CN102093998B - Preparation method of antibacterial peptide cecropin feed additive - Google Patents
Preparation method of antibacterial peptide cecropin feed additive Download PDFInfo
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Abstract
The invention relates to a preparation method of an antibacterial peptide cecropin feed additive, belonging to the technical field of gene engineering. The preparation method of the antibacterial peptide cecropin feed additive comprises the following steps of: (1) carrying out enzyme cutting to an antibacterial peptide cecropin gene and a multicopy integration expression vector respectively by Not I, after connecting by DNA ligase, converting E.coli TOP10 bacterial strains, coating an LB (Luria-Bertani) flat plate, carrying out enzyme cutting verification by the Not I, and selecting and naming a transformant inversely inserted into a cecA1 fragment as pYIP-cecA1; (2) carrying out enzyme cutting to the pYIP-cecA1 prepared in step (1) by using Hpa I, recovering big fragments, adding the pYIP-cecA1 into 80 muL of yeast electricity transformation competent cells, carrying out electric shock on a mixture, and screening the mixture by the YEPD (Yeast Extract Peptone Dex) flat plate of G418 to obtain the yeast cell for expressing the cecropin; and (3) culturing the yeast cell for expressing the cecropin, which is prepared in step (2), and drying to obtain the cecropin feed additive. According to the preparation method, the production cost of the cecropin can be lowered, and a good foundation is laid for popularizing the cecropin to serve as the feed additive to be antibiotics substitute for feeds.
Description
Technical field
The present invention relates to a kind of preparation method of antibacterial peptide cecropin fodder additives, belong to gene engineering technology field.
Background technology
Livestock and poultry and culture fishery are occupied critical role in national economy.The high-density breeding mode also causes the aquaculture organism disease to be grown when improving output, causes the tremendous economic loss.Generally use microbiotic to prevent and treat disease at present, but problems such as drug residue that is caused and pathogenic bacteria resistance, harm humans health and environment, and become the major reason that restriction animal husbandry and fishery article export.In people today to the food safety growing interest, seek microbiotic substitute safely and efficiently, the development healthy aquaculture has become the research direction of feed subject.At present, the microbiotic substitute mainly through inhibition or kill pathogenic bacteria, improve intestinal microflora, enhance immunity power, to reach the purpose of prophyiaxis and promoting growth.
Antibacterial peptide is one type of small peptide with anti-microbial activity, extensively is present in organic sphere.The antibacterial peptide of biological endogenous property can be induced synthetic through infectation of bacteria, is playing an important role aspect the invasion of body opposing cause of disease, especially the biological important defence composition of lack of specific immunologic function.Cecropin (Cecropins) is stronger a kind of of anti-microbial activity in 5 types of insect antimicrobial peptides, and its has a broad antifungal spectrum can not only also can kill a part of fungi by killing bacteria; Its bactericidal mechanism is to rely on passage through the electromotive force that changes on the bacterial cell membrane; Cause the intracellular organic matter leakage and cause bacterial death; These are different through the biosynthesizing of blocking-up macromole with microbiotic, be difficult for producing resistant organism, and various antibiotic resistant bacterias are also had lethal effect; Characteristics such as it also has that molecular weight is little, good water solubility and non-immunogenicity; It has than heat-flash stability, can also keep certain vigor in 10 minutes 100 ℃ of heating, is more suitable for the high temperature process process of feed; And after artificial hydrochloric acid in gastric juice processing, activity remains unchanged; It finds in chick that to the person poultry harmless Cecropins also has somatotrophic effect.Cecropins not only can be substituted feeding antibiotic as fodder additives, kill digestive tube cause of disease bacterium, reach the effect of prophyiaxis and promoting growth, but also can be used as the sanitas use of feed, be convenient to feed and store.
