CN102816220B - The bio-modification technique of ricin - Google Patents
The bio-modification technique of ricin Download PDFInfo
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- CN102816220B CN102816220B CN201210316220.6A CN201210316220A CN102816220B CN 102816220 B CN102816220 B CN 102816220B CN 201210316220 A CN201210316220 A CN 201210316220A CN 102816220 B CN102816220 B CN 102816220B
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- ricin
- maggot
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Abstract
The bio-modification technique of ricin, belongs to biological technical field.The present invention's castor seeds is raw material, through protein extraction, bio-modification, reduces the toxicity of ricin, expands the purposes of ricin.The object of the invention is to be achieved through the following technical solutions: 1, castor seeds leaches oil, 2, ricin extracts, 3, inductive formation ricin maggot ovum; 4, the training of ricin larva is grown; 5, castor-oil plant maggot is cultivated; 6, ricin maggot is separated, cleans and deactivation; 7, the drying of ricin maggot and packaging.The bio-modification technological process of ricin of the present invention is easy to control, and production efficiency is high, energy-conservation, non-environmental-pollution, is applicable to suitability for industrialized production.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of bio-modification technique of ricin.
Background technology
Ricin is glycoprotein heterodimer, and the protein be made up of holotoxin, malicious class element, lectin three kinds of materials, its molecular formula is-[C
8h
8n
2o
2]
n-, molecular weight 64000, ricin is white powder or crystalline solids, is insoluble to the organic solvents such as ethanol, ether, chloroform, toluene, is dissolved in diluted acid or salts solution.
Ricin is the one that Ricin toxic is the strongest, all poisonous to various Mammals, but nontoxic or toxicity is lower to insect.
The method removing ricin has: gene detoxification adopts to import has the gene of higher homology can strengthen the silence of native gene with native gene; Adopt methoxy poly (ethylene glycol) and polyethyleneglycol modified ricin; Heating reduces toxicity.
ZL200810072473.7 discloses a kind of year ricin toxin A chain ultrasonic microbubble and preparation method of high-efficiency low-toxicity, and present method is that the employing ricin A chain aqueous solution adds PLGA-PEG-PLGA solution and polyvinyl alcohol water solution high-speed stirring is prepared into ultrasonic microvesicle for carrying ricin A chain.
200410018033.5 disclose a kind of method utilizing Ricin to kill and wound specific tumors cell, present method utilizes gene recombination technology specific expressed ricin toxin B chain albumen in tumour cell, inject toxin A catenin simultaneously, thus in tumor tissues, form activated toxin, killing tumor cell.
201010552949.4 structure and the alternatively vaccine antigens disclosing ricin A chain density body, protein provided by the invention is the mutant mRTA obtained by point mutation, is by the recombinant expressed ricin A chain mutant protein obtained.
The research of China castor-oil plant is in rank first, and annual output is comparatively large, but due to containing toxicant, limits the application of castor-oil plant.
Summary of the invention
The object of this invention is to provide a kind of biological improving technique of ricin, is raw material with castor seeds, through protein extraction, bio-modification, reduces the toxicity of ricin, expands the purposes of ricin.
