CN104830891A - An efficient preparing method of a banded krait antibacterial peptide Cath-BF30 - Google Patents
An efficient preparing method of a banded krait antibacterial peptide Cath-BF30 Download PDFInfo
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Abstract
An efficient preparing method of a banded krait antibacterial peptide Cath-BF30 is disclosed. The method includes following steps: designing and synthesizing a Cath-BF30 gene having a sequence of SEQ ID NO.1; cloning the synthesized gene segment into a pGAPZalpha vector by utilization of double-enzyme restriction with Xho I and Xba I to obtain a recombinant expression plasmid pGAPZalpha-Cath-BF30; transforming a pichia pastoris cell by the recombinant expression plasmid to obtain an engineering bacterium SMD-pGAPZalpha-30; allowing the engineering bacterium to ferment under a condition having a pH value lower than 5; filtering fermentation supernatant by a filter membrane having a size of 0.45 mum to remove impurities; and separating and purifying by adoption of an SP Sepharose FF column. The method can achieve efficient secretory expression and can ensure that a secretion product is provided with a natural N-terminal sequence. The secretory expression product has activity for inhibiting propionibacterium acnes. The Cath-BF30 of high purity can be obtained only by one step of ion exchange from the fermentation supernatant.
Description
Technical field
The invention belongs to polypeptide drugs technical field.More specifically, the high efficiency preparation method of a kind of Bungarus fasciatus antibacterial peptide Cath-BF30 is related to.
Background technology
Acne is a kind of common pilosebaceous inflammations dermatoses, caused by anaerobism propionibacterium acnes, mainly adopt antibiotic therapy at present, but curative effect is undesirable, and can cause generation and the allergy of resistance bacterium.Recently research finds that the antibacterial peptide Cath-BF be separated from krait venom effectively can suppress the growth of propionibacterium acnes and other acne infection bacterium in mouse model, and suppresses related inflammatory hyperplasia, therefore in acne control, has important using value.The antibacterial peptide Cath-BF that should be separated from krait venom belongs to first by the vertebrate origin antibacterial peptide reported, and the Cath-BF of natural separation is the polypeptide of 30 amino-acid residue compositions, therefore can be denoted as Bungarus fasciatus antibacterial peptide Cath-BF30.
In real work, due to venin-derived limited, therefrom prepare antibacterial peptide Cath-BF30 yield low, cost is high, and chemosynthesis antibacterial peptide exists the high problem of cost equally, synthesis difficulty, expensive, is also difficult to apply.Therefore, set up a kind of high-efficiency method for producing applying of Cath-BF is had important practical significance.
Summary of the invention
The technical problem to be solved in the present invention is the deficiency overcoming existing Bungarus fasciatus antibacterial peptide Cath-BF30 technology of preparing, a kind of method efficiently preparing Bungarus fasciatus antibacterial peptide Cath-BF30 is provided, successfully construct the efficient constitutive and secretive expression system of pichia spp and the expression product separation purification method of Bungarus fasciatus antibacterial peptide Cath-BF30, and experiment demonstrates and prepares gained 30 PEPC ath-BF and have Propiobacterium inhibit activities.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The high efficiency preparation method of a kind of Bungarus fasciatus antibacterial peptide Cath-BF30, first design Cath-BF30 gene order as shown in SEQ ID NO.1, recycling expression vector establishment recombinant bacterial strain, after engineering bacteria ferments under the condition of pH lower than 5, carry out separation and purification through strong cation SP Sepharose FF post, obtain Bungarus fasciatus antibacterial peptide Cath-BF30.
The concrete steps of above-mentioned high efficiency preparation method are as follows:
S1. design Cath-BF30 gene, sequence as shown in SEQ ID NO.1, and synthesizes this gene fragment; Specifically adopt the encoding sequence of pichia spp preferred codons design Cath-BF30, head and the tail add restriction enzyme site, protection base, terminator codon etc. respectively, adopt primer Overlap extension PCR method (Overlap extension PCR) to synthesize goal gene;
S2. Cath-BF30 cloning vector is built: utilize
xhoi with
xbai double digestion, (Cath-BF30 goal gene and the secreting signal peptide MF-α of synthesis merge rear clone in the downstream of promotor GAP the Cath-BF30 gene fragment clone of synthesis to be entered Bichi yeast system pGAPZ α carrier, so that constitutive and secretive expression N holds the antibacterial peptide for native sequences do the condition of basic carbon source at glucose under), obtain recombinant expression plasmid pGAPZ α-Cath-BF30;
S3. build Cath-BF30 Pichia yeast engineering: the method transformed by electricity, recombinant expression plasmid pGAPZ α-Cath-BF30 is transformed Pichia pastoris, obtain engineering bacteria SMD-pGAPZ α-30;
S4. ferment: at pH lower than under 5,30 DEG C of conditions, be that the YPD substratum of carbon source ferments with glucose, 72h is cultivated in 200rmp concussion;
S5. purifying: fermentation supernatant, after 0.45 μm of membrane filtration removing impurity, adopts strong cation SP Sepharose FF post to carry out separation and purification.
Wherein, step S2 builds in cloning vector process, and Cath-BF30 gene fragment and the double digestion product of pGAPZ α carrier are connected according to the ratio of 4:1, and the condition of described connection is 16 DEG C and connects 9 hours.
Can embodiment as one, transform described in step S3 is the method utilizing electricity to transform, the transformant obtained carries out bleomycin screening (by Antibiotics of Low Concentration to the setting-out of high density microbiotic) and the antibacterial spot screening of propionibacterium successively, positive through PCR qualification again, obtain engineering bacteria SMD-pGAPZ α-30.
