CN104829703B - A kind of recombination Bungarus fasciatus antibacterial peptide Cath-BF34 and its high efficiency preparation method - Google Patents
A kind of recombination Bungarus fasciatus antibacterial peptide Cath-BF34 and its high efficiency preparation method Download PDFInfo
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Abstract
本发明公开了一种重组金环蛇抗菌肽Cath‑BF34及其高效制备方法。抗菌肽Cath‑BF34的氨基酸序列如SEQ ID NO.1所示，其高效制备方法是首先设计Cath‑BF34基因序列如SEQ ID NO.2所示，再利用表达载体pGAPzα构建重组工程菌，工程菌在pH低于5的条件下进行发酵后，经强阳离子SP Sepharose FF柱进行分离纯化，得到金环蛇抗菌肽Cath‑BF34。该方法可实现抗菌肽的高效分泌表达，并保证分泌产物具有天然N端序列，表达产物具有抑制痤疮杆菌的活性，而且发酵上清只需一步离子交换即可获得高纯度Cath‑BF34，对金环蛇抗菌肽Cath‑BF的研究和进一步推广应用具有重要的意义。
The invention discloses a recombinant golden ring snake antibacterial peptide Cath-BF34 and an efficient preparation method thereof. The amino acid sequence of the antibacterial peptide Cath-BF34 is shown in SEQ ID NO.1, and its efficient preparation method is to first design the Cath-BF34 gene sequence as shown in SEQ ID NO.2, and then use the expression vector pGAPzα to construct recombinant engineering bacteria, engineering bacteria After fermentation under the condition of pH lower than 5, it is separated and purified by a strong cation SP Sepharose FF column to obtain the antibacterial peptide Cath-BF34 of golden ring snake. The method can realize the efficient secretion and expression of antimicrobial peptides, and ensure that the secreted product has a natural N-terminal sequence, the expression product has the activity of inhibiting Bacillus acnes, and the fermentation supernatant only needs one step of ion exchange to obtain high-purity Cath-BF34. The research and further promotion and application of the antibacterial peptide Cath‑BF is of great significance.
Description
Technical field
The invention belongs to polypeptide drugs technical fields.More particularly, to a kind of recombination Bungarus fasciatus antibacterial peptide Cath-BF34
And its high efficiency preparation method.
Background technique
Acne is a kind of common pilosebaceous inflammations dermatoses, mainly caused by anaerobic propionibacterium acnes,
Antibiotic treatment is mainly used at present, but curative effect is undesirable, and can lead to the generation and hypersensitivity of resistance bacterium.Recently it studies
It was found that can effectively inhibit propionibacterium acnes and other in mouse model from the antibacterial peptide Cath-BF separated in krait venom
The growth of acne infection bacterium, and inhibit related inflammatory hyperplasia, therefore there is important application value in acne prevention and treatment.It should be from gold
The antibacterial peptide Cath-BF separated in krait snake venom belongs to first vertebrate origin antibacterial peptide being reported, naturally isolated
Cath-BF is the polypeptide of 30 amino acid residues composition, therefore can be denoted as Bungarus fasciatus antibacterial peptide Cath-BF30.
But coded product deduction is carried out according to the cDNA sequence of Bungarus fasciatus antibacterial peptide Cath-BF, the antibacterial peptide is in N-terminal
Should be there are also 4 amino acid residues, i.e. natural length should be 34 amino acid residues, be denoted as Bungarus fasciatus antibacterial peptide Cath-
BF34, but the antibacterial peptide for being not yet found the structure type at present whether there is and whether there is or not bacteriostatic activities.
In actual operation, due to venin-derived limited, it is low therefrom to prepare antibacterial peptide Cath-BF30 yield, at high cost, more
It is the presence for not finding Bungarus fasciatus antibacterial peptide Cath-BF34；And chemical synthesis antibacterial peptide equally exists problem at high cost, synthesis
Difficulty, it is expensive, it is also difficult to promote and apply.Therefore, carry out the research for Bungarus fasciatus antibacterial peptide Cath-BF34, explore one
The method that kind efficiently prepares natural Cath-BF34 can not only increase a newcomer for Bungarus fasciatus antibacterial peptide Cath-BF, for
The popularization and application of Cath-BF also have important practical significance.
Summary of the invention
The technical problem to be solved by the present invention is to overcome existing Bungarus fasciatus antibacterial peptide Cath-BF correlative study and technologies of preparing
Deficiency, a kind of new Bungarus fasciatus antibacterial peptide Cath-BF34 and its high efficiency preparation method are provided, banked krait antibacterial is successfully constructed
The efficient constitutive and secretive expression system of the Pichia pastoris of Peptide C ath-BF34 and expression product isolation and purification method, and experiments have shown that
34 Peptide C ath-BF of preparation gained have Propiobacterium inhibitory activity.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of recombination Bungarus fasciatus antibacterial peptide Cath-BF34, amino acid sequence is as shown in SEQ ID NO.1.
A kind of high efficiency preparation method of above-mentioned recombination Bungarus fasciatus antibacterial peptide Cath-BF34 is to design Cath-BF34 base first
Because sequence is as shown in SEQ ID NO.2, expression vector establishment recombination engineering is recycled, engineering bacteria is under conditions of pH is lower than 5
After being fermented, is isolated and purified through strong cation SP Sepharose FF column, obtain Bungarus fasciatus antibacterial peptide Cath-BF34.