At present, have the report of in pichia spp, expressing fruit bat Cecropin B.But the Pichia anomala expression heterologous protein needs methanol induction, and residual methyl alcohol possibly bring toxic action.Yeast saccharomyces cerevisiae be U.S. food and drug administration announce can be used as fodder additives and to unique yeast quasi-microorganism of person poultry harmless.Yeast saccharomyces cerevisiae has sophisticated allogeneic gene expression system, and itself is high protein feed still, and yeast cells wall still is a kind of immunopotentiating agent.Therefore, yeast saccharomyces cerevisiae is more suitable for being used for producing Cecropins as fodder additives.
Summary of the invention
The present invention is directed to the deficiency of prior art, a kind of preparation method of antibacterial peptide cecropin fodder additives is provided.
A kind of preparation method of antibacterial peptide cecropin fodder additives, step is following:
(1) antibacterial peptide cecropin gene (shown in SEQ ID NO.14) and multi-copy integration expression vector (shown in SEQ IDNO.1) are cut with Not I enzyme respectively; After connecting with dna ligase then; Transformed E .coli TOP10 bacterial strain, coating LB (100 μ g/mL penbritin) flat board utilizes Not I to carry out enzyme and cuts checking; Through analyzing order-checking, choose the segmental transformant called after of reverse insertion cecA1 pYIP-cecA1;
(2) pYIP-cecA1 that step (1) is made cuts with Hpa I enzyme, reclaim big fragment after, join 80 μ L yeast electricity transformed competence colibacillus cell, after the mixture electric shock,, obtain to express the yeast cell of cecropin through the dull and stereotyped screening of the YEPD of G418;
(3) yeast cell of the expression cecropin that makes of culturing step (2) through dry, makes the cecropin fodder additives.
The electric shock time that electricity transforms in the above-mentioned steps (2) is 3.5～4.0ms.
Culture condition in the above-mentioned steps (3): in the YEPD of no selective pressure substratum, cultivated 2～3 days for 28～30 ℃.
Yeast in the above-mentioned steps (2) is the wild-type yeast saccharomyces cerevisiae ABXL-1D with flocculence, and bacterial classification is available from U.S. ATCC company.
Described multi-copy integration preparation of expression vectors method can number be referring to one Chinese patent application: " 201010531234.0 ", denomination of invention is: " record in the application of a kind of multi-copy integration expression vector and preparation method thereof and application in expressing Bovinelactoferrin ".
Yeast saccharomyces cerevisiae is nontoxic to the mankind, is confirmed as harmless expression system by Food and Drug Administration (FDA), and expression product can directly be used as fodder additives, and the cell wall constituent β of yeast saccharomyces cerevisiae-1,3 VISOSE also is a kind of immunostimulant.So the present invention utilizes strong flocculation type yeast saccharomyces cerevisiae constitutive expression cecropin; In industrial production, do not need separating technology; Complicated protein purification technology; Utilize lower-cost fermention medium can carry out fermentative prodn, collect yeast after fermentation finishes, can directly process the yeast dry powder formulations and use as fodder additives.
In order to reduce the production cost of reorganization cecropin; Simplify production technique; The wild-type yeast saccharomyces cerevisiae ABXL-1D integrative gene expression cecropin that this research selection has flocculence, but present commercial yeast saccharomyces cerevisiae integrating expression vector mostly utilizes nutrient defect type mark as selection markers and integration site, needs the auxotrophy yeast saccharomyces cerevisiae as the host; And the copy number that obtains after integrating is lower, is not suitable for carrying out suitability for industrialized production.There are some researches show that the rDNA gene of yeast saccharomyces cerevisiae also can be used as integration site, and the copy number of recombinant protein gene is brought up to more than 50 or 50.The present invention makes up and contains rDNA as integration site, and the G418 resistance is a selection markers, and the strong composition type expression promoter PGK of yeast is the novel yeast saccharomyces cerevisiae multi-copy integration expression vector of promotor.In addition, Cecropins has stronger anti-microbial activity, and its antibacterial mechanisms is different from microbiotic, should not produce resistant organism, itself is again little peptide, finally can be degraded safe noresidue in vivo.In addition, the thermostability of Cecropins is very good, remains unchanged at its anti-microbial activity of boiling water treating 30min, is suitable for as the sanitas in the course of processing of silage very much.