Preferably, the concrete grammar of step S3 structure engineering bacteria is as follows:
S31. by after recombinant expression plasmid pGAPZ α-Cath-BF30 linearizing, electric transforming protein deficient host pichia spp SMD1168 competent cell;
S32. after electricity transforms, first screen the resistance transformant of bleomycin, then the culture supernatant of getting the resistance transformant of bleomycin carries out the antibacterial spot screening of propionibacterium, chooses the transformant strong to the bacteriostatic activity of propionibacterium;
S33. strong to the bacteriostatic activity of S32 screening transformant carries out PCR qualification, and identify that whether it is containing Cath-BF30 goal gene fragment, positive strain is engineering bacteria SMD-pGAPZ α-30.
Preferably, the inoculum size of fermenting described in step S4 is 1 ~ 5v/v %; The condition of described fermentation be pH=4 ~ 5,28 ~ 30 DEG C, 170 ~ 200rmp shakes cultivation 70 ~ 72h; Described YPD substratum take glucose as the YPD substratum of carbon source.
More preferably, the inoculum size of described fermentation is 1%, and the pH of described fermentation is 4, and leavening temperature is 30 DEG C.
Fermentation supernatant described in step S5 needs first after the centrifugal 20 ~ 30min of 4000 ~ 5000rmp, gets supernatant, is diluted with water to and balance liquid same electrical conductance, then removes impurity with 0.45 μm of membrane filtration; Described balance liquid is the balance liquid of balance SP Sepharose FF post; Described SP Sepharose FF post is strong cation SP Sepharose FF post.
In addition, after step S4 has fermented, fermentation supernatant 0.45 μm of membrane filtration impurity, freezingly vacuumize drying, and dissolve freeze-drying sample with distilled water, obtain antibacterial peptide Cath-BF30 crude samples, 96 well plate method observe crude samples to the growth-inhibiting effect of propionibacterium acnes anaerobic cultures, namely activation analysis is carried out to produced antibacterial peptide Cath-BF30, after confirming that it has bacteriostatic activity, then carry out the purifying of step S5.This step is significant when large-scale practical application.
In addition, preferably, recombinant expression plasmid pGAPZ α-Cath-BF30 described in step S31 utilizes
blni enzyme makes its linearizing, and the plasmid of total Linearization is carried out phenol chloroform, make its reach electricity transform needed for concentration.
The electric shock condition that described in step S31, electricity transforms is 1.5kv, 25 μ F, 200 Europe; Converted product incubation conditions is 30 DEG C of incubation 1h.
More preferably, the concrete operations that described in step S31, electricity transforms are as follows:
Linearizing pGAPZ α-Cath-BF30 plasmid is added in pichia spp SMD1168 competent cell, mix gently, be placed at 10min in the quartz transition cup of the 0.2cm of precooling in advance on ice, transform cup and should be soaked in 75% alcohol before doing conversion prerequisite, and sterilize half an hour under ultraviolet lamp; Dry the water transforming cup outer wall, be put in electric conversion instrument groove, 1.5kv, 25 μ F, 200 Europe, electric shock, add the sorbyl alcohol mixing of 1mL precooling immediately, converted product is placed in 30 DEG C of incubation 1h.
In addition, more specifically, described in step S4, the method concrete operations of engineering bacteria SMD-pGAPZ α-30 large scale fermentation are as follows:
(1) single colony inoculation of picking engineering bacteria SMD-pGAPZ α-30 is in YPD substratum, and 30 DEG C of concussions are spent the night, and obtains seed liquor;
(2) with 1v/v% inoculum size, seed liquor be inoculated in YPD substratum, 30 DEG C of concussions are spent the night;
(3) carry out high density fermentation, working volume is 5L, rotating speed, air flow, dissolved oxygen, pH(optimal pH 4 ~ 5) and feed supplement (monitoring dissolved oxygen, the flow feeding when the sugar consumption in basic medium is complete) be all that adjustment is set by master control system;
(4) thalline weight in wet base is surveyed every 6h;
(5) 72h closed cans, centrifuging and taking supernatant.
This institute builds the pichia spp system that engineering bacteria is constitutive expression system, need not add methyl alcohol during the fermentation to induce, only need stream to add glucose, feed supplement is associated with dissolved oxygen, parameter as shown in Figure 9, X-coordinate is fermentation time, and ordinate zou is relative value, as dissolved oxygen is 100% to the maximum, rotating speed is 700 to the maximum, represent with 70 in the drawings, temperature is within the scope of 28 ~ 30 DEG C, and pH is between 4 ~ 5.When dissolved oxygen start to drop to a certain degree start to rise time, this experiment is 18th hour, and the sugar in basic medium runs out of, now need flow feeding, control dissolved oxygen about 30%, within every six hours, survey a bacterium weight in wet base, as shown in Figure 10, to ferment closed cans to 72h.
The Bungarus fasciatus antibacterial peptide Cath-BF30 prepared according to above-mentioned high efficiency preparation method is also within protection scope of the present invention.
