CN104531670A - Compound mutation method for inosine-producing strain - Google Patents
Compound mutation method for inosine-producing strain Download PDFInfo
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- CN104531670A CN104531670A CN201510032287.0A CN201510032287A CN104531670A CN 104531670 A CN104531670 A CN 104531670A CN 201510032287 A CN201510032287 A CN 201510032287A CN 104531670 A CN104531670 A CN 104531670A
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Abstract
The invention discloses a compound mutation method for an inosine-producing strain. Bacillus subtilis protoplast is subjected to atmospheric and room temperature plasma mutation. The compound mutation method comprises the following steps: (1) activation culture of the strain; (2) shake flask liquid culture of the strain; (3) enzymolysis and wall digestion of thallus; (4) atmospheric temperature plasma mutation; (5) regeneration culture of protoplast; (6) 24-well plate screening; and (7) shake flask fermentation and rescreening. After the strain is subjected to passage for fifty times, the genetic characters are stable. Compared with the original strain, the mutant strain has the advantages that the inosine-producing amount is increased from 60g/L to 72g/L, the conversion rate is increased by 20% and the significant effect is achieved.
Description
Technical field
The invention belongs to the mutagenic and breeding technical field of microbial strains, particularly relate to a kind of method of protoplastis and atmospheric pressure at room plasma body mutagenesis compound seed selection Inosin Producing Mutant, seed selection can obtain the bacterial strain of high yield inosine productive rate according to this method.
Background technology
Inosine (Inosine) is also known as inosine, and molecular formula is C
10h
12n
4o
5, molecular weight 268.23.Inosine has very important purposes in fields such as medicine and food: inosine is the integral part of ATP in body, coenzyme A, Yeast Nucleic Acid and thymus nucleic acid, participates in substance metabolism and the energy metabolism of body.Inosine cell membrane has good permeability, directly can enter cell, inosine changes t-inosinic acid and Triphosaden in vivo into, participate in energy metabolism and the protein synthesis of cell, improve the activity of various enzyme, particularly coenzyme A and pyruvic oxidase, thus make cell proceed metabolism under anoxic condition, activation liver function, promotes the recovery of damaged liver.When acute hepatitis, chronic hepatitis, liver cirrhosis cause dysfunction of liver, inosine can be used as an auxiliary hepatoprotective and uses, and also can be used for rescuing hepatic coma.In food, it is good flavour volatile in food, and its content is usually as one of characteristic index weighing food " freshness "; He is the raw material of 5`-t-inosinic acid in foodstuffs industry, is also the nutrition fragrance adding agent of various seasoning food (such as monosodium glutamate, soy sauce etc.) simultaneously.
When inosine is produced in industrialization of fermenting, easily there is Character instability in bacterial strain, causes abnormal fermentation, brings sky high cost and huge financial loss.For obtaining the superior strain of stabilization characteristics of genetics, needing to carry out mutagenic and breeding to corresponding bacterial strain and being applied to industrialization to produce, bringing stable economic benefit for producing.
Mutagenic and breeding is conventional Microbial Breeding method.Routine mutagenesis breeding mainly adopts chemical mutagen (as ethyl sulfate, nitrosoguanidine etc.) or physical mutagenesis (as ultraviolet, X-ray etc.) directly to carry out mutagenic treatment to bacterium, but because thalline has cell walls, the susceptibility of thalline to mutagenic compound can be reduced to a certain extent, be difficult to obtain desirable Mutagenic Effect.And protoplastis is as mutagenesis object, its superiority is the protoplastis after sloughing cell walls, and mutagenic compound more easily enter bacterium internal structure, can act on genetic material in cell rapidly, therefore the probability that morphs of genetic material is larger, efficiency is higher, and Mutagenic Effect can be more obvious.Classic mutagenesis breeding method is tool toxicity or carinogenicity substantially, all has harm to people and environment.And the mutagenesis of atmospheric pressure at room plasma body is a kind of novel microorganism different from classic mutagenesis breeding method substantially organizes induced-mutation technique, this technical equipment is simple, easy to operate, do not need vacuum condition, low and the active particle of plasma jet temperature is evenly distributed, and is safe from harm to environment and people.
Summary of the invention
For above-mentioned technological deficiency, the subtilis mutagenic breeding method that the invention provides that a kind of equipment is simple, easy to operate, efficiency of inducing mutation is high a kind of.