Beneficial effect:
1, multi-copy integration expression vector of the present invention is a skeleton with the YIPlac204 plasmid, and PGK is a promotor, and the G418 resistance is a selection markers, and rDNA is an integration site, is the yeast multi-copy integration type expression vector that is applicable to that suitability for industrialized production is used.
2, the present invention adopts the host bacterium of the wild-type yeast saccharomyces cerevisiae of strong flocculence as recombinant protein, and successful expression reorganization cecropin.The S. cervisiae of strong flocculence is when suitability for industrialized production; Can make the yeast cell that is dispersed in the nutrient solution assemble the generation aggegation each other, form flocculation particle and sedimentation, thereby help the effective separation of yeast cell with nutrient solution; Can simplify production technique greatly, reduce cost.
3, the present invention adopts composing type strong promoter PGK efficiently to start Recombinant Protein Expression, and the feed supplement CONTROL PROCESS has been simplified operation when having improved the expression rate of reorganization and having deducted suitability for industrialized production.
4, the present invention utilizes wild-type yeast saccharomyces cerevisiae successful expression antibacterial peptide cecropin first; Thereby and utilize the higher multi-copy integration expression vector of biological safety to improve the copy number of cecropin in the genes of brewing yeast group and improve cecropin in Expression in Saccharomyces Cerevisiae efficient, the reorganization cecropin does not need purifying just can directly use as fodder additives.Utilize this reorganization cecropin saccharomyces cerevisiae engineered yeast to carry out the fermentative prodn of cecropin, reduce the production cost of cecropin, thereby lay good basis for the popularization cecropin becomes the feeding antibiotic substitute as fodder additives.
Description of drawings
Fig. 1 CecropinA1 mRNA sequence is with the comparison figure of the CecropinA1 artificial synthesized sequence that designs according to its aminoacid sequence;
Fig. 2 pYIP-cecA1 bacterium liquid PCR checking
Wherein swimming lane 1:pYIP-cecA1 bacterium liquid PCR checking; Swimming lane 2:DL2000 Marker
Fig. 3 yeast saccharomyces cerevisiae transforms daughter bacteria liquid PCR checking;
Wherein: 1 swimming lane: S.cerevisiae ABXL-1D bacterium liquid PCR result, 2 swimming lanes: S.cerevisiae ABXL-1D reorganization cecA1 transforms daughter bacteria liquid PCR result, 3 swimming lanes: DL2000 Marker;
The sub-SDS-PAGE of Fig. 4 recombinant conversion detects the protein expression situation;
Wherein: 1 swimming lane: the transformant of 1.CecropinA1 transformed saccharomyces cerevisiae ABXL-1D, 2 swimming lanes: small molecular weight protein Marker, 3 swimming lanes: contrast yeast saccharomyces cerevisiae ABXL-1D.The arrow indication is reorganization CecropinA1 band among the figure.
Embodiment
Below in conjunction with embodiment and accompanying drawing content of the present invention is done further explanation, but institute of the present invention protection domain is not limited thereto;
Plasmid pPIC9K is available from Invitrogen company, and to be the Jin Site bio tech ltd to cecA1 carry out being inserted among the pUC57 behind the synthetic plasmid pUC57-cecA1 obtains.
The acquisition of antibacterial peptide cecropin gene:
The codon-bias of yeast saccharomyces cerevisiae and fruit bat exists than big-difference; In order to make drosophila melanogaster antimicrobial peptide cecropin gene in yeast saccharomyces cerevisiae, obtain expressing more efficiently; Therefore according to yeast saccharomyces cerevisiae codon-bias table; To convert the nucleotide sequence that is adapted at expressing the yeast saccharomyces cerevisiae to from the drosophila melanogaster antimicrobial peptide cecropinA1 aminoacid sequence that NCBI finds, also will consider the problem such as GC content, secondary structure of whole nucleotide sequence simultaneously, avoid the translation process premature termination.CecropinA1 mRNA sequence is seen Fig. 1 with the comparison of the CecropinA1 artificial synthesized sequence that designs according to its aminoacid sequence.