The present invention adopts genetic engineering means to prepare Cath-BF30 first, and establishes efficient preparation method.Adopt the efficient expression engineering of pichia spp constitutive and secretive expression system constructing Cath-BF30.Pichia spp used (Pichia pastoris) system is the most ripe exogenous gene high-efficient expressed system, and genetic background is clear, can high density fermentation, thus has good industrial performance.Its constitutive expression system is the strong promoter pGAP based on phosphoglyceraldehy-de dehydrogenase, and without the need to adopting methanol induction as AOX1 promotor, the production for people's product is safer.In addition, Cath-BF30 is as micromolecule polypeptide, and be difficult to independent expression, secreting, expressing can avoid little peptide by intracellular protein enzyme liberating, and the easy purifying of product.Meanwhile, the present invention prepares the bacteriostatic activity of gained antibacterial peptide Cath-BF30 by vitro study, and establishes high-efficiency fermenting technique and separation purification method, for the further Application and Development of Cath-BF30 lays a solid foundation.
The Cath-BF30 high efficiency preparation method that the present invention sets up is that contriver is explored by great many of experiments and studied and draws.Those skilled in the art are fully aware of, build in the process of engineering bacteria utilizing gene engineering method, the design of gene order is very crucial, the specific gene order designed is unique due to it, and the factors such as the selection on expression vector, the selection on restriction enzyme site all can affect the success or not of engineering bacteria structure, the efficiency of structure and quality.The technical barrier that first the present invention runs into is exactly gene order problem, although Bungarus fasciatus antibacterial peptide Cath-BF sequencing and analyzing is known, but as foreign gene be directly used in express in Bichi yeast system time, because the existence of a large amount of rare codon and can not high expression, therefore, our gene order of Cath-BF that adopted pichia spp preference codon to redesign, and through a large amount of adjustment and checking, again in conjunction with suitable restriction enzyme site, protection base, terminator codon etc., finally draw the Cath-BF30 gene order as shown in SEQ ID NO.1.
In addition, when by goal gene Direct Cloning when the multiple clone site of commercial expression vector pGAP-z α, the N end of expression product can have more several non-natural aminoacid sequence, affects antibacterial peptide active, also easily causes immune response for during human body.Secretion signal peptide sequence MF-α and antibacterial peptide Cath-BF gene are directly merged by gene chemical synthesis by the present invention, the XbaI site in the XhoI site and multiple clone site that are positioned at MF-α upstream in recycling pGAP-z α is cloned, the correct secreting, expressing of antibacterial peptide can be guaranteed like this, and secreting, expressing product has natural N terminal sequence, is extremely conducive to follow-up separation and purification.
In zymotechnique, although Pichia yeast is widely used in the production of foreign protein, but be not just can determine technological condition for fermentation according to the Guide Book of routine, because the growth required for different destination gene expressions is different from expression condition, the ability of different foreign protein opposing Host Strains proteasome degradations is different.The present invention is found by large quantifier elimination, the engineering bacteria SMD-pGAPZ α-30 of constructed antibacterial peptide Cath-BF must could high expression under the condition of pH lower than 5, and could reduce degraded under this pH condition, therefore during fermentation, the control of pH becomes the key being fermented into merit.PH controls well, antibacterial peptide ability high expression; Control bad, just can not even not express by high expression.Up to now, adopt bacillus coli gene engineering expression system, subtilis expression system expresses having of antibacterial peptide, but the antibacterial peptide expressed also needs cutting or sex change, renaturation, cutting just to have activity.In addition, yet there are no adopt yeast particularly pichia yeast expression system prepare the report of Cath-BF, one of them major reason is exactly that processing condition do not find out, and when large scale fermentation, expression level is low or do not express.
In separation and purification, as everyone knows, the downstream technique such as separation and purification are the bottlenecks of biotechnology industry, namely technological difficulties place.Different protein or polypeptide are all personalized, do not have a kind of universal method to can be used for the purifying of all proteins.Prior art, in purifying process, generally can adopt two or more chromatographies could obtain high purity.And the present invention adopts a step ion exchange method namely to obtain highly purified antibacterial peptide Cath-BF30, this high-efficiency purifying method makes the manpower of Bungarus fasciatus antibacterial peptide Cath-BF, time, production cost greatly reduce.
The novel process preparing antibacterial peptide Cath-BF30 efficiently that the present invention sets up mainly comprises the structure of efficient expression engineering, the foundation of the zymotechnique of high expression and the foundation of high efficiency separation purifying process, three is indispensable, provides solid theoretical basis to the further genralrlization application of Bungarus fasciatus antibacterial peptide Cath-BF.
Research of the present invention mainly achieves following result:
(1) successful design has cloned Cath-BF30 encoding sequence, and this sequence is based on the design of pichia spp preference codon, and N end and the seamless fusion in secreting signal peptide MF α downstream, can ensure that secretory product is natural N end sequence.
(2) obtain the pichia spp efficient secretory expression bacterial strain of Cath-BF30, secreting, expressing product has the activity suppressing Propiobacterium.
(3) engineering bacteria SMD-pGAPZ α-30 secretion level under medium pH 4.0 condition is the highest, product is the most stable, when pH is more than 5, culture supernatant anti-microbial activity is low, and the phase almost can't detect anti-microbial activity after incubation, show that needs cultivate one's ability under lower pH condition and effectively resist the Degradation of host cell proteins enzyme.
(4) engineering bacteria SMD-pGAPZ α-30 cultured continuously in the YPD substratum doing carbon source with glucose, and when taking carbon source control device, growth and the efficient secretory expression of higher density can be realized, final cell density reaches 160g/L(weight in wet base) more than, antibacterial peptide secretion level reaches more than 100mg/L.
(5) fermentation supernatant can obtain the Cath-BF30 of purity more than 95% through SP Sepharose FF post single step purification.