The object of the invention is to be realized by technical scheme: 1, castor seeds leaches oil, 2, ricin extracts, 3, inductive formation ricin maggot ovum; 4, the training of ricin larva is grown; 5, castor-oil plant maggot is cultivated; 6, ricin maggot is separated, cleans and deactivation; 7, the drying of ricin maggot and packaging.Concrete steps are as follows:
(1) castor seeds leach oil: castor seeds shells, pulverize after leach oil, castor seed meal is for subsequent use;
(2) ricin extracts: be that raw material extracts ricin with castor seed meal, that is: castor seed meal adds deionized water, adjusts pH=5.0-7.0 with damping fluid, and control temperature is 25-35 DEG C, and the time is 1-4h, adds ethanol or (NH after being uniformly dissolved
4)
2sO
4generate precipitation, be then separated solid liquid phase, collect solids, it is for subsequent use that solids is dried into castor protein powder;
(3) inductive formation ricin maggot ovum: get 100g cavings, 10-20g castor protein powder, 5-10g peptone, 5-8gKH
2pO
4mix, spray 10-20ml pure water, keep temperature 25-45 DEG C, time 48-120h, generate worm's ovum;
(4) training of ricin larva is grown: get 200-300g castor protein powder, 10-20g extraction from yeast powder, 10-15g peptone, 10-20gKH
2pO
4, 2-5gNaCl, 1-5mL ammoniacal liquor, adding the mixing of 1.0-2.0L pure water becomes ricin children maggot nutrient solution, is sub-packed in 20-30 Erlenmeyer flask, every bottle is respectively charged into the worm's ovum trained before 0.2-1.0g and grow, shaking table 1-10 time/min, cultivates 12-48h, generates containing ricin children maggot nutrient solution;
(5) castor-oil plant maggot is cultivated: get 2000-5000g castor protein powder, 500-800g extraction from yeast powder, 12-15gNH
4cl, 0.1-0.5gNaCl, 50-100gNa
2hPO
4, 0.2-1.0gCaCl
2, 10-15L deionized water and stirring evenly becomes maggot and cultivates liquid, with the process of Gauss's magnetic induction, add 1.0-2.0L and cultivate containing ricin children maggot nutrient solution, the ricin maggot cultivated becomes milk yellow by oyster white;
(6) ricin maggot is separated, cleans and deactivation: ricin maggot is put into separating machine and is separated worm liquid, use pure water rinsing, then use heat water-spraying's ricin maggot, ricin maggot is inactivated;
(7) ricin maggot is dry: the ricin maggot of deactivation is dry, obtains ricin maggot finished product.
Ricin is main toxic ingredient in castor-oil plant, 86KDa is increased to by 64KDa by bio-modification recombinant protein relative molecular weight, and become animal proteinum characteristic by vegetable-protein characteristic transition, Mouse Acute Toxicity experimental result shows, Ricin LD is 2.5-7.5 μ g/kg, the plain LD of castor-oil plant through bio-modification is 25-28mg/kg, still there is similar cytotoxicity, because restructuring ricin matter had both remained original performance, turn eliminate specific toxicities, therefore expand the purposes of ricin.The bio-modification technological process of ricin of the present invention is easy to control, and production efficiency is high, energy-conservation, non-environmental-pollution, is applicable to suitability for industrialized production.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.
Embodiment 1:
(1) dry castor seeds, slough shell with caster bean sheller, be then rolled into cake, be crushed to 0.2-0.5mm particle with pulverizer with 400-600r/min.
(2) 1000g castor seeds particle adds 2-10L No. 6 petroleum naphthas leachings, temperature 30-50 DEG C, pressure 0.1-0.5MPa, time 2-5 hour.Leach Castor oil and do oil purification process, residue castor seed meal is as ricin raw material for standby.
(3) castor seed meal dries 1-3h at 30-50 DEG C, removes dissolvent residual.
(4) 1000g oven dry castor seed meal adds 2-10L deionized water, and adjust pH=5.8-7 with phosphate buffered saline buffer, temperature 25-35 DEG C, time 2-4h, is uniformly dissolved, and adds 100-200g (NH
4)
2sO
4generate precipitation, place 1-2h, be then separated with 750-1250r/min with separating centrifuge, collect solids.
(5) solids Vacuumdrier is at 35-55 DEG C, vacuum tightness 0.5-2MPa, dries to water content 5-8% one-tenth ricin for subsequent use.
(6) 100g cavings, 10-20g castor protein powder, 5-10g peptone, 5-8gKH is got
2pO
4mix, spray 10-20ml pure water, keep temperature 25-45 DEG C, time 48-120h, generate worm's ovum, sift out worm's ovum with aperture 0.1-2.0mm sieve for subsequent use.
(7) 200-300g castor protein powder, 10-20g extraction from yeast powder, 10-15g peptone, 10-20gKH is got
2pO
4, 2-5gNaCl, 1-5mL ammoniacal liquor, adding the mixing of 1.0-2.0L pure water becomes ricin children maggot nutrient solution, is sub-packed in 20-30 Erlenmeyer flask, every bottle is respectively charged into the worm's ovum trained before 0.2-1.0g and grow, shaking table 1-10 time/min, cultivates 12-48h, generates containing ricin children maggot nutrient solution.