Behind Cecropin A1 nucleotide sequence (cecA1) the two ends introducing Not I restriction enzyme site of the sequence that designs shown in SEQ ID NO.14, transfer to the Jin Site bio tech ltd and carry out synthetic.The said firm will synthesize good fragment and insert among the pUC57, and check order, and the sequencing result feedback sequence is synthetic errorless.
The structure of multi-copy integration expression vector
(I) structure of carrier framework pYB-1:
(i) the segmental amplification of Bgl: with YIPlac204 is template, and primer b1 and b2 are primer, utilizes round pcr amplification Bgl fragment and introduces Aat II respectively, EcoR I and Bgl II restriction enzyme site at its two ends.Primer sequence is following: underscore partly is the recognition site of Bgl II, and dash area is the recognition site of Aat II, and the sequence in the framework is the recognition site of EcoR I.
Primer b1:5 '
AGATCTATTCTTGCCACGACTCATCTCC 3 '; (SEQ ID NO.2)
Primer b2:5 '
AGATCTGATTCCGATGCTGACTTGCTG 3 '; (SEQ ID NO.3)
The PCR reaction conditions is 94 ℃ of 2min, 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 30sec, cycle repeats 34 times, 72 ℃ of 10min.PCR obtains the dna fragmentation of 280bp size; This fragment purification is reclaimed the back connect with the pGM-T carrier, Transformed E .coli TOP10 bacterial strain, coating contains on LB (the 100 μ g/mL penbritin) flat board of IPTG and X-gal; Carry out blue hickie screening picking white colony; Shake and carry out bacterium liquid PCR (with bacterium liquid is template, and T7 and SP6 universal primer are primer) after bacterium spends the night and obtain about 460bp size fragment, promptly the Bgl fragment of 280bp is added on the carrier distance of about 180bp between T7 and SP6 primer binding site.This conforms to expection, explains that the Bgl fragment is with the successful connection of T carrier.
(ii) being connected of Bgl fragment and YIPlac204: the Bgl fragment is scaled off with Aat II and EcoR I enzyme from the T carrier, cut glue and reclaim.The restriction enzyme site that Aat II and EcoR I are arranged on the carrier YIPlac204; With this carrier with Aat II and EcoR I double digestion after, reclaim big fragment, and its Bgl fragment of cutting with enzyme connected; Connect liquid Transformed E .coliTOP10 bacterial strain, coating LB (100 μ g/mL penbritin) flat board.After the some transformants of picking carry out a small amount of plasmid preparation, cut with Bgl II enzyme and to verify, obtain size and be about the big fragment of 2.2kb and the small segment of 256bp.Same expection conforms to, and explains that the Bgl fragment successfully is connected to become the skeleton of expression vector with the fragment of YIPlac204, with its called after pYB-1.
(II) insertion of promotor terminator element
1.8kb PGK promotor and terminator fragment (PGKp+t) are template with plasmid pMA91, utilize high-fidelity PCR polysaccharase primeSTAR to carry out pcr amplification and obtain, reaction conditions is 98 ℃ of 10sec; 61 ℃ of 15sec; 72 ℃ of 120sec, cycle repeats 34 times, 72 ℃ of 10min; Primer is all introduced BamH I restriction enzyme site (underscore)
Pgk1:CGC
GGATCCAAGCTTTCTAACTGATCTATC (SEQ ID NO.4) and
Pgk2:CGC
GGATCCCTTTAACGAACGCAGAATTTTGGAG (SEQ ID NO.5), the reaction product size is about 1836bp.