The present invention has following beneficial effect:
The invention discloses the high efficiency preparation method of a kind of Bungarus fasciatus antibacterial peptide Cath-BF30, be first adopt pichia spp preference codon to design Cath-BF30 gene order as shown in SEQ ID NO.1, again goal gene and secreting signal peptide partial gene sequence are merged rear clone to expression vector pGAPz α, build constitutive and secretive expression recombinant bacterial strain, after engineering bacteria ferments under the condition of pH lower than 5, carry out separation and purification through strong cation SP Sepharose FF post, obtain Bungarus fasciatus antibacterial peptide Cath-BF30.There is following significant progress:
1) adopt gene engineering method to establish the high-efficiency method for producing of Bungarus fasciatus antibacterial peptide Cath-BF30 first, and ensure that secretory product is natural N end sequence, prepare gained Bungarus fasciatus antibacterial peptide Cath-BF30 and there is the activity suppressing Propiobacterium;
2) the method can realize high expression secretion, and only needs a step ion-exchange can obtain highly purified Cath-BF30(purity higher than more than 95%);
3) efficient expression system of the Cath-BF30 set up does not rely on methanol induction, for human body safety.
The method of the present invention not only can realize the efficient secretory expression of antibacterial peptide, and ensure that secretory product has natural N end sequence, expression product has the activity suppressing Propiobacterium, and fermentation supernatant only needs a step ion-exchange can obtain high-purity C ath-BF30, the research of Bungarus fasciatus antibacterial peptide Cath-BF and further genralrlization application are had great importance.
Accompanying drawing explanation
Fig. 1 is Bungarus fasciatus antibacterial peptide Cath-BF30 gene order.
Fig. 2 is antibacterial peptide Cath-BF30 goal gene synthesis strategy.
Fig. 3 is Cath-BF30 goal gene amplified production electroresis appraisal.
Fig. 4 is cloning vector pGAPZ α-Cath-BF30 schematic diagram.
Fig. 5 is cloning vector pGAPZ α-Cath-BF30 electroresis appraisal figure.
Fig. 6 is high density antibiotic-screening height copy transformant.
Fig. 7 is that pichia spp transformant PCR identifies, M:DNA Marker; 1-7: different transformant goal gene amplified production.
Fig. 8 be Cath-BF30 transformants secrete product to the growth-inhibiting of propionibacterium acnes, 1-6: different transformants secrete product; SMD: unconverted Host Strains secretory product.
Fig. 9 is engineering bacteria SMD-pGAPZ α-30 large scale fermentation Parameters variation figure.
Figure 10 is the engineering bacteria growth curve under engineering bacteria SMD-pGAPZ α-30 fermentation condition.
Figure 11 is SP sepharose FF cation exchange column Cath-BF30 expression product, M: low molecular weight protein (LMWP) Marker, 1: fermentation supernatant, 2-3: elution peak point sample.
Embodiment
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention and material are commercial.
the Design and synthesis of embodiment 1 Bungarus fasciatus antibacterial peptide Cath-BF30 goal gene
1, gene order design
According to the aminoacid sequence of the 30 PEPC ath-BF that known snake venom antibacterial peptide Cath-BF30 gene (as open in patent 200810058976.9) is translated, select pichia spp preferred codons, translated out, select
xhoi site with
xbai site is as its restriction enzyme site, and head and the tail add protection base, the bases such as terminator codon respectively, and design Cath-BF30 goal gene, the Cath-BF30 goal gene sequence designed is as shown in SEQ ID NO.1.
In addition, as shown in Figure 1, synthesized full length gene 113bp, comprises 30 amino acid and translates the sequence, terminator codon, protection base, restriction enzyme site etc. of coming according to pichia spp preferred codons.
2, goal gene synthesis
The Cath-BF30 goal gene sequence designed is divided into three sections of base sequences having tumor-necrosis factor glycoproteins each other, adopts primer Overlap extension PCR method (Overlap extension PCR) to synthesize goal gene whole section of sequence.
Synthesis strategy as shown in Figure 2.Three sections of sequences template each other between two, reflection system (50 μ L): 2 × PCR Mix 25 μ L, P1 2 μ L, P2 2 μ L, P3 2 μ L, ddH
2o 19 μ L.Mixing, on brief centrifugation machine centrifugal several seconds, 94 DEG C of denaturation 5min, annealing temperature was 55 DEG C, cycle index 25 times, and elongating temperature is 72 DEG C, gets PCR primer 2 μ L, 2% agarose gel electrophoresis, and record is taken pictures.PCR primer is reclaimed the clean recovery of test kit with clean, namely obtains Cath-BF30 goal gene fragment ,-20 DEG C of preservations.
Result obtains the specific amplification products of the same size with theory, 2% agarose gel electrophoresis detected result as shown in Figure 3, the fragment that in electrophorogram, visible molecular weight is consistent.
the structure of embodiment 2 Bungarus fasciatus antibacterial peptide Cath-BF30 cloning vector
1, genetically engineered operation is carried out according to Molecular Cloning: A Laboratory Directory Method, and the Cath-BF30 goal gene synthesize PCR and vector plasmid PGAPZa use respectively
xhoi with
xbai double digestion, then connect conversion.Specific procedure is as follows:
(1) acquisition of vector plasmid PGAPZa: 37 DEG C, 170rmp, incubated overnight contains the intestinal bacteria of PGAPZa plasmid, extracts plasmid, gel electrophoresis ,-20 DEG C of preservations with the little extraction reagent kit of plasmid.