Specific step is as follows for above-mentioned high efficiency preparation method:
S1. Cath-BF34 gene is designed, sequence synthesizes the genetic fragment as shown in SEQ ID NO.2；Specifically adopt
With the coded sequence of Pichia pastoris preferred codons design Cath-BF34, head and the tail add restriction enzyme site, terminator codon respectively
Deng using primer Overlap extension PCR method (Overlap extension PCR) synthesis target gene；
S2. it constructs Cath-BF34 cloning vector: utilizingXho I withXbaI double digestion, by the Cath-BF34 gene of synthesis
Segment is cloned into Bichi yeast system pGAPZ α carrier, and (the Cath-BF34 target gene of synthesis is merged with secreting signal peptide MF- α
Rear clone is in the downstream of promoter GAP, so that constitutive and secretive expression N-terminal is natural under conditions of glucose does basic carbon source
The antibacterial peptide of sequence), obtain recombinant expression plasmid pGAPZ α-Cath-BF34；
S3. Cath-BF34 Pichia yeast engineering is constructed: by the method for electrotransformation, by recombinant expression plasmid pGAPZ α-
Cath-BF34 converts Pichia pastoris, obtains engineering bacteria SMD-pGAPZ α -34；
S4. it ferments: under the conditions of pH is lower than 5,30 DEG C, fermenting by the YPD culture medium of carbon source of glucose, 200rmp
Shake culture 72h；
S5. purify: fermentation supernatant is after 0.45 μm of membrane filtration removes impurity, using strong cation SP Sepharose
FF column is isolated and purified.
Wherein, during step S2 constructs cloning vector, the double digestion of Cath-BF34 genetic fragment and pGAPZ α carrier is produced
Object is attached according to the ratio of 4:1, and the condition of the connection is 16 DEG C and connects 9 hours.
As a kind of implementable solution, conversion described in step S3 is the method using electrotransformation, and the transformant of acquisition is successively
Bleomycin screening (being drawn lines from Antibiotics of Low Concentration to high concentration antibiotic) and the antibacterial spot screening of Propionibacterium are carried out, using
PCR identification is positive, obtains engineering bacteria SMD-pGAPZ α -34.
Preferably, the specific method is as follows for step S3 building engineering bacteria:
S31. after recombinant expression plasmid pGAPZ α-Cath-BF34 being linearized, electrotransformation protease-deficient host Bi Chi
Yeast SMD1168 competent cell；
S32. after electrotransformation, the resistance transformant of bleomycin is first screened, then take the training of the resistance transformant of bleomycin
It supports supernatant and carries out the antibacterial spot screening of Propionibacterium, choose the transformant strong to the bacteriostatic activity of Propionibacterium；
S33. the transformant strong to the bacteriostatic activity of S32 screening carries out PCR identification, identifies whether it contains Cath-BF34
Target gene fragment, positive strain are engineering bacteria SMD-pGAPZ α -34.
Preferably, the inoculum concentration of fermentation described in step S4 is 1~5v/v %；The condition of the fermentation be pH=4~5,28~
30 DEG C, 170~200rmp shake culture, 70~72h；The YPD culture medium is the YPD culture medium using glucose as carbon source.
It is highly preferred that the inoculum concentration of the fermentation is 1%, the pH of the fermentation is 4, and fermentation temperature is 30 DEG C.
After fermentation supernatant described in step S5 needs to first pass through 4000~5000rmp centrifugation, 20~30min, supernatant is taken, water is added
It is diluted to conductivity identical as equilibrium liquid, then removes impurity with 0.45 μm of membrane filtration；The equilibrium liquid is balance SP
The equilibrium liquid of Sepharose FF column；The SP Sepharose FF column is strong cation SP Sepharose FF column.
In addition, step S4 is after fermentation, 0.45 μm of membrane filtration impurity of fermentation supernatant freezes vacuum drying, and
Freeze-drying sample is dissolved with distilled water, obtains antibacterial peptide Cath-BF34 crude samples, 96 well plate methods observe crude samples to propionibacterium acnes
The growth inhibition effect of anaerobic cultures carries out activity analysis to generated antibacterial peptide Cath-BF34, confirm that it has suppression
After bacterium activity, then carry out the purifying of step S5.The step is significant in large-scale practical application.
Preferably, recombinant expression plasmid pGAPZ α-Cath-BF34 described in step S31 is utilizedBlnI enzyme makes its linearisation,
And the plasmid of total Linearization is subjected to phenol chloroform, concentration needed for reaching electrotransformation；
The electric shock condition of electrotransformation described in step S31 is 1.5kv, 25 μ F, 200 Europe；Converted product incubation conditions are 30 DEG C
Incubate 1h.
It is highly preferred that the concrete operations of electrotransformation described in step S31 are as follows:
PGAPZ α-Cath-BF34 the plasmid of linearisation is added in Pichia pastoris SMD1168 competent cell, is gently mixed
It is even, it is placed in 10min in the quartz transition cup for the 0.2cm being pre-chilled in advance on ice, conversion cup should be soaked in advance before converting
75% alcohol, and half an hour is sterilized under ultraviolet lamp；The water for drying conversion cup outer wall, is put in electric converter slot, 1.5kv, 25 μ
F, 200 Europe, electric shock, the sorbierite that 1mL pre-cooling is added immediately mix, and converted product is placed in 30 DEG C of incubation 1h.
In addition, more specifically, the method concrete operations of engineering bacteria SMD-pGAPZ α -34 large scale fermentation described in step S4 such as
Under:
(1) single colonie of picking engineering bacteria SMD-pGAPZ α -34 is seeded in YPD culture medium, and 30 DEG C of concussions overnight, obtain
Seed liquor；
(2) with 1v/v% inoculum concentration, seed liquor is inoculated in YPD culture medium, 30 DEG C of concussions are overnight；
(3) high density fermentation, working volume 5L, revolving speed, ventilatory capacity, dissolved oxygen, pH(optimal pH 4~5 are carried out) and mend
Material (monitoring dissolved oxygen, the flow feeding when the sugar consumption in basal medium is complete) is all to be arranged to adjust by master control system；
(4) thallus weight in wet base is surveyed every 6h；
(5) 72h closed cans, centrifuging and taking supernatant.
Engineering bacteria constructed by this research is the Pichia pastoris system of constitutive expression system, does not have to add first during the fermentation
Alcohol is induced, and need to only be flowed and be added glucose, and feed supplement is associated with dissolved oxygen, and parameter is as shown in Fig. 9, and abscissa is fermentation time,
Ordinate is relative value, and if dissolved oxygen is up to 100%, revolving speed is up to 700, is indicated in figure with 70, and temperature is 28 ~ 30 DEG C
In range, pH is between 4 ~ 5.When dissolved oxygen, which is begun to decline, to be begun to ramp up to a certain extent, this experiment is the 18th hour, base
Sugar in basal culture medium has run out of, and needs flow feeding at this time, control dissolved oxygen 30% or so, survey primary by every six hours
Bacterium weight in wet base, as shown in Fig. 10, fermentation to closed cans after 72h.