Add the A end reaction with carrying out end after the recovery of PCR product; And then, connecting liquid Transformed E .coli TOP10 bacterial strain with the pGM-T connection, coating contains on LB (the 100 μ g/mL penbritin) flat board of IPTG and X-gal; Carry out the some white colonies of blue hickie screening picking; Prepare in a small amount and carry out enzyme with EcoR I behind the plasmid and cut checking, obtain the T carrier segments of about 3051bp and the PGKp+t fragment of about 1836bp, expecting together conforms to explains that this fragment inserted in the T carrier.The transformant that filters out is sent to order-checking, and sequencing result shows that through the sequence alignment analysis PGKp+t fragment sequence is identical with the PGKp+t on the pMA91 plasmid.
Utilize BamH I and Bgl II characteristics for isocaudarner; To scale off from T carrier enzyme with BamH I; Cut glue and reclaim the PGKp+t fragment obtain with Bgl II single endonuclease digestion pYB-1; Cut glue and reclaim the fragment connection that obtains, connect liquid Transformed E .coliTOP10 bacterial strain, coating LB (100 μ g/mL penbritin) flat board.The some transformants of picking utilize Hind III to carry out enzyme and cut checking after carrying out the preparation of a small amount of plasmid.If PGKp+t fragment forward inserts the Bgl II site of pYB-1, then, Hind III enzyme will obtain an electrophoretic band after cutting, and reverse insertion then two bands can occur, and size is about 2230bp and 1960bp respectively.The transformant of choosing reverse insertion is with its called after pYB-2.
(III) the unitary insertion of integration site rDNA
Yeast genes group camber multiple rDNA unit is the first-selected sequence that makes up high copy integrating expression vector homologous recombination district.Each rDNA unit reaches about 9.1kb, is made up of transcriptional domain and some nontranscribed domains of 5S, 5.8S, 25S and 18S rRNA.There are some researches show, be used to mediate the rDNA zone of integration, should not comprise the initial transcriptional domain of its RNA polymerase I, when being integrated in the regional external source segment size of rDNA near the rDNA element length simultaneously, its stability in mitotic division is the highest.According to this requirement, S.cerevisiae rDNA sequence is analyzed, confirm required 2.2kb segment, its relative position in the rDNA unit.This fragment contains plasmid and is used for the linear Hpa I single endonuclease digestion site that transforms.
In order to reduce the diffusion of the beta-lactam enzyme coding gene that can give the bacterium amicillin resistance; Increase the biological safety of recombinant protein engineering bacteria as fodder additives; The rDNA fragment of selecting is punished into two sections from Hpa I carries out PCR and is cloned into the pYB-2; So just can be when transformed yeast, thus utilize Hpa I linearized vector to remove beta-lactam enzyme coding gene Ampr.
According to these two sections rDNA sequences Design primers
rDNA1F：
TTTCCTCTGGC?TTCACCCTATT?(SEQ?ID?NO.6)，
rDNA1R：
CCTGTTTGAGCGTCATTTCCTTCT?(SEQ?ID?NO.7)，
rDNA2F：
GACGTCACCTAAAACGACCGTACTTGCAT?(SEQ?ID?NO.8)，
rDNA2R：
GACGTC?GAAACTCACCAGGTCCAGACACA?(SEQ?ID?NO.9)，
Primer is all introduced restriction enzyme site (Aat II underscore, Pst I shade).With yeast saccharomyces cerevisiae ABXL-1D karyomit(e) is template, and above-mentioned primer carries out pcr amplification rDNA1 and rDNA2 fragment for the upstream and downstream primer.The PCR reaction conditions is: 95 ℃ of 5min, 94 ℃ of 30sec, 61 ℃ of 30sec, 72 ℃ of 100sec, cycle repeats 34 times, 72 ℃ of 10min.The size of PCR reaction product rDNA1 and rDNA2 is about 1134bp and 1216bp respectively.RDNA2 and pYB-2 are cut with Aat II enzyme, cut and connect after glue purification reclaims fragment, connect liquid Transformed E .coli TOP10 bacterial strain, coating LB (100 μ g/mL penbritin) flat board.After the some transformants of picking carry out the preparation of a small amount of plasmid; Utilize Hind III and Hpa I to carry out enzyme and cut checking, enzyme is cut the back and is produced three bands, and size is about 2156bp respectively; The transformant of 1856bp and 960bp is the transformant that the rDNA2 fragment is oppositely inserted pYB-2, with its called after pYB-3.