(2) double digestion: vector plasmid PGAPZa and Cath-BF30 gene are used respectively
xhoi with
xbai double digestion, 37 DEG C of water-bath 2h, gel electrophoresis, whether double digestion is complete to observe it, if double digestion is complete all for object fragment and carrier, double digestion product is reclaimed test kit with DNA glue and reclaims, gel electrophoresis again, observe band brightness and determine its content.
(3) connect and transform: by double digestion product vector plasmid PGAPZa and the Cath-BF30 that the reclaims ratio according to 1:4,16 DEG C connect 9 hours, proceeded to DH5a competent cell, the preparation of E. coli competent see concrete document, ice bath 30min, 42 DEG C of water-bath exactly 90s, ice bath 10min again, add 1mL low salt LB medium 37 DEG C and place 1h, getting 100 μ L, to coat containing Zeicon bleomycin be on the less salt LB flat board of 25 μ g/mL, and 16h cultivated by 37 DEG C of incubators.
(4) qualification of recombinant expression plasmid: picking 10 ~ 20 recons, to containing in the liquid low salt LB medium of 25 μ g/mL bleomycins, extract plasmid, carry out plasmid size, PCR colony identification, and enzyme cut qualification.The recon 3 screened through preliminary evaluation sends to the order-checking of Guangzhou Hua Da genome company.The recon called after that sequencing result is correct: pGAPZ α-Cath-BF30.
The method of above-mentioned structure cloning vector is based on Bichi yeast system pGAPZ α carrier, object fragment and secreting signal peptide MF-α merge rear clone in the downstream of promotor GAP, so that constitutive and secretive expression N holds the antibacterial peptide for native sequences do the condition of basic carbon source at glucose under.
The schematic diagram of the cloning vector pGAPZ α-Cath-BF30 2, built as shown in Figure 4.PCR primer warp
xhoi and
xbai double digestion rear clone is in the corresponding site of pGAPZ α, through sequential analysis qualification, the object fragment sequence of cloning and the encoding sequence of Cath-BF completely the same, the expression vector that obtains is named as pGAPZ α-Cath-BF30, carrier size is 3200bp, as shown in Figure 5.
embodiment 3 builds engineering bacteria SMD-pGAPZ α-30
1, recombinant expression plasmid pGAPZ α-Cath-BF30 linearizing
Extract pGAPZ α-Cath-BF30 plasmid in a large number with the large extraction reagent kit of plasmid, and use
blni enzyme makes its linearizing.200 μ L linearizing systems are: 10 μ L
blni, 40 μ L pGAPZ α-Cath-BF30(about 20 μ g), 20 μ L 10 × buffer, 130 μ L ddH
2o.After abundant mixing, 37 DEG C of enzymes cut through night, get 2 μ L digestion products and do 1% agarose gel electrophoresis, observe its whether total Linearization.
2, the plasmid of total Linearization is carried out phenol chloroform, make it reach electricity and transform desired concentration, concrete steps are as follows:
(1) enzyme is cut in system 200 μ L and add 300 μ L ddH
2o, makes its cumulative volume be 500 μ L.
(2) add the phenol chloroform of 500 μ L once, 12000rpm, 10min, carefully get the upper strata in layering.
(3) add the 3M sodium-acetate of 1/10 volume in the upper layer, PH is 5.2, and the dehydrated alcohol of the prior precooling of 2.5 times of volumes, piping and druming mixing, be placed in-20 DEG C and place 1h, 12000rpm, 10min, abandon supernatant, and by 75% washing with alcohol twice, be placed in super clean bench and dry up, with 10 μ L ddH
2o dissolves on bottom and wall and precipitates ,-20 DEG C of preservations.
3, the competent preparation of SMD1168 pichia spp
(1) by being placed in-20 DEG C of frozen protease-deficient host pichia spp SMD1168 cell transfering loop setting-outs in YPD solid plate, be inverted, 30 DEG C of incubator 72h.
(2) picking saccharomyces albicans list bacterium colony is in 5mL liquid YPD test tube, 30 DEG C, and 200rpm shakes overnight incubation.
(3) get 0.5mL overnight culture in the Erlenmeyer flask containing 50mL YPD liquid nutrient medium, 30 DEG C of concussions are cultured to OD
600be between 1.3 ~ 1.5
(4) bacterium liquid is placed in 50mL sterile centrifugation tube, 2500rpm, 4 DEG C of centrifugal 10min, abandons supernatant and collect thalline.
(5) blow and beat bacterial sediment gently with the distilled water 50mL of pre-cold sterilization, after mixing, 1500rpm, 4 DEG C of centrifugal 10min, abandon supernatant.
(6) blow and beat bacterial sediment gently with the distilled water 25mL of pre-cold sterilization, after mixing, 1500rpm, 4 DEG C of centrifugal 10min, abandon supernatant.
(7) blow and beat bacterial sediment gently with the 1M sorbyl alcohol 10mL of pre-cold sterilization, after mixing, 1500rpm, 4 DEG C of centrifugal 10min, abandon supernatant.
(8) precipitate with the 1M sorbyl alcohol re-suspended cell of pre-cold sterilization, make its cell concn reach OD
600be 100, be competent cell, competent cell is placed in 4 DEG C, uses as early as possible.
4, electricity transforms
(1) 10 μ L linearizing recombinant expression plasmid pGAPZ α-Cath-BF30 is added in the competence that 80 μ L have prepared, blow and beat mixing gently, be placed at 10min in the quartz transition cup of the 0.2cm of precooling in advance on ice, transform cup and should soak 75% alcohol before doing conversion prerequisite, and sterilize half an hour under ultraviolet lamp.