For solving the problems of the technologies described above, technical scheme provided by the invention is: the composite mutagenesis method of a kind of inosine production bacterium, and it is on subtilis protoplastis, carry out the mutagenesis of atmospheric pressure at room plasma body.It comprises the following steps: (1) bacterial strain activation culture; (2) bacterial strain shaking flask liquid culture; (3) bacterial enzyme frees wall; (4) atmospheric pressure at room plasma body mutagenesis; (5) protoplast regeneration is cultivated; (6) 24 orifice plate screenings; (7) shake flask fermentation sieves again.
Further: in the composite mutagenesis method of above-mentioned inosine production bacterium, described step (3) bacterial enzyme frees wall: take the logarithm phase bacterium liquid 10 ± 2mL, and the centrifugal 10 ± 2min of 4000 ± 50r/min, abandons supernatant liquor, centrifugal after thalline SMM liquid is washed 2 ~ 3 times, abandon supernatant liquor; Again thalline is suspended in 5mL ± 1SMM liquid, then 5 ± 1mL lysozyme soln is added, mixing, after being placed in 36 ± 3 DEG C of water bath heat preservation process 30 ± 2min, centrifugal 10 ± the 2min of 4000 ± 50r/min, abandon supernatant liquor, ooze SMM liquid washing 2 ~ 3 removing N,O-Diacetylmuramidases with high, protoplastis is suspended in 5 ± 1mL SMM liquid for subsequent use; SMM liquid preparation (mol/L): sucrose 0.5 ± 0.05, maleic acid 0.02 ± 0.002, MgCl20.02 ± 0.002, pure water is prepared, and pH6.5 ± 0.2,121 DEG C of sterilizing 15 ± 1min, 4 ± 0.5 DEG C save backup.Lysozyme soln is prepared: take N,O-Diacetylmuramidase, be dissolved in SMM liquid, enzyme concn is 100g/L, and 0.22 μM of sterilizing filter is degerming, and-20 ± 1 DEG C saves backup.Logarithmic phase is also known as exponential phase.This one-phase outstanding feature is that bacterial count increases with geometricprogression, for time stablize, the increase of number of bacteria and the increase of protoplasma total amount, with increase being all proportionate property of bacterium liquid turbidity.
Described step (4) atmospheric pressure at room plasma body mutagenesis: it is 0.4-0.8 that protoplast suspension SMM liquid is diluted to OD value, drawing 10 ± 1 μ L is placed on slide glass, atmospheric pressure at room plasma body mutagenesis process 120 ± 10s, working conditions is: irradiation distance 2+1mm, gas flow 10 ± 1slpm, irradiation power 120 ± 10W.OD is the abbreviation of optical delnsity (optical density(OD)), represents the optical density(OD) that detected material sponges.
Protoplastis SMM liquid serial dilutions after described step (5) protoplast regeneration is cultivated and got mutagenesis with without mutagenesis (contrast) is 10
-1~ 10
-8, 10
-1~ 10
-8represent extension rate.Draw 0.1 ± 0.01mL/ ware protoplastis diluent in regenerated solids substratum, carry out regeneration cultivation, culture condition 36 ± 3 DEG C cultivates 20 ± 2h, and front 4 ± 1h rotating speed is 100 ± 10rpm, 4-20h rotating speed is 180 ± 10rpm.(mL/ ware represents the volume of protoplastis added by a culture dish), protoplast regeneration culture medium prescription (g/L): glucose 20 ± 2, potassium primary phosphate 1.5 ± 0.2, yeast powder 2 ± 0.2, agar 20 ± 2, preparation is dissolved, pH7.0-7.2,121 ± 5 DEG C of sterilizing 15 ± 1min with SMM liquid.
In the composite mutagenesis method of above-mentioned inosine production bacterium, it also comprises the detection of step (8) mitotic stability: by the glycerine kind activation culture of cryopreservation, this is the first-generation; Choose a ring bacterium from first-generation inclined-plane kind transfering loop and be transferred to new slant culture, this is the s-generation; Choose a ring bacterium from s-generation inclined-plane kind transfering loop and be transferred to new slant culture, this is the third generation; By that analogy, reached for the 50th generation, by 50 generation bacterial strain carry out shake flask fermentation test, and detect fermented liquid inosine productive rate.