PYB-3 and rDNA1 are cut with Pst I enzyme, cut and connect after glue purification reclaims fragment, connect liquid Transformed E .coli TOP10 bacterial strain, coating LB (100 μ g/ml penbritin) flat board.The some transformants of picking utilize Hpa I to carry out enzyme and cut checking after carrying out the preparation of a small amount of plasmid, and enzyme is cut the back and produced two bands, and the transformant that size is about 4089bp and 2450bp respectively is the transformant that the rDNA1 fragment is oppositely inserted pYB-3, with its called after pYB-4.
(IV) structure of pYB-5
Because contain Bgl II site in the G418 resistant gene, therefore, the cloning site Bgl II in the PGKp+t fragment is then unavailable, need change cloning site in addition.Through after the sequential analysis, learn not have Not I site on carrier and the cecA1, and Not I frequency of occurrences in genome is low, so the segmental Bgl II of PGKp+t site is replaced as Not I site.The Not fragment that is used to replace restriction enzyme site obtains through PCR, and this PCR is template with YIPlac204, and BamH I (underscore) and Not I site (shade) are introduced in two ends
NotF:CGC
GGATCC 
ATTCTTGCCACGACTCATCTCC (SEQ ID NO.10) and
notR：CGC
GGATCC 
GATTCCGATGCTGACTTGCTG?(SEQ?ID?NO.11)
Be primer, condition is: 95 ℃ of 2min, and 94 ℃ of 30sec, 61 ℃ of 30sec, 72 ℃ of 30sec, cycle repeats 34 times, 72 ℃ of 10min, the PCR reaction obtains the Not fragment that size is about 256bp.The Not fragment is cut with the BamHI enzyme, and pYB-4 cuts with Bgl II enzyme, connects after the purifying and recovering, connects liquid Transformed E .coli TOP10 bacterial strain, coating LB (100 μ g/mL penbritin) flat board.The some transformants of picking utilize Not I to carry out enzyme and cut checking after carrying out the preparation of a small amount of plasmid.Select the segmental transformant of the Not that discharges about 256bp, with its called after pYB-5.
(the v) insertion of G418 resistant gene
With plasmid pUG6 is template, utilizes high-fidelity PCR polysaccharase primeSTAR to carry out the KanMX gene that pcr amplification obtains about 1.4Kb, and the PCR reaction conditions is 98 ℃ of 10sec; 58 ℃ of 15sec; 72 ℃ of 120sec, cycle repeats 34 times, 72 ℃ of 10min; Primer is all introduced BamH I restriction enzyme site (underscore), and primer sequence is:
KanF:
GGATCCTAGGTCTAGAGATCTGTTTAGCTTGC (SEQ ID NO.12) and
KanR:
GGATCCATTAAGGGTTCTCGAGAGCTCG (SEQ ID NO.13), the reaction product size is about 1456bp.
Add the A end reaction with carrying out end after the recovery of PCR product; And then, connecting liquid Transformed E .coli TOP10 bacterial strain with the pGM-T connection, coating contains on LB (the 100 μ g/mL penbritin) flat board of IPTG and X-gal; Carry out the some white colonies of blue hickie screening picking; Prepare in a small amount and carry out enzyme with EcoR I behind the plasmid and cut checking, obtain the T carrier segments of about 3051bp and the KanMX fragment of about 1472bp, expecting together conforms to explains that this fragment inserted in the T carrier.The transformant that filters out is sent to order-checking, and sequencing result shows that through the