(2) dry the water transforming cup outer wall, be put in BioRad electricity conversion instrument groove, 1.5kv, 25 μ F, 200 Europe, electric shock, adds the sorbyl alcohol mixing of 1mL precooling immediately.
(3) converted product is placed in 30 DEG C of incubation 1h.
(4) by electric converted product in the centrifugal 2min of 1000rpm, stay part supernatant mix with precipitation, be applied on the YPDS flat board containing 100 μ g/mL bleomycin zeicon, 30 DEG C of incubations 3 ~ 5 days, the growth of observation transformant.
5, bleomycin zeicon screens high copy recon
The recon of the high copy of screening first will by transformant by Antibiotics of Low Concentration to the setting-out of high density microbiotic, specific as follows:
(1) the transformant setting-out on use sterilizing toothpick picking 100 μ g/mL zeicon microbiotic flat board is on 500 μ g/mL zeicon solid plates, and each bacterium colony setting-out length is about 1cm, is inverted, and 2 ~ 3d cultivated by 30 DEG C of incubators
(2) the transformant setting-out on use sterilizing toothpick picking 500 μ g/mL zeicon microbiotic flat board is on 1000 μ g/mL zeicon solid plates, and each bacterium colony setting-out length is about 1cm, is inverted, and 2 ~ 3d cultivated by 30 DEG C of incubators
(3) the transformant setting-out on use sterilizing toothpick picking 1000 μ g/mL zeicon microbiotic flat board is on 2000 μ g/mL zeicon solid plates, and each bacterium colony setting-out length is about 1cm, is inverted, and 2 ~ 3d cultivated by 30 DEG C of incubators
(4) transformant on picking 2000 μ g/mL zeicon solid plate, in 3mL YPDS liquid nutrient medium, 30 DEG C of concussion overnight incubation, numbering ,-20 DEG C of glycerine conservations.
This experiment is 100 μ g/mL to 500 μ g/mL with concentration gradient, 500 μ g/mL to 1000 μ g/mL, the bleomycin zeicon of 1000 μ g/mL to 2000 μ g/mL screens, finally obtain a few plant height copy transformant, as shown in Figure 6, a few strain transformant being frozen cooking method by boiling, carrying out PCR, observe PCR primer and have obvious object band, as shown in Figure 7.
6, the antibacterial spot screening of propionibacterium
Collect culture supernatant, 96 well plate method observe supernatant to the growth-inhibiting effect of propionibacterium acnes anaerobic cultures, utilize the antibacterial spot of propionibacterium to choose the strong transformant of supernatant bacteriostatic activity.
7, identify through PCR again
(1) by frozen yeast (transformant after above-mentioned bleomycin zeicon and the antibacterial spot screening of propionibacterium) activated overnight, get 1mL bacterium liquid, the centrifugal 2min of 10000rpm, abandons supernatant.
(2) with 500 μ L PBS washed cells, the centrifugal 2min of 10000rpm, washes twice, abandons supernatant.
The TE dissolution precipitation of (3) 100 μ L PH8.0, in boiling water boiling 10min, be placed in-20 DEG C freezing.
(4) cell that taking-up-20 DEG C is frozen, again boil 10min, the centrifugal 5min of 12000rpm, gets supernatant as pcr template.
(5) 20 μ L PCR reaction systems: 2 × TAQ enzyme MIX 10 μ L, upstream primer 2 μ L, downstream primer 2 μ L, template 2 μ L, ddH
2o 4 μ L.
(6) response procedures: 94 DEG C of 5min denaturations, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations, 72 DEG C of 10min, get 5 μ L PCR primer and carry out agarose gel electrophoresis, observe whether there is object clip size band.
(7) choose the positive strain that PCR identified, expression product activity identification is carried out to it.
8, engineering bacterium expression its lytic activity qualification
(1) acquisition of antibacterial peptide Cath-BF30 crude samples
Get SMD-pGAPZ α-30 glycerine kind 500 μ L in 50mL YPD liquid nutrient medium, 30 DEG C, 72h is cultivated in 200rmp concussion;
50mL culture is placed in centrifuge tube, 10000rmp, centrifugal 5min, gets supernatant, abandons precipitation.
By 50mL supernatant 0.45 μm of membrane filtration impurity, freezingly vacuumize drying, and dissolve freeze-drying samples with 500 μ L distilled waters, i.e. antibacterial peptide crude samples.
(2) fenestra method qualification antibacterial peptide crude samples is active
1) propionibacterium acnes activation: the frozen propionibacterium acnes in-20 DEG C is pressed 1% inoculum size in liquid FT substratum, incubation activation in 37 DEG C of anaerobism bags.
2) propionibacterium acnes slat chain conveyor: get 70 μ L activation products in super clean bench, coat ox brain heart infusion solid plate.
3) get antibacterial peptide crude samples 30 μ L on the punching scraps of paper (the circle scraps of paper), the scraps of paper are affixed on the planar surface containing Propiobacterium, 37 DEG C of quiescent culture one day (spending the night) after being placed in anaerobism bag, observe inhibition zone size.
4) result as shown in Figure 8, and SMD is that the supernatant liquor freeze-drying that unconverted pichia spp ferments three days concentrates sample, and can find out by comparison, transformant has good bacteriostatic activity.