The subtilis glycerine kind of-72 ± 2 DEG C of cryopreservations is transferred to slant medium and carries out activation culture by described step (1) bacterial strain activation culture, cultivates 20-24h for 34-37 DEG C; Slant culture based formulas (g/L): peptone 3 ± 0.3, extractum carnis 5 ± 0.5, yeast extract paste 5 ± 0.5, agar 20 ± 2, pH7.0-7.2,121 ± 5 DEG C of sterilizing 15 ± 1min.
Described step (2) bacterial strain shaking flask liquid culture: from the inclined-plane kind that activation culture is good, being inoculated into transfering loop picking one ring lawn is equipped with in 500 ± 20mL triangular flask of 50 ± 2mL seed culture medium, 34-37 DEG C, 180 ± 10rpm cultivates 16 ± 1h, then getting 1 ± 0.1mL bacterium liquid transfers into 50 ± 2mL fresh seeds substratum, 34-37 DEG C, 180 ± 10rpm cultivate 5 ± 0.5h and enter logarithmic phase; Seed culture based formulas (g/L): glucose 20 ± 2, potassium primary phosphate 1 ± 0.1, yeast powder 1 ± 0.1, urea 2 ± 0.2, pH7.0-7.2,121 ± 5 DEG C of sterilizing 15 ± 1min.
Described step (6) 24 orifice plate screening: the single bacterium colony grown on protoplast regeneration substratum 24 orifice plates are carried out just screening.A. seed culture: each hole fills 2 ± 0.2mL seed culture medium, inoculate 1 mutagenesis individual plant, 34-37 DEG C, 180 ± 10rpm cultivate 16 ± 1h.B. fermentation culture: each hole fills 0.9 ± 0.05mL fermention medium, inoculation 0.1 ± 0.01mL, 37 ± 2 DEG C, 300 ± 10rpm cultivates 60 ± 2h.Screen the high individual plant of productive rate by the inosine productive rate detecting each individual plant and carry out preservation.Fermentative medium formula (g/L): glucose 130 ± 5, yeast powder 25 ± 2, potassium primary phosphate 2.5 ± 0.5, ammonium sulfate 10 ± 1, magnesium sulfate 1.5 ± 0.2, peptone 12 ± 1, guanine 0.1 ± 0.01, monosodium glutamate 20 ± 2, pH7.0-7.2,121 ± 5 DEG C of sterilizing 15 ± 1min.
Described step (7) shake flask fermentation sieves again: the individual plant be improved the product glycosides of primary dcreening operation preservation carries out shake flask fermentation and sieves again.A. seed culture: the bottled 50 ± 3mL seed culture medium of 500mL triangle, temperature 34-37 DEG C, reciprocating shaking flask amplitude 7 ± 1cm, rotating speed 80 ± 5rpm, cultivate 16-24h.B. fermentation culture: the bottled 20 ± 2mL fermented liquid of 500mL triangle, seed inoculum size is 10%, temperature 37 ± 2 DEG C, reciprocal shaker amplitude 7 ± 1cm, rotating speed 110 ± 10rpm, cultivates 24 ± 1h.
Further again: in the composite mutagenesis method of above-mentioned inosine production bacterium, described inosine productive rate measuring method is the straight survey method of 24 orifice plate screening, and shake flask fermentation sieves paper using chromatography method again.Described straight survey method be 24 orifice plates in the centrifugal 10 ± 1min of 3500 ± 500rpm, after getting supernatant liquor dilution 2 ~ 3 times, the uv-absorbing light value detected under 260 ± 10nm wavelength by multi-functional microplate reader.Described paper chromatography method is that fermented liquid is in the centrifugal 15 ± 2min of 10000 ± 500rpm, after getting supernatant liquor dilution 2 ~ 3 times, 10 ± 1 μ L points are got on filter paper with point needle, chromatographic solution is propyl carbinol: acetic acid: water=4:1:1, after launching 2 ~ 3, inosine spot is cut water-bath 70 ± 5 DEG C of constant temperature in hydrochloric acid elutriant and soak 30 ± 5min, 260nm wavelength detecting uv-absorbing light value.