The Bungarus fasciatus antibacterial peptide Cath-BF34 being prepared according to above-mentioned high efficiency preparation method is also in the scope of the present invention
Within.
First demonstration that the presence of Bungarus fasciatus antibacterial peptide Cath-BF34, and genetic engineering means are used for the first time
Cath-BF34 is prepared, and establishes efficient preparation method.Cath- is constructed using Pichia pastoris constitutive and secretive expression system
The efficient expression engineering of BF34.Pichia pastoris (Pichia pastoris) system used is most mature exogenous gene high-efficient
Expression system, genetic background understands, can high density fermentation, thus there is good industrial performance.Its constitutive expression system
It is the strong promoter pGAP based on glyceraldehyde phosphate dehydrogenase, without using methanol induction as AOX1 promoter, is used for people
It is safer with the production of product.In addition, Cath-BF34 as micromolecule polypeptide, is difficult independent expression, secreting, expressing can be to avoid
Small peptide is degraded by intracellular protein enzyme, and product is easy purifying.Meanwhile present invention antibacterial peptide as obtained by vitro study preparation
The bacteriostatic activity of Cath-BF34, and high-efficiency fermenting technique and isolation and purification method are established, for further opening for Cath-BF34
Hair application lays a solid foundation.
Cath-BF34 high efficiency preparation method established by the present invention is that inventor is explored by many experiments and research obtains
's.Those skilled in the art are fully aware of, and during using gene engineering method building engineering bacteria, gene order is set
Meter be it is very crucial, designed specific gene order due to its uniqueness, to the selection of expression vector, to restriction enzyme site
The factors such as selection can all influence the efficiency and quality of the success or not of engineering bacteria building, building.The skill that the present invention initially encounters
Art problem is exactly gene order problem, although Bungarus fasciatus antibacterial peptide Cath-BF sequencing and analyzing is it is known that as outer
Source gene is directly used in when expressing in Bichi yeast system, because of a large amount of rare codons presence and be unable to high efficient expression, because
This, we have redesigned the gene order of Cath-BF using Pichia pastoris preference codon, and by largely adjusting and testing
Card is finally obtained as shown in SEQ ID NO.2 in conjunction with suitable restriction enzyme site, protection base, terminator codon etc.
Cath-BF34 gene order.
In addition, when by target gene Direct Cloning when the multiple cloning sites of commercial expression vector pGAP-z α, expression product
N-terminal can have more several non-natural amino acid sequences, influence antibacterial peptide activity, also easily caused when for human body immune anti-
It answers.The present invention is directly merged secretion signal peptide sequence MF- α with antibacterial peptide Cath-BF gene by gene chemical synthesis, is recycled
It is cloned in pGAP-z α positioned at the site XhoI of the upstream MF- α and the site XbaI of multiple cloning sites, may insure to resist in this way
The correct secreting, expressing of bacterium peptide, and secreting, expressing product has natural N-terminal sequence, is advantageous to subsequent isolate and purify.
It is not according to routine although Pichia yeast is widely used in the production of foreign protein in terms of zymotechnique
Guide Book just can determine that technological condition for fermentation because growth required for different destination gene expression with expression condition not
Together, different foreign proteins resists the ability difference of host strain proteasome degradation.The present invention is by a large amount of the study found that institute's structure
The engineering bacteria SMD-pGAPZ α -34 of the antibacterial peptide Cath-BF built must pH be lower than 5 under conditions of could high efficient expression, and
It is successfully crucial to become fermentation for the control of pH when just can be reduced degradation under the conditions of the pH, therefore fermenting.PH controls well, antibacterial
Peptide could high efficient expression；Control it is bad, cannot high efficient expression even do not express.So far, using bacillus coli gene work
Journey expression system, bacillus subtilis expression system also need to cut come the antibacterial peptide expressing having for antibacterial peptide, but expressing
Cut or be denaturalized, renaturation, cutting just it is active.In addition, yet there are no using yeast especially Pichia anomala expression system
Controlling is for the report of Cath-BF, and one of major reason is exactly that process conditions are not found out, the table in large scale fermentation
Up to horizontal low or do not express.
Isolating and purifying aspect, it is well known that isolate and purify the bottleneck for waiting downstream techniques to be biotechnology industry, also
It is technological difficulties place.Different protein or polypeptide be all it is personalized, can be used for all albumen without a kind of universal method
The purifying of matter.The prior art generally can could obtain high purity using two or more chromatographies in terms of purifying process.And this
Invention is the antibacterial peptide Cath-BF34 for obtaining high-purity using a step ion-exchange, this high-efficiency purifying method makes gold
Manpower, time, the production cost of krait antibacterial peptide Cath-BF substantially reduces.
It mainly includes efficient expression engineering that the present invention established, which efficiently prepares the new process of antibacterial peptide Cath-BF34,
Building, high efficient expression zymotechnique foundation and efficiently separate the foundation of purifying process, three is indispensable, anti-to banked krait
The further genralrlization application of bacterium Peptide C ath-BF provides solid theoretical basis.
Research of the invention mainly achieves following result:
(1) successful design has cloned Cath-BF34 coded sequence, which is designed based on Pichia pastoris preference codon, N
End with the downstream secreting signal peptide MF α is seamless merges, it is ensured that secretory product is natural N end sequence.
(2) the Pichia pastoris efficient secretory expression bacterial strain of Cath-BF34 is obtained, secreting, expressing product, which has, inhibits acne
The activity of bacillus.
(3) engineering bacteria SMD-pGAPZ α -34 secretion level highest under the conditions of medium pH 4.0, product is most stable, works as pH
Culture supernatant antibacterial activity is low when being 5 or more, and after incubation the phase be nearly no detectable antibacterial activity, demonstrate the need in lower pH
Under the conditions of cultivate one's ability and be effective against the degradation of host cell proteins enzyme.