(3) 96 orifice plates identify the thick Product Activity of antibacterial peptide again
Above-mentioned scraps of paper fenestra method can observe the suppression of antibacterial peptide to Propiobacterium intuitively, but can not the height of antibacterial efficiency between more different transformant under equal inhibition zone, therefore 96 well plate method are selected, under same bacteria concentration, do medicine gradient from low to high, namely getting 100 μ L antibacterial peptide crude samples mixes as first sample concentration with 100 μ L FT liquid nutrient mediums, getting first time compound sample 100 μ L mixes as second sample concentration with 100 μ L FT liquid nutrient mediums again, by that analogy, doubling dilution is to the 7th concentration.
The propionibacterium acnes of activation is diluted to 0.5 Maxwell concentration, gets 10 μ L bacterium liquid and be seeded to above-mentioned seven concentration respectively, contrast the mixed solution do not inoculated for comparable sodium.
Be placed in 37 DEG C of quiescent culture 48h after anaerobism bag, survey 570nm photoabsorption by microplate reader, measure its OD value, the blank of each concentration should be and do not add this medicine of Propiobacterium and the mixture of substratum, but not blank cultures, because the thick product of antibacterial peptide has background color.
Two OD values are subtracted each other, and are the growth whether this drug level produces Propiobacterium, as mark, judge minimal inhibitory concentration.Result is as shown in table 1.Consider, select No. 2 bacterial strains as optimum bacterial strain, called after SMD-pGAPZ α-30.
Table 1 different concns Cath-BF secreting, expressing product is to propionibacterium acnes stand density (OD
570) impact
Note: OD in table
570=OD
570 add Propiobacterium-OD
570 do not add Propiobacterium
embodiment 4 engineering bacteria SMD-pGAPZ α-30 ferments
1, pH value is observed the impact of Cath-BF secreting, expressing product
Carry out under shaking flask level, with phosphate buffered saline buffer regulate pH to 4.0,5.0,6.0 and without phosphate buffered saline buffer regulate pH 6.8 YPD substratum, by 1%(v/v) inoculate engineering bacteria SMD-pGAPZ α-30 seed liquor, 28 DEG C, 170rpm shaking culture 96h, get 0h, 24h, 48h, 72h, 96h sample, measures the pH corresponding to it, cell density OD
600with inhibition zone size, determine expression product optimal pH.
Result is as shown in table 2, result shows, the pH of engineering bacteria SMD-pGAPZ α-30 culture all presents remarkable decline after cultivation 24 h, significantly rise again to cultivating the middle and later periods, when pH value is in 4.0 ~ 5.0, the bacteriostatic activity of culture supernatant sample is higher, and when pH higher than 5.0 time almost can't detect the anti-microbial activity of culture supernatant.
In addition, when culture pH maintains 4.0, even if the supernatant cultivated after 96h still has higher bacteriostatic activity.Result shows, the size height correlation of the secreting, expressing level of Cath-BF and product stability and Medium's PH Value, and optimum pH is about 4.0, for next step large scale fermentation provides pH condition.
Table 2 Medium's PH Value grows engineering bacteria SMD-pGAPZ α-30, the impact of expression product bacteriostatic activity
Note: NB is not with damping fluid; ND is non-detected value; OD is OD
600institute's measured value; RI is relative bacteriostatic activity.
2, the fermentation of snake venom antibacterial peptide engineering bacteria
(1) picking list colony inoculation is in 3mL YPD substratum, and 30 DEG C of concussions are spent the night;
(2) with 1%(v/v) inoculum size is inoculated in containing in 200mL YPD substratum, and 30 DEG C of concussions are spent the night;
(3) carry out high density fermentation, working volume is 5L, rotating speed, air flow, dissolved oxygen, pH(optimal pH 4 ~ 5) and feed supplement (monitoring dissolved oxygen, the flow feeding when the sugar consumption in basic medium is complete) be all that adjustment is set by master control system;
(4) thalline weight in wet base is surveyed every 6h;
(5) 72h closed cans, centrifuging and taking supernatant.
This institute builds the pichia spp system that engineering bacteria is constitutive expression system, need not add methyl alcohol during the fermentation to induce, only need stream to add glucose, feed supplement is associated with dissolved oxygen, parameter as shown in Figure 9, X-coordinate is fermentation time, and ordinate zou is relative value, as dissolved oxygen is 100% to the maximum, rotating speed is 700 to the maximum, represent with 70 in the drawings, temperature is within the scope of 28 ~ 30 DEG C, and pH is between 4 ~ 5.When dissolved oxygen start to drop to a certain degree start to rise time, this experiment is 18th hour, and the sugar in basic medium runs out of, now need flow feeding, control dissolved oxygen about 30%, within every six hours, survey a bacterium weight in wet base, as shown in Figure 10, to ferment closed cans to 72h.
embodiment 5 engineering bacteria SMD-pGAPZ α-30 tunning purifying
1, the iso-electric point of Bungarus fasciatus antibacterial peptide Cath-BF30 is strong basicity (pI=11.2), according to this feature, and the expression product that we select strong cation-exchanging resin SP sepharose FF to come in separation and purification fermentation supernatant.Concrete steps are as follows:
(1) sample preparation: by tunning at the centrifugal 20min of 4000rmp, get supernatant, is diluted with water to and balance liquid same electrical conductance, 0.45 μm of membrane filtration removing impurity.
(2) balance: balance SP strong cation post with 60mL 20mM PB.
(3) loading: 1000mL sample is with 2mL/min speed loading.
(4) wash-out: with the high salt PBS wash-out containing 1M NACL, collects peak point sample.
(5) sds gel electrophoresis: get peak point sample three layers of glue protein electrophoresis, decolouring, dyeing, observes electrophoretic band.