(8) 2000-5000g castor protein powder, 500-800g extraction from yeast powder, 12-15gNH is got
4cl, 0.1-0.5gNaCl, 50-100gNa
2hPO
4, 0.2-1.0gCaCl
2, 10-15L deionized water and stirring is even.With 10000-15000 Gauss magnetic induction process 3-5min, add 1.0-2.0L after processing 2 times and cultivate containing ricin children maggot nutrient solution.
Cultivate parameter: temperature 25-35 DEG C, pH=5.5-8, stirring velocity 1-10r/min, air flow 0.5-2L/min, time 72-120h.
The ricin maggot cultivated becomes milk yellow by oyster white, and cultivating process completes.
(9) put into separating machine together and be separated worm liquid by cultivating liquid and the ricin maggot cultivated with rotating speed 550-1250r/min, with 10-20L pure water rinsing 2-3 time, with 80-100 DEG C of heat water-spraying's ricin maggot 1-5min, ricin maggot is inactivated.
(10) the ricin maggot of deactivation with vacuum freeze drier temperature-30-50 DEG C, time 12-24h, vacuum tightness 10-100kPa are dried to ricin maggot moisture content 5-8% becomes ricin maggot finished product, its ricin maggot content is 95%.
(11) ricin maggot plastic-aluminum vacuum packaging bag vacuum packaging.
Embodiment 2:
(1) dry castor seeds, slough shell with caster bean sheller, be then crushed to 0.2-0.5mm particle with pulverizer with 400-600r/min.
(2) 1000g castor seeds particle expeller to be squeezed out Castor oil and castor seed meal cake with 10-60r/min, and castor seeds grouts are for subsequent use.
(3) 1000g castor seeds grouts add 2-10L deionized water, with 0.2mol/L Sodium phosphate dibasic-0.1mol/L citrate buffer solution adjustment pH=5.0-6.0, control temperature 25-35 DEG C, time 180-210min, stirring velocity 10-30r/min, adds 5-20L ethanol and generates precipitation, place 1-2h, then be separated solid liquid phase with separating centrifuge with 750-1250r/min, collect solids.
(4) solids Vacuumdrier is at 35-55 DEG C, vacuum tightness 0.5-2MPa, dries to water content 5-8% one-tenth castor protein powder for subsequent use.
(5) 100g cavings, 10-20g castor protein powder, 5-10g peptone, 5-8gKH is got
2pO
4mix, spray 10-20ml pure water, keep temperature 25-45 DEG C, time 48-120h, generate worm's ovum, sift out worm's ovum with aperture 0.1-2.0mm sieve for subsequent use.
(6) 200-300g castor protein powder, 10-20g extraction from yeast powder, 10-15g peptone, 10-20gKH is got
2pO
4, 2-5gNaCl, 1-5mL ammoniacal liquor, add the mixing of 1.0-2.0L pure water, be sub-packed in 20-30 Erlenmeyer flask, every bottle is respectively charged into the worm's ovum trained before 0.2-1.0g and grow, shaking table 1-10 time/min, cultivates 12-48h, generates and contains the young maggot nutrient solution of ricin.
(7) 2000-5000g castor protein powder, 500-800g extraction from yeast powder, 12-15gNH is got
4cl, 0.1-0.5gNaCl, 50-100gNa
2hPO
4, 0.2-1.0gCaCl
2, 10-15L deionized water and stirring is even.With 10000-15000 Gauss magnetic induction process 3-5min, add 1.0-2.0L after processing 2 times and cultivate containing ricin children maggot nutrient solution.
Cultivate parameter: temperature 25-35 DEG C, pH=5.5-8, stirring velocity 1-10r/min, air flow 0.5-2L/min, time 72-120h.
The ricin maggot cultivated becomes milk yellow by oyster white, and cultivating process completes.
(8) put into separating machine together and be separated worm liquid by cultivating liquid and the ricin maggot cultivated with rotating speed 550-1250r/min, with 10-20L pure water rinsing 2-3 time, with 80-100 DEG C of heat water-spraying's ricin maggot 1-5min, ricin maggot is inactivated.
(9) the ricin maggot of deactivation with vacuum freeze drier temperature-30-50 DEG C, time 12-24h, vacuum tightness 10-100kPa are dried to ricin maggot moisture content 5-8% becomes ricin maggot finished product, its ricin maggot content is 93%.
(10) ricin maggot plastic-aluminum vacuum packaging bag vacuum packaging.