Compared with prior art, beneficial effect of the present invention is as follows: one is the protoplastis after sloughing cell walls, and mutagenic compound more easily enter bacterium internal structure, can act on genetic material in cell rapidly, the probability that genetic material morphs is larger, efficiency is higher, and Mutagenic Effect can be more obvious; Two be atmospheric pressure at room plasma body mutagenesis technical equipment simple, easy to operate, do not need vacuum condition, low and the active particle of plasma jet temperature is evenly distributed, environment and people are safe from harm: three be through mutagenesis after, obtain the mutagenic strain that inosine productive rate significantly improves, there is good genetic stability, produce glycosides concentration and bring up to 72g/L from 60g/L, transformation efficiency improves 20%, Be very effective.
Embodiment
Embodiment 1: the mutagenic breeding method of subtilis protoplastis
(1) bacterial strain activation culture: the subtilis glycerine kind of-72 DEG C of cryopreservations is transferred to slant medium and carries out activation culture, 34-37 DEG C of constant temperature culture 20-24h; Slant culture based formulas (g/L): peptone 3, extractum carnis 5, yeast extract paste 5, agar 20, pH7.0-7.2,121 DEG C of sterilizing 15min.
(2) bacterial strain shaking flask liquid culture: from the inclined-plane kind that activation culture is good, be inoculated into transfering loop picking one ring lawn and be equipped with in the 500mL triangular flask of 50mL seed culture medium, 34-37 DEG C, 180rpm cultivate 16h.Then getting 1mL bacterium liquid transfers into 50mL fresh seeds substratum, and 34-37 DEG C, 180rpm cultivate 5h and enter logarithmic phase.Seed culture based formulas (g/L): glucose 20, potassium primary phosphate 1, yeast powder 1, urea 2, pH7.0-7.2,121 DEG C of sterilizing 15min.
(3) bacterial enzyme frees wall: take the logarithm later stage bacterium liquid 10mL, and the centrifugal 10min of 4000r/min, abandons supernatant, centrifugal after thalline SMM liquid is washed 2 times, abandons supernatant liquor.Thalline is suspended in 5mL SMM liquid again, then adds 5mL lysozyme soln, mixing, after being placed in 36 DEG C of water bath heat preservation process 30min, the centrifugal 10min of 4000r/min, abandons supernatant liquor, ooze SMM liquid with height to wash 2 times and dezymotize, protoplastis is suspended in 5mL SMM liquid for subsequent use.SMM liquid preparation (mol/L): sucrose 0.5, maleic acid 0.02, MgCl
20.02, pure water is prepared, and pH6.5,121 DEG C of sterilizing 15min, 4 DEG C save backup.Lysozyme soln is prepared: take N,O-Diacetylmuramidase, be dissolved in SMM liquid, enzyme concn is 100g/L, and 0.22 μM of sterilizing filter is degerming, and-20 DEG C save backup.
(4) atmospheric pressure at room plasma body mutagenesis: protoplast suspension SMM liquid is diluted to finite concentration, drawing 10 μ L is placed on slide glass, atmospheric pressure at room plasma body mutagenesis process 120s, and working conditions is: irradiation distance 2mm, gas flow 10slpm, irradiation power 120W.
(5) protoplast regeneration is cultivated: after getting mutagenesis and without the protoplastis SMM liquid serial dilutions of mutagenesis (contrast), draw 0.1mL/ ware protoplastis diluent in regenerated solids substratum, carry out regeneration cultivation, culture condition 36 DEG C cultivates 20h, front 4h rotating speed is 100rpm, 4-20h rotating speed is 180rpm.Protoplast regeneration culture medium prescription (g/L): glucose 20, potassium primary phosphate 1.5, yeast powder 2, agar 20, dissolves preparation with SMM liquid, pH7.0-7.2,121 DEG C of sterilizing 15min.
(6) 24 orifice plate screenings: the single bacterium colony grown on protoplast regeneration substratum 24 orifice plates are carried out just screening.A. seed culture: each hole dress 2mL seed culture medium, inoculate 1 mutagenesis individual plant, 34-37 DEG C, 180rpm cultivate 16h.B. fermentation culture: each hole dress 0.9mL fermention medium, inoculation 0.1mL, 37 DEG C, 300rpm cultivates 60h.Screen the high individual plant of productive rate by the inosine productive rate detecting each individual plant and carry out preservation.Fermentative medium formula (g/L): glucose 130, yeast powder 25, potassium primary phosphate 2.5, ammonium sulfate 10, magnesium sulfate 1.5, peptone 12, guanine 0.1, monosodium glutamate 20, pH7.0-7.2,121 DEG C of sterilizing 15min.