(4) engineering bacteria SMD-pGAPZ α -34 is continuously cultivated in the YPD culture medium for doing carbon source with glucose, and takes carbon
, it can be achieved that growth and the efficient secretory expression of higher density, final cell density reach 160g/L(weight in wet base when the control means of source)
More than, antibacterial peptide secretion level reaches 100mg/L or more.
(5) fermentation supernatant purifies the Cath-BF34 that can get 95% or more purity by one step of SP Sepharose FF column.
The invention has the following advantages:
The invention discloses a kind of recombination Bungarus fasciatus antibacterial peptide Cath-BF34 and its high efficiency preparation methods.Antibacterial peptide Cath-
For the amino acid sequence of BF34 as shown in SEQ ID NO.1, high efficiency preparation method is first using Pichia pastoris preference codon
The Cath-BF34 gene order of design is as shown in SEQ ID NO.2, then by target gene and secreting signal peptide partial gene sequence
Rear clone is merged to expression vector pGAPz α, constructs constitutive and secretive expression recombination engineering, engineering bacteria is lower than 5 condition in pH
Under fermented after, isolated and purified through strong cation SP Sepharose FF column, obtain Bungarus fasciatus antibacterial peptide Cath-
BF34.With following significant progress:
1) for the first time experiments have shown that the presence of 34 Peptide C ath-BF, and successful purification obtains Cath-BF34, and demonstrates its tool
There is the activity for inhibiting Propiobacterium；
2) high-efficiency method for producing of Bungarus fasciatus antibacterial peptide Cath-BF34 is established using gene engineering method for the first time, and is protected
Card secretory product is natural N end sequence, and preparation gained Bungarus fasciatus antibacterial peptide Cath-BF34 has the activity for inhibiting Propiobacterium；
3) this method can realize that high efficient expression is secreted, and a step ion exchange is only needed to can be obtained high-purity
Cath-BF34(purity is higher than 95% or more)；
4) efficient expression system of the Cath-BF34 established does not depend on methanol induction, is used for human body safety.
This method of the present invention can not only realize the efficient secretory expression of antibacterial peptide, and guarantee that secretory product has day
Right N-terminal sequence, expression product has the activity for inhibiting Propiobacterium, and fermentation supernatant only needs a step ion exchange can be obtained
High-purity C ath-BF34, research and further genralrlization application to Bungarus fasciatus antibacterial peptide Cath-BF have great importance.
Detailed description of the invention
Fig. 1 is Bungarus fasciatus antibacterial peptide Cath-BF34 gene order.
Fig. 2 is antibacterial peptide Cath-BF34 target gene synthetic strategy.
Fig. 3 is Cath-BF34 target gene amplified production electroresis appraisal, M:DNA marker；1:Cath-BF30；2:
Cath-BF34。
Fig. 4 is cloning vector pGAPZ α-Cath-BF34 schematic diagram.
Fig. 5 is cloning vector pGAPZ α-Cath-BF34 electrophoretic identification, M:DNA marker；1:pGAPZ α-Cath-
BF34；2:pGAPZ α-Cath-BF30 cloning vector
Fig. 6 is that high concentration antibiotic-screening height copies transformant.
Fig. 7 is Pichia pastoris transformant PCR identification, M:DNA Marker；1-7: different transformant target gene amplifications produce
Object.
Fig. 8 is growth inhibition of the Cath-BF34 transformant secretory product to propionibacterium acnes, 1-6: different transformants
Secretory product；SMD: unconverted host strain secretory product.
Fig. 9 is engineering bacteria SMD-pGAPZ α -34 large scale fermentation Parameters variation figure.
Figure 10 is the engineering bacteria growth curve under engineering bacteria SMD-pGAPZ α -34 fermentation condition.
Figure 11 is SP sepharose FF cation exchange column Cath-BF34 expression product, M: low molecular weight egg
White Marker, 1: fermentation supernatant, 2-3: eluting peak point sample.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the method and apparatus that the present invention uses is the art conventional method and sets
It is standby.
Unless stated otherwise, agents useful for same and material of the present invention are commercially available.
The design and synthesis of 1 Bungarus fasciatus antibacterial peptide Cath-BF34 target gene of embodiment
1, gene order designs
It is translated according to known snake venom antibacterial peptide Cath-BF30 gene (disclosing in such as patent 200810058976.9) is theoretical
34 Peptide C ath-BF amino acid sequence (whether naturally occurring not confirming before making the present invention), select Pichia pastoris preference it is close
Numeral is translated and, and is selectedXhoI site withXbaI site is as its restriction enzyme site, and head and the tail are respectively plus protection alkali
Base, the bases such as terminator codon, designs Cath-BF34 target gene, and the Cath-BF34 objective gene sequence designed is such as
Shown in SEQ ID NO.2.
In addition, as shown in Fig. 1, synthesized full length gene 125bp, including 34 amino acid are according to Pichia pastoris preference
Sequence, terminator codon, protection base, restriction enzyme site that codon translation comes etc..
2, target gene synthesizes
The Cath-BF34 objective gene sequence designed is divided into three sections of base sequences for having repetitive sequence each other,
Whole section of sequence of target gene is synthesized using primer Overlap extension PCR method (Overlap extension PCR).
Synthetic strategy is as shown in Fig. 2.Three sections of sequences template each other two-by-two, reflects system (50 μ L): 2 × PCR Mix 25
μ L, P1 2 μ L, P2 2 μ L, P3 2 μ L, ddH2O 19μL.It mixes, is centrifuged on brief centrifugation machine several seconds, 94 DEG C of initial denaturations
5min, annealing temperature are 55 DEG C, and cycle-index 25 times, elongating temperature is 72 DEG C, takes 2 μ L of PCR product, 2% Ago-Gel electricity
Swimming, and record and take pictures.PCR product is arrived into Cath-BF34 target gene fragment with cleaning QIAquick Gel Extraction Kit cleaning recycling ,-
20 DEG C of preservations.
As a result it obtains and theoretical specific amplification products of the same size, 2% agarose gel electrophoresis testing result is for example attached
Shown in Fig. 3, the visible consistent segment of molecular weight in electrophoretogram.