Claims (8)
1. the bio-modification technique of ricin, is characterized in that described bio-modification technique is as follows:
(1) castor seeds leach oil: castor seeds shells, pulverize after leach oil, castor seed meal is for subsequent use;
(2) ricin extracts: be that raw material extracts ricin with castor seed meal;
(3) inductive formation ricin maggot ovum: get 100g cavings, 10-20g castor protein powder, 5-10g peptone, 5-8gKH
2pO
4mix, spray 10-20ml pure water, keep temperature 25-45 DEG C, time 48-120h, generate worm's ovum;
(4) training of ricin larva is grown: get 200-300g castor protein powder, 10-20g extraction from yeast powder, 10-15g peptone, 10-20gKH
2pO
4, 2-5gNaCl, 1-5mL ammoniacal liquor, adding the mixing of 1.0-2.0L pure water becomes ricin children maggot nutrient solution, is sub-packed in 20-30 Erlenmeyer flask, every bottle is respectively charged into the worm's ovum trained before 0.2-1.0g and grow, shaking table 1-10 time/min, cultivates 12-48h, generates containing ricin children maggot nutrient solution;
(5) castor-oil plant maggot is cultivated: get 2000-5000g castor protein powder, 500-800g extraction from yeast powder, 12-15gNH
4cl, 0.1-0.5gNaCl, 50-100gNa
2hPO
4, 0.2-1.0gCaCl
2, 10-15L deionized water and stirring evenly becomes maggot and cultivates liquid, with the process of Gauss's magnetic induction, add 1.0-2.0L and cultivate containing ricin children maggot nutrient solution, the ricin maggot cultivated becomes milk yellow by oyster white;
(6) ricin maggot be separated, cleaning and deactivation: put into separating machine with the ricin maggot cultivated be separated worm liquid by cultivating liquid, use pure water rinsing, then use heat water-spraying's ricin maggot, ricin maggot is inactivated;
(7) ricin maggot is dry: the ricin maggot of deactivation is dry, obtains ricin maggot finished product.
2. the bio-modification technique of ricin according to claim 1, is characterized in that in described step (1), castor seeds is crushed to 0.2-0.5mm particle.
3. the bio-modification technique of ricin according to claim 1, it is characterized in that in described step (2), castor seed meal adds deionized water, adjusts pH=5.8-7 with phosphate buffered saline buffer, temperature 25-35 DEG C, time 2-4h, is uniformly dissolved, and adds (NH
4)
2sO
4generate precipitation, place 1-2h, be then separated with 750-1250r/min with separating centrifuge, collect solids, it is for subsequent use that solids is dried into castor protein powder.
4. the bio-modification technique of ricin according to claim 1, it is characterized in that in described step (2), castor seeds grouts add deionized water, with the adjustment of 0.2mol/L Sodium phosphate dibasic-0.1mol/L citrate buffer solution pH=5.0-6.0, control temperature 25-35 DEG C, time 180-210min, add ethanol and generate precipitation, place 1-2h, be then separated solid liquid phase with separating centrifuge with 750-1250r/min, collect solids, it is for subsequent use that solids is dried into castor protein powder.
5. the bio-modification technique of the ricin according to claim 3 or 4, is characterized in that described solids Vacuumdrier is at 35-55 DEG C, vacuum tightness 0.5-2MPa, dries to water content 5-8% one-tenth castor protein powder for subsequent use.
6. the bio-modification technique of ricin according to claim 1, is characterized in that in described step (3), worm's ovum aperture 0.1-2.0mm sieve.
7. the bio-modification technique of ricin according to claim 1, is characterized in that in described step (5), cultivates parameter: temperature 25-35 DEG C, pH=5.5-8, stirring velocity 1-10r/min, air flow 0.5-2L/min, time 72-120h.
8. the bio-modification technique of ricin according to claim 1, it is characterized in that in described step (7), with vacuum freeze drier temperature-30-50 DEG C, time 12-24h, vacuum tightness 10-100kPa are dried to ricin maggot moisture content 5-8% becomes ricin maggot finished product.
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家蝇蛆粉对小鼠急性毒性及致突变性作用;黎运西等;《中国公共卫生》;20101231;第26 卷(第11 期);第1416-1417页 * |
蓖麻毒蛋白的研究与应用进展;陈志慧等;《化学世界》;20051231(第5期);第309-312、272页 * |
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