(7) shake flask fermentation sieves again: the individual plant be improved the product glycosides of primary dcreening operation preservation carries out shake flask fermentation and sieves again.A. seed culture: the bottled 50mL seed culture medium of 500mL triangle, temperature 34-37 DEG C, reciprocating shaking flask amplitude 7cm, rotating speed 80rpm, cultivate 16-24h.B. fermentation culture: the bottled 20mL fermented liquid of 500mL triangle, seed inoculum size is 10%, temperature 37 DEG C, reciprocal shaker amplitude 7cm, rotating speed 110rpm, cultivates 24h.
Embodiment 2: the mensuration of inosine productive rate
Inosine productive rate measuring method is the straight survey method of 24 orifice plate screening, and shake flask fermentation sieves paper using chromatography method again.
Straight survey method is, 24 orifice plates, in the centrifugal 10min of 3500rpm, are got after supernatant liquor dilutes 2 times, the uv-absorbing light value detected under 260nm wavelength by multi-functional microplate reader.
Paper chromatography method is, fermented liquid is in the centrifugal 15min of 10000rpm, get after supernatant liquor dilutes 2 times, 10 μ L points are got on filter paper with point needle, chromatographic solution is propyl carbinol: acetic acid: water=4:1:1, after launching 2h, inosine spot is cut water-bath 70 DEG C of constant temperature in hydrochloric acid elutriant and soak 30min, 260nm wavelength detecting uv-absorbing light value.
Embodiment 3: genetic stability is tested
By the glycerine kind activation culture of cryopreservation, this is the first-generation; Choose a ring bacterium from first-generation inclined-plane kind transfering loop and be transferred to new slant culture, this is the s-generation; Choose a ring bacterium from s-generation inclined-plane kind transfering loop and be transferred to new slant culture, this is the third generation; By that analogy, reached for the 50 generation.Will wherein seven generation bacterial strain carry out shake flask fermentation test, detect fermented liquid inosine productive rate by embodiment 2 method, and detect with efficient liquid-phase chromatography method and correct, seven generation its inosine productive rate of bacterial strain all at about 70-74g/L.
Claims (10)
1. a composite mutagenesis method for inosine production bacterium, is characterized in that: it is on subtilis protoplastis, carry out the mutagenesis of atmospheric pressure at room plasma body.
2. the composite mutagenesis method of inosine production bacterium according to claim 1, comprise the following steps: (1) bacterial strain activation culture, (2) bacterial strain shaking flask liquid culture, (3) bacterial enzyme frees wall, (4) atmospheric pressure at room plasma body mutagenesis, (5) protoplast regeneration is cultivated, (6) 24 orifice plate screenings, and (7) shake flask fermentation sieves again; Step (1) is subtilis to the bacterium in (7).
3. the composite mutagenesis method of inosine production bacterium according to claim 2, is characterized in that:
Described step (3) bacterial enzyme frees wall: take the logarithm phase bacterium liquid 10 ± 2mL, and the centrifugal 10 ± 2min of 4000 ± 50r/min, abandons supernatant liquor, centrifugal after thalline SMM liquid is washed 2 ~ 3 times, abandons supernatant liquor; Again thalline is suspended in 5mL ± 1SMM liquid, then 5 ± 1mL lysozyme soln is added, mixing, after being placed in 36 ± 3 DEG C of water bath heat preservation process 30 ± 2min, centrifugal 10 ± the 2min of 4000 ± 50r/min, abandon supernatant liquor, ooze SMM liquid washing 2 ~ 3 removing N,O-Diacetylmuramidases with high, protoplastis is suspended in 5 ± 1mL SMM liquid for subsequent use;
Described step (4) atmospheric pressure at room plasma body mutagenesis: it is 0.4-0.8 that protoplast suspension SMM liquid is diluted to OD value, drawing 10 ± 1 μ L is placed on slide glass, atmospheric pressure at room plasma body mutagenesis process 120 ± 10s, working conditions is: irradiation distance 2+1mm, gas flow 10 ± 1slpm, irradiation power 120 ± 10W.
4. the composite mutagenesis method of inosine production bacterium according to claim 3, is characterized in that: it is as follows that described height oozes the configuration of SMM liquid: sucrose 0.5 ± 0.05mol/L, maleic acid 0.02 ± 0.002mol/L, MgCL
20.02 ± 0.002mol/L, pure water configuration pH 6.5.