The building of 2 Bungarus fasciatus antibacterial peptide Cath-BF34 cloning vector of embodiment
1, genetic engineering operation is carried out according to Molecular Cloning:A Laboratory guide method, by the base of the Cath-BF34 mesh of PCR synthesis
Because being used respectively with vector plasmid PGAPZaXhoI withXbaI double digestion reconnects conversion.Specific procedure is as follows:
(1) acquisition of vector plasmid PGAPZa: 37 DEG C, 170rmp is incubated overnight the Escherichia coli containing PGAPZa plasmid,
Plasmid, gel electrophoresis, -20 DEG C of preservations are extracted with the small extraction reagent kit of plasmid.
(2) double digestion: vector plasmid PGAPZa and Cath-BF34 gene is used respectivelyXhoI withXbaI double digestion, 37
DEG C water-bath 2h, gel electrophoresis, observing it, whether double digestion is complete, if all double digestion is complete for target fragment and carrier, by double enzymes
The recycling of product DNA plastic recovery kit, again gel electrophoresis are cut, observation band brightness determines its content.
(3) connection and conversion: by the double enzyme digestion product vector plasmid PGAPZa and Cath-BF34 of recycling according to the ratio of 1:4
Example, 16 DEG C connect 9 hours, are transferred to DH5a competent cell, the preparation of E. coli competent is referring to specific document, ice bath
30min, 42 DEG C of water-baths exactly 90s, ice bath 10min, is added 37 DEG C of placements 1h of 1mL low salt LB medium again, takes 100 μ L coating
In on the less salt LB plate for being 25 μ g/mL containing Zeicon bleomycin, 37 DEG C of incubators cultivate 16h.
(4) identification of recombinant expression plasmid: 10~20 recons of picking are low to the liquid containing 25 μ g/mL bleomycins
In salt LB culture medium, plasmid is extracted, carries out plasmid size, PCR colony identification and digestion identification.Preliminary Identification is passed through in screening
Recon 3 send to Guangzhou Huada gene company sequencing.The correct recon name of sequencing result are as follows: pGAPZ α-Cath-
BF34。
The method of above-mentioned building cloning vector based on Bichi yeast system pGAPZ α carrier, believe by target fragment and secretion
Number peptide MF- α fusion rear clone is in the downstream of promoter GAP, so that composing type is secreted under conditions of glucose does basic carbon source
Express the antibacterial peptide that N-terminal is native sequences.
2, the schematic diagram of the cloning vector pGAPZ α-Cath-BF34 constructed is as shown in Fig. 4.PCR product warpXhoI andXbaI double digestion rear clone is analyzed and identified in the corresponding site of pGAPZ α by sequence, the target fragment sequence cloned with
The coded sequence of Cath-BF is completely the same, and obtained expression vector is named as pGAPZ α-Cath-BF34, and carrier size is
3200bp, as shown in Fig. 5.
Embodiment 3 constructs engineering bacteria SMD-pGAPZ α -34
1, recombinant expression plasmid pGAPZ α-Cath-BF34 is linearized
PGAPZ α-Cath-BF34 plasmid is largely extracted with the big extraction reagent kit of plasmid, is used in combinationBlnI enzyme makes its linearisation.
200 μ L linearize system are as follows: 10 μ L BlnAbout 20 μ g of I, 40 μ L pGAPZ α-Cath-BF34(), 20 μ L 10 × buffer, 130
μL ddH2O.After mixing well, 37 DEG C of digestions are stayed overnight, and are taken 2 μ L digestion products to do 1% agarose gel electrophoresis, whether are observed it
Total Linearization.
2, the plasmid of total Linearization is subjected to phenol chloroform, reaches the desired concentration of electrotransformation, specific steps
It is as follows:
(1) 300 μ L ddH will be added in 200 μ L of digestion system2O makes 500 μ L of its total volume.
(2) phenol chloroform that 500 μ L are added is primary, and 12000rpm, 10min carefully take the upper layer in layering.
(3) the 3M sodium acetate of 1/10 volume, the nothing of PH 5.2 and 2.5 times of volumes being pre-chilled in advance are added in the upper layer
Water-ethanol, piping and druming mix, and are placed in -20 DEG C of placements 1h, 12000rpm, 10min, abandon supernatant, and twice with 75% ethanol washing, set
It is dried up in super-clean bench, with 10 μ L ddH2It is precipitated on the dissolution bottom O and wall, -20 DEG C of preservations.
3, the preparation of SMD1168 Pichia pastoris competence
(1) be placed on the setting-out of -20 DEG C of protease-deficient host's Pichia pastoris SMD1168 cell oeses frozen in
YPD solid plate is inverted, 30 DEG C of incubator 72h.
(2) picking saccharomyces albicans single bacterium is fallen in 5mL liquid YPD test tube, and 30 DEG C, 200rpm shake culture is stayed overnight.
(3) take 0.5mL overnight culture in the conical flask containing 50mL YPD fluid nutrient medium, 30 DEG C of shake cultures are extremely
OD600Between 1.3~1.5
(4) bacterium solution is placed in 50mL sterile centrifugation tube, 2500rpm, 4 DEG C of centrifugation 10min, abandons supernatant and collects thallus.
(5) bacterial sediment is gently blown and beaten with the distilled water 50mL of pre- cold sterilization, after mixing, 1500rpm, 4 DEG C of centrifugations
10min abandons supernatant.
(6) bacterial sediment is gently blown and beaten with the distilled water 25mL of pre- cold sterilization, after mixing, 1500rpm, 4 DEG C of centrifugations
10min abandons supernatant.
(7) bacterial sediment is gently blown and beaten with the 1M sorbierite 10mL of pre- cold sterilization, after mixing, 1500rpm, 4 DEG C of centrifugations
10min abandons supernatant.