2, result shows SP strong cation post energy better purifying snake venom antibacterial peptide, as shown in Figure 11.
Bungarus fasciatus antibacterial peptide Cath-BF30 colleges and universities preparation method of the present invention, can obtain the antibacterial peptide of purity more than 95% through a step cation exchange chromatography purifying.
SEQUENCE LISTING
<110> Guangdong Pharmaceutical University
The high efficiency preparation method of a <120> Bungarus fasciatus antibacterial peptide Cath-BF30
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 113
<212> DNA
<213> Cath-BF30 gene order
<400> 1
gtgcctcgag aagttcttca gaaagttgaa gaagagcgtc aagaagagag ctaaggagtt 60
cttcaagaag ccaagagtca tcggtgtctc catcccattc tgatctagag atg 113
Claims (10)
1. the high efficiency preparation method of a Bungarus fasciatus antibacterial peptide Cath-BF30, it is characterized in that, first Cath-BF30 gene order is designed as shown in SEQ ID NO.1, recycling expression vector establishment recombinant bacterial strain, after engineering bacteria ferments under the condition of pH lower than 5, carry out separation and purification through strong cation SP Sepharose FF post, obtain Bungarus fasciatus antibacterial peptide Cath-BF30.
2. high efficiency preparation method according to claim 1, it is characterized in that, concrete steps are as follows:
S1. design Cath-BF30 gene, sequence as shown in SEQ ID NO.1, and synthesizes this gene fragment;
S2. cloning vector is built: utilize
xhoi with
xbai double digestion, enters pGAPZ α carrier by the Cath-BF30 gene fragment clone of synthesis, obtains recombinant expression plasmid pGAPZ α-Cath-BF30;
S3. build engineering bacteria: recombinant expression plasmid pGAPZ α-Cath-BF30 is transformed Pichia pastoris, obtain engineering bacteria SMD-pGAPZ α-30;
S4. engineering bacterium fermentation: under the condition of pH lower than 5, ferments with YPD substratum;
S5. purifying: fermentation supernatant, after 0.45 μm of membrane filtration removing impurity, adopts SP Sepharose FF post to carry out separation and purification.
3. high efficiency preparation method according to claim 2, it is characterized in that, step S2 builds in cloning vector process, and Cath-BF30 gene fragment and the double digestion product of pGAPZ α carrier are connected according to the ratio of 4:1, and the condition of described connection is 16 DEG C and connects 9 hours.
4. high efficiency preparation method according to claim 2, it is characterized in that, transforming described in step S3 is the method utilizing electricity to transform, and the transformant of acquisition carries out bleomycin screening and the antibacterial spot screening of propionibacterium successively, positive through PCR qualification again, obtain engineering bacteria SMD-pGAPZ α-30.
5. high efficiency preparation method according to claim 2, is characterized in that, the concrete grammar that step S3 builds engineering bacteria is as follows:
S31. by after recombinant expression plasmid pGAPZ α-Cath-BF30 linearizing, electric transforming protein deficient host pichia spp SMD1168 competent cell;
S32. after electricity transforms, first screen the resistance transformant of bleomycin, then the culture supernatant of getting the resistance transformant of bleomycin carries out the antibacterial spot screening of propionibacterium, chooses the transformant strong to the bacteriostatic activity of propionibacterium;
S33. strong to the bacteriostatic activity of S32 screening transformant carries out PCR qualification, and identify that whether it is containing Cath-BF30 goal gene fragment, positive strain is engineering bacteria SMD-pGAPZ α-30.
6. high efficiency preparation method according to claim 2, it is characterized in that, the inoculum size of fermenting described in step S4 is 1 ~ 5v/v%; The condition of described fermentation be pH=4 ~ 5,28 ~ 30 DEG C, 170 ~ 200rmp shakes cultivation 70 ~ 72h; Described YPD substratum take glucose as the YPD substratum of carbon source.
7. high efficiency preparation method according to claim 2, it is characterized in that, after step S4 has fermented, fermentation supernatant 0.45 μm of membrane filtration impurity, freezingly vacuumizes drying, and dissolves freeze-drying sample with distilled water, obtain antibacterial peptide Cath-BF30 crude samples, observe crude samples to the growth-inhibiting effect of propionibacterium acnes, after confirming that it has bacteriostatic activity, then carry out the purifying of step S5;
Fermentation supernatant described in step S5 needs first after the centrifugal 20 ~ 30min of 4000 ~ 5000rmp, gets supernatant, is diluted with water to and balance liquid same electrical conductance, then removes impurity with 0.45 μm of membrane filtration; Described balance liquid is the balance liquid of balance SP Sepharose FF post; Described SP Sepharose FF post is strong cation SP Sepharose FF post.
8. high efficiency preparation method according to claim 5, it is characterized in that, recombinant expression plasmid pGAPZ α-Cath-BF30 described in step S31 utilizes
blni enzyme makes its linearizing, and the plasmid of total Linearization is carried out phenol chloroform, make its reach electricity transform needed for concentration;
The electric shock condition that described in step S31, electricity transforms is 1.5kv, 25 μ F, 200 Europe; Converted product incubation conditions is 30 DEG C of incubation 1h.
9. high efficiency preparation method according to claim 6, it is characterized in that, the inoculum size of described fermentation is 1 v/v %, and the pH of described fermentation is 4, and leavening temperature is 30 DEG C.
10. the Bungarus fasciatus antibacterial peptide Cath-BF30 that high efficiency preparation method prepares according to claim 1 or 2.
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