5. the composite mutagenesis method of inosine production bacterium according to claim 4, is characterized in that:
Protoplastis SMM liquid serial dilutions after described step (5) protoplast regeneration is cultivated and got mutagenesis with without mutagenesis is 10
-1~ 10
-8, draw 0.1 ± 0.01mL/ ware protoplastis diluent in regenerated solids substratum, carry out regeneration cultivation, culture condition 36 ± 3 DEG C cultivates 20 ± 2h, and front 4 ± 1h rotating speed is 100 ± 10rpm, 4-20h rotating speed is 180 ± 10rpm.
6. the composite mutagenesis method of inosine production bacterium according to claim 5, is characterized in that: it also comprises the detection of step (8) mitotic stability: by the glycerine kind activation culture of cryopreservation, this is the first-generation; Choose a ring bacterium from first-generation inclined-plane kind transfering loop and be transferred to new slant culture, this is the s-generation; Choose a ring bacterium from s-generation inclined-plane kind transfering loop and be transferred to new slant culture, this is the third generation; By that analogy, reached for the 50th generation, by 50 generation bacterial strain carry out shake flask fermentation test, and detect fermented liquid inosine productive rate.
7. the composite mutagenesis method of the inosine production bacterium according to any one of claim 2-5, is characterized in that:
The subtilis glycerine kind of-72 ± 2 DEG C of cryopreservations is transferred to slant medium and carries out activation culture by described step (1) bacterial strain activation culture, cultivates 20-24h for 34-37 DEG C;
Described step (2) bacterial strain shaking flask liquid culture: from the inclined-plane kind that activation culture is good, being inoculated into transfering loop picking one ring lawn is equipped with in 500 ± 20mL triangular flask of 50 ± 2mL seed culture medium, 34-37 DEG C, 180 ± 10rpm cultivates 16 ± 1h, then getting 1 ± 0.1mL bacterium liquid transfers into 50 ± 2mL fresh seeds substratum, 34-37 DEG C, 180 ± 10rpm cultivate 5 ± 0.5h and enter logarithmic phase;
Described step (6) 24 orifice plate screening: the single bacterium colony grown on protoplast regeneration substratum 24 orifice plates are carried out just screening;
Described step (7) shake flask fermentation sieves again: the individual plant be improved the product glycosides of primary dcreening operation preservation carries out shake flask fermentation and sieves again.
8. the composite mutagenesis method of inosine production bacterium according to claim 7, is characterized in that: described inosine productive rate measuring method is the straight survey method of 24 orifice plate screening, and shake flask fermentation sieves paper using chromatography method again.
9. the composite mutagenesis method of inosine production bacterium according to claim 8, it is characterized in that: described straight survey method is that 24 orifice plates are in the centrifugal 10 ± 1min of 3500 ± 500rpm, after getting supernatant liquor dilution 2 ~ 3 times, the uv-absorbing light value detected under 260 ± 10nm wavelength by multi-functional microplate reader.
10. the composite mutagenesis method of inosine production bacterium according to claim 8, it is characterized in that: described paper chromatography method is that fermented liquid is in the centrifugal 15 ± 2min of 10000 ± 500rpm, after getting supernatant liquor dilution 2 ~ 3 times, 10 ± 1 μ L points are got on filter paper with point needle, chromatographic solution is propyl carbinol: acetic acid: water=4:1:1, after launching 2 ~ 3, inosine spot is cut water-bath 70 ± 5 DEG C of constant temperature in hydrochloric acid elutriant and soak 30 ± 5min, 260nm wavelength detecting uv-absorbing light value.
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| CN115820597A (en) * | 2022-12-08 | 2023-03-21 | 深度进化(广州)生物技术有限公司 | Preparation method of modified molecular enzyme and molecular immunochromatography detection method thereof |
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Effective date of registration: 20170505 Address after: 151100, eight North Street, Zhaodong, Heilongjiang, Suihua Applicant after: Zhaodong Xinghu Biotechnology Co. Ltd. Address before: 526040 Duanzhou City, Guangdong Province workers and peasants North Road, No. 67, No. Applicant before: Xinghu Biotech Co., Ltd., Zhaoqing City, Guangdong Prov. |
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Application publication date: 20150422 |