(8) cell precipitation is resuspended with the 1M sorbierite of pre- cold sterilization, its cell concentration is made to reach OD600It is 100, as
Competent cell, competent cell are placed in 4 DEG C, use as early as possible.
4, electrotransformation
(1) impression that 80 μ L have been prepared is added in the recombinant expression plasmid pGAPZ α-Cath-BF34 of 10 μ L linearisation
In state, gently piping and druming is mixed, and is placed in 10min in the quartz transition cup for the 0.2cm being pre-chilled in advance on ice, and conversion cup should turn
75% alcohol is impregnated in advance before changing, and sterilizes half an hour under ultraviolet lamp.
(2) water for drying conversion cup outer wall, is put in BioRad electric converter slot, 1.5kv, 25 μ F, 200 Europe, shocks by electricity,
The sorbierite that 1mL pre-cooling is added immediately mixes.
(3) converted product is placed in 30 DEG C of incubation 1h.
(4) electrotransformation product is centrifuged 2min in 1000rpm, stays part supernatant to mix with precipitating, is applied to containing 100 μ g/mL
On the YPDS plate of bleomycin zeicon, 30 DEG C are incubated 3~5 days, observe the growth of transformant.
5, the high copy recon of bleomycin zeicon screening
The recon of the high copy of screening first has to draw lines transformant from Antibiotics of Low Concentration to high concentration antibiotic, specifically
It is as follows:
(1) it is drawn lines with the transformant on 100 μ g/mL zeicon antibiotic plate of sterilizing toothpick picking in 500 μ g/mL
On zeicon solid plate, each bacterium colony setting-out length is 1cm or so, is inverted, 30 DEG C of 2~3d of incubator culture
(2) it is drawn lines with the transformant on 500 μ g/mL zeicon antibiotic plate of sterilizing toothpick picking in 1000 μ g/mL
On zeicon solid plate, each bacterium colony setting-out length is 1cm or so, is inverted, 30 DEG C of 2~3d of incubator culture
(3) it is drawn lines with the transformant on 1000 μ g/mL zeicon antibiotic plate of sterilizing toothpick picking in 2000 μ g/mL
On zeicon solid plate, each bacterium colony setting-out length is 1cm or so, is inverted, 30 DEG C of 2~3d of incubator culture
(4) transformant on 2000 μ g/mL zeicon solid plate of picking, in 3mL YPDS fluid nutrient medium, 30 DEG C
Shake culture is stayed overnight, number, -20 DEG C of glycerol conservations.
This experiment is with concentration gradient for 100 μ g/mL to 500 μ g/mL, 500 μ g/mL to 1000 μ g/mL, 1000 μ g/mL
Bleomycin zeicon to 2000 μ g/mL is screened, and is finally obtained a few plant height copy transformants and is as shown in Fig. 6 turned several plants
Beggar carries out PCR, observes that PCR product has obvious purpose band, see attached drawing 7 by boiling jelly cooking method.
6, the antibacterial spot screening of Propionibacterium
Collect culture supernatant, 96 well plate methods observe supernatant to the growth inhibition effects of propionibacterium acnes anaerobic cultures,
The strong transformant of supernatant bacteriostatic activity is chosen using the antibacterial spot screening of Propionibacterium.
7, it is identified again through PCR
(1) by the saccharomycete frozen (transformant after above-mentioned bleomycin zeicon and Propionibacterium antibacterial spot screening) mistake
Night activation takes 1mL bacterium solution, and 10000rpm is centrifuged 2min, abandons supernatant.
(2) cell is washed with 500 μ L PBS, 10000rpm is centrifuged 2min, washes twice, and abandons supernatant.
The TE of (3) 100 μ L PH8.0 dissolves precipitating, in boiling water boiling 10min, is placed in -20 DEG C of freezings.
(4) -20 DEG C of cells frozen are taken out, boil 10min again, 12000rpm is centrifuged 5min, takes supernatant as PCR mould
Plate.
(5) 20 μ L PCR reaction systems: 2 × TAQ enzyme MIX, 10 μ L, 2 μ L of upstream primer, 2 μ L of downstream primer, 2 μ L of template,
ddH2O 4μL。
(6) response procedures: 94 DEG C of 5min initial denaturations, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations, 72 DEG C
10min takes 5 μ L PCR products to carry out agarose gel electrophoresis, has seen whether purpose clip size band.
(7) positive strain that PCR was identified is chosen, expression product activity identification is carried out to it.
8, engineering bacterium expression lytic activity is identified
(1) acquisition of antibacterial peptide Cath-BF34 crude samples
Take 500 μ L of SMD-pGAPZ α -34 glycerol stock in 50mL YPD fluid nutrient medium, 30 DEG C of 200rmp shake cultures
72h；
50mL culture is placed in centrifuge tube, 10000rmp, is centrifuged 5min, take supernatant, abandons precipitating.
By 0.45 μm of membrane filtration impurity of 50mL supernatant, vacuum drying is freezed, and is dissolved and is lyophilized with 500 μ L distilled waters
Sample, i.e. antibacterial peptide crude samples.
(2) speculum method identification antibacterial peptide crude samples activity
1) propionibacterium acnes activate: by freeze in -20 DEG C propionibacterium acnes by 1% inoculum concentration in liquid FT cultivate
In base, activation is incubated in 37 DEG C of anaerobism bags.
2) propionibacterium acnes culture: 70 μ L activation products are taken in super-clean bench, are coated on ox brain heart infusion solid plate.
3) it takes 30 μ L of antibacterial peptide crude samples on the punching scraps of paper (the circle scraps of paper), the scraps of paper is affixed on to the plate table containing Propiobacterium
Face is placed in after anaerobism bag 37 DEG C stationary culture one day (overnight), observes inhibition zone size.
4) result is as shown in Fig. 8, and SMD is the supernatant freeze-drying concentration sample that unconverted Pichia pastoris ferments three days, can
To find out in contrast, transformant has good bacteriostatic activity.
