, follow-up immunization is 4 weeks at interval.
5, according to the preparation method of claim 1 or 2 or 3 or 4 described multivalence anti-venin yolk antibodies, it is characterized in that: when described antibody ovum gallinaceum is the multivalence anti-venin yolk antibody ovum gallinaceum, described step (3) is extracted preparation multivalence anti-venin yolk antibody method: adopt the yolk of the egg that water dilution method directly gives birth to from the hen after two kinds of venom antigen immunity of injection at least and extract multivalence anti-venin yolk antibody, wherein the ratio of deionized water and egg yolk liquid is 5～12: 1, transfer pH to 4～8,4 ℃ placement 6～12h; 4 ℃ centrifugal, and rotating speed is 15min/19000g; Get the supernatant liquor ultrafiltration and concentration and can obtain multivalence anti-venin yolk antibody solution; Described strength of solution 15～25mg/ml.
6, according to the preparation method of claim 1 or 2 or 3 or 4 described multivalence anti-venin yolk antibodies, it is characterized in that: when described antibody ovum gallinaceum is unit price anti-venin yolk antibody ovum gallinaceum, described step (3) is extracted preparation multivalence anti-venin yolk antibody method: adopt water dilution method to extract different unit price anti-venin yolk antibodies respectively from different egg yolk liquid, wherein the ratio of deionized water and egg yolk liquid is 5～12: 1, transfer pH to 4～8,4 ℃ placement 6～12h; 4 ℃ centrifugal, and rotating speed is 15min/19000g; Get the supernatant liquor ultrafiltration and concentration and obtain 15～25mg/ml unit price anti-venin yolk antibody solution; Different unit price anti-venin yolk antibody equal portions with gained mix then, obtain multivalence anti-venin yolk antibody solution.
7, according to the preparation method of claim 5 or 6 described multivalence anti-venin yolk antibodies, it is characterized in that: the multivalence anti-venin yolk antibody of described extraction gained is further purified, its purification step is: add saturated ammonium sulphate solution in the IgY aqueous solution, reach 40%～60% saturation ratio to ammonium sulfate in IgY solution; Placed 2 hours for 4 ℃ then, centrifugal, abandon supernatant, precipitation is dissolved in PBS, ultrafiltration desalination; With the solution of gained, adopting DEAE Sepharos FF post then, is respectively 7.0 0.075,0.15 and 0.3mol/L phosphate buffered saline buffer gradient elution with pH, collects purpose solution, ultrafiltration desalination, concentrated, freeze-drying, 4 ℃ of preservations.
8, the preparation method of multivalence anti-venin yolk antibody according to claim 7 is characterized in that: the saturation ratio that described saturated ammonium sulphate solution adds at IgY solution is 50%.
9, the preparation method of multivalence anti-venin yolk antibody according to claim 1 is characterized in that: described step (1) utilizes the snake venom venon extractor to gather snake venom, and venom dilutes in 1: 4 ratio adding distil water, and is centrifugal, and low-temperature vacuum drying is a lyophilized powder; Snake poison lyophilized powder is dissolved in the phosphate buffered liquor, adds 0.25% glutaraldehyde after placing 2h under 30 ℃ the temperature, utilize SephadexG-25 chromatography desalination, measure snake venom LD
50, and fully emulsified and make antigen with freund adjuvant by 1: 1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013004020A1 (en) * | 2011-07-07 | 2013-01-10 | 深圳市宝舜泰生物医药股份有限公司 | Pseudomonas aeruginosa (pa-msha) strain igy and preparation method and use thereof |
| CN103554257A (en) * | 2013-11-20 | 2014-02-05 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Anti-schistosoma japonicum Sjp40 chicken egg-yolk antibodies, and preparation method and application thereof |
| CN103910798A (en) * | 2014-04-24 | 2014-07-09 | 上海赛伦生物技术有限公司 | Antibody for resisting vipera berus toxin and long-noded pit viper toxin and preparation method and application thereof |
| CN106243224A (en) * | 2016-08-05 | 2016-12-21 | 安徽威尔试剂盒科技有限责任公司 | One utilizes Agkistrodon acutus venom differential protein to prepare sero-fast method and application thereof |
-
2007
- 2007-04-28 CN CN 200710027790 patent/CN101045749A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013004020A1 (en) * | 2011-07-07 | 2013-01-10 | 深圳市宝舜泰生物医药股份有限公司 | Pseudomonas aeruginosa (pa-msha) strain igy and preparation method and use thereof |
| CN103554257A (en) * | 2013-11-20 | 2014-02-05 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Anti-schistosoma japonicum Sjp40 chicken egg-yolk antibodies, and preparation method and application thereof |
| CN103910798A (en) * | 2014-04-24 | 2014-07-09 | 上海赛伦生物技术有限公司 | Antibody for resisting vipera berus toxin and long-noded pit viper toxin and preparation method and application thereof |
| CN106243224A (en) * | 2016-08-05 | 2016-12-21 | 安徽威尔试剂盒科技有限责任公司 | One utilizes Agkistrodon acutus venom differential protein to prepare sero-fast method and application thereof |
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Open date: 20071003 |