(3) 96 orifice plates identify antibacterial peptide crude product activity again
Above-mentioned scraps of paper speculum method can intuitively observe inhibition of the antibacterial peptide to Propiobacterium, but in same inhibition zone
Down can not between more different transformants antibacterial efficiency height, therefore select 96 well plate methods, under same bacteria concentration, by
It is low to high to do medicine gradient, that is, take 100 μ L antibacterial peptide crude samples to mix with 100 μ L FT fluid nutrient mediums dense as first sample
Degree takes 100 μ L of first time aggregate sample to be mixed again with 100 μ L FT fluid nutrient mediums as second sample concentration, with such
It pushes away, doubling dilution to the 7th concentration.
The propionibacterium acnes of activation are diluted to 0.5 Maxwell concentration, take 10 μ L bacterium solutions be seeded to respectively above-mentioned seven it is dense
Degree, compares the mixed liquor not being inoculated with for comparable sodium.
37 DEG C of stationary culture 48h after anaerobism bag are placed in, 570nm light absorption is surveyed with microplate reader, measures its OD value, each medicine
The blank control of concentration, which should be, is not added the medicine of Propiobacterium and the mixture of culture medium, rather than blank cultures, because of antibacterial
Peptide crude product has background color.
Two OD values are subtracted each other, and the growth that as whether the drug concentration generates Propiobacterium judges minimum as mark
Mlc.It the results are shown in Table 1.Comprehensively consider, selects No. 2 bacterial strains as optimal bacterial strain, be named as SMD-pGAPZ α -34.
1 various concentration Cath-BF secreting, expressing product of table is to propionibacterium acnes stand density (OD570) influence
Note: OD in table570 = OD570 add Propiobacterium—OD570 are not added Propiobacterium
4 engineering bacteria SMD-pGAPZ α -34 of embodiment fermentation
1, pH value influences to observe on Cath-BF secreting, expressing product
It is carried out in the case where shaking flask is horizontal, adjusts pH to 4.0 with phosphate buffer, 5.0,6.0 and slow without perphosphate
The 6.8 YPD culture medium of pH that fliud flushing is adjusted, by 1%(v/v) inoculation engineering bacteria SMD-pGAPZ α -34 seed liquor, 28 DEG C, 170rpm
Shaken cultivation 96h takes 0h, and for 24 hours, 48h, 72h, 96h sample measure pH, the cell density OD corresponding to it600It is big with inhibition zone
It is small, determine expression product optimal pH.
The results are shown in Table 2, the results show that the pH of engineering bacteria SMD-pGAPZ α -34 culture is in after cultivating 24 h
Now it is remarkably decreased, until the culture middle and later periods significantly rises again, the bacteriostatic activity of culture supernatant sample when pH value is in 4.0~5.0
It is higher, and the antibacterial activity of culture supernatant is nearly no detectable when pH is higher than 5.0.
In addition, when culture pH maintains 4.0, even if the supernatant after culture 96h still has higher bacteriostatic activity.As a result
Show that the size of the horizontal secreting, expressing of Cath-BF and product stability and Medium's PH Value is highly relevant, optimum pH is
4.0 or so, pH condition is provided for next step large scale fermentation.
2 Medium's PH Value of table is to engineering bacteria SMD-pGAPZ α -34 growth, the influence of expression product bacteriostatic activity
Note: NB is that buffer is not added；ND is non-detected value；OD is OD600 Institute's measured value；RI is opposite bacteriostatic activity.
2, the fermentation of snake venom antibacterial peptide engineering bacteria
(1) picking single colonie is seeded in 3mL YPD culture medium, and 30 DEG C of concussions are overnight；
(2) with 1%(v/v) inoculum concentration is inoculated in the culture medium of YPD containing 200mL, and 30 DEG C of concussions are overnight；
(3) high density fermentation, working volume 5L, revolving speed, ventilatory capacity, dissolved oxygen, pH(optimal pH 4~5 are carried out) and mend
Material (monitoring dissolved oxygen, the flow feeding when the sugar consumption in basal medium is complete) is all to be arranged to adjust by master control system；
(4) thallus weight in wet base is surveyed every 6h；
(5) 72h closed cans, centrifuging and taking supernatant.
Engineering bacteria constructed by this research is the Pichia pastoris system of constitutive expression system, does not have to add first during the fermentation
Alcohol is induced, and need to only be flowed and be added glucose, and feed supplement is associated with dissolved oxygen, and parameter is as shown in Fig. 9, and abscissa is fermentation time,
Ordinate is relative value, and if dissolved oxygen is up to 100%, revolving speed is up to 700, is indicated in figure with 70, and temperature is 28~30 DEG C
In range, pH is between 4~5.When dissolved oxygen, which is begun to decline, to be begun to ramp up to a certain extent, this experiment is the 18th hour,
Sugar in basal medium has run out of, and needs flow feeding at this time, for control dissolved oxygen 30% or so, every six hours survey one
Secondary bacterium weight in wet base, as shown in Fig. 10, fermentation to closed cans after 72h.
The purifying of 5 engineering bacteria SMD-pGAPZ α -34 tunning of embodiment
1, the isoelectric point of Bungarus fasciatus antibacterial peptide Cath-BF34 is strong basicity (pI=11.2), according to this feature, Wo Menxuan
The expression product in fermentation supernatant is isolated and purified with strong cation-exchanging resin SP sepharose FF.Specific step is as follows:
(1) sample treatment: tunning is centrifuged 20min in 4000rmp, takes supernatant, is diluted with water to and balance liquid phase
Same conductivity, 0.45 μm of membrane filtration remove impurity.
(2) it balances: balancing SP strong cation column with 60mL 20mM PB
(3) loading: 1000mL sample is with 2mL/min speed loading
(4) it elutes: being eluted with the PBS with high salt of the NACL containing 1M, collect peak point sample.
(5) sds gel electrophoresis: taking three layers of glue protein electrophoresis of peak point sample, decolourizes, and electrophoretic band is observed in dyeing.
2, the result shows that SP strong cation column can preferably purify snake venom antibacterial peptide, as shown in Fig. 11.
Bungarus fasciatus antibacterial peptide Cath-BF34 colleges and universities preparation method of the invention, by a step cation exchange chromatography
Purifying can be obtained the antibacterial peptide that purity is more than 95%.
SEQUENCE LISTING
<110>Guangdong Pharmaceutical University
<120>a kind of recombination Bungarus fasciatus antibacterial peptide Cath-BF34 and its high efficiency preparation method
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 34
<212> PRT
<213>Cath-BF34 amino acid sequence
<400> 1
Lys Arg Phe Lys Lys Phe Phe Arg Lys Leu Lys Lys Ser Val Lys Lys
1 5 10 15
Arg Ala Lys Glu Phe Phe Lys Lys Pro Arg Val Ile Gly Val Ser Ile
20 25 30
Pro Phe
<210> 2
<211> 125
<212> DNA
<213>Cath-BF34 gene order
<400> 2
gtgcctcgag aagagattca agaagttctt cagaaagttg aagaagagcg tcaagaagag 60
agctaaggag ttcttcaaga agccaagagt catcggtgtc tccatcccat tctgatctag 120
agatg 125
Claims (4)
1. a kind of preparation method for recombinating Bungarus fasciatus antibacterial peptide Cath-BF34, which is characterized in that specific step is as follows:
S1. Cath-BF34 gene is designed, sequence synthesizes the genetic fragment as shown in SEQ ID NO.2；
S2. it constructs cloning vector: utilizingXhoI withXbaI double digestion enters the Cath-BF34 gene fragment clone of synthesis
PGAPZ α carrier obtains recombinant expression plasmid pGAPZ α-Cath-BF34；
S3. it constructs engineering bacteria: recombinant expression plasmid pGAPZ α-Cath-BF34 being converted into Pichia pastoris, obtains engineering bacteria
SMD-pGAPZα-34；
S4. it engineering bacterium fermentation: under conditions of pH is lower than 5, is fermented with YPD culture medium；
S5. purify: fermentation supernatant is separated after 0.45 μm of membrane filtration removes impurity using SP Sepharose FF column
Purifying；
Wherein, the specific method is as follows for step S3 building engineering bacteria:
S31. after recombinant expression plasmid pGAPZ α-Cath-BF34 being linearized, electrotransformation protease-deficient host's Pichia pastoris
SMD1168 competent cell；
S32. after electrotransformation, the resistance transformant of bleomycin is first screened, then take in the culture of the resistance transformant of bleomycin
It is clear to carry out the antibacterial spot screening of Propionibacterium, choose the transformant strong to the bacteriostatic activity of Propionibacterium；
S33. the transformant strong to the bacteriostatic activity of S32 screening carries out PCR identification, identifies whether it contains Cath-BF34 purpose
Genetic fragment, positive strain are engineering bacteria SMD-pGAPZ α -34；
Recombinant expression plasmid pGAPZ α-Cath-BF34 described in step S31 is utilizedBlnI enzyme makes its linearisation, and will be fairly linear
The plasmid of change carries out phenol chloroform, concentration needed for reaching electrotransformation；
The electric shock condition of electrotransformation described in step S31 is 1.5kv, 25 μ F, 200 Europe；Converted product incubation conditions are 30 DEG C of incubations
1h；
The inoculum concentration of fermentation described in step S4 is 1~5v/v%；The condition of the fermentation be pH=4~5,28~30 DEG C, 170~
70~72h of 200rmp shake culture；The YPD culture medium is the YPD culture medium using glucose as carbon source；
After fermentation, 0.45 μm of membrane filtration impurity of fermentation supernatant freezes vacuum drying to step S4, and molten with distilled water
Defrosting dry sample obtains antibacterial peptide Cath-BF34 crude samples, observes crude samples to the growth inhibition effect of propionibacterium acnes, really
After recognizing it with bacteriostatic activity, then carry out the purifying of step S5；
After fermentation supernatant described in step S5 needs to first pass through 4000~5000rmp centrifugation, 20~30min, supernatant is taken, is diluted with water
Extremely conductivity identical as equilibrium liquid, then impurity is removed with 0.45 μm of membrane filtration；The equilibrium liquid is balance SP Sepharose
The equilibrium liquid of FF column；The SP Sepharose FF column is strong cation SP Sepharose FF column.
2. preparation method according to claim 1, which is characterized in that during step S2 constructs cloning vector, Cath-BF34
Genetic fragment and the double enzyme digestion product of pGAPZ α carrier are attached according to the ratio of 4:1, and the condition of the connection is 16 DEG C
Connection 9 hours.
3. preparation method according to claim 1, which is characterized in that conversion described in step S3 is the method using electrotransformation,
The transformant of acquisition successively carries out bleomycin screening and the antibacterial spot screening of Propionibacterium, identifies the positive using PCR, obtains work
Journey bacterium SMD-pGAPZ α -34.
4. preparation method according to claim 1, which is characterized in that the inoculum concentration of the fermentation is 1 v/v %, the fermentation
PH be 4, fermentation temperature be 30 DEG C.
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| CN101845454A (en) * | 2010-04-16 | 2010-09-29 | 刘德虎 | Method for expressing pseudoplectania nigrella mature peptide in recombinant pichia pastoris |
| CN103045636A (en) * | 2012-12-17 | 2013-04-17 | 中国农业科学院饲料研究所 | Method for efficiently expressing antibacterial peptide NZ2114 in recombinant pichia pastoris |
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| CN103045636A (en) * | 2012-12-17 | 2013-04-17 | 中国农业科学院饲料研究所 | Method for efficiently expressing antibacterial peptide NZ2114 in recombinant pichia pastoris |
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| Title |
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| Recombinant expression of human cathelicidin(hCAP18/LL-37) in Pichia pastoris;In-Pyo Hong等;《Biotechnology letters》;20070131;第29卷(第1期);第74-75页、摘要 |
| 利用SUMO融合技术在枯草芽孢杆菌中重组表达抗菌肽cathelicidin-BF及其生物学活性研究;栾超;《中国博士学位论文全文数据库 基础科学辑》;20150115(第1期);第二至五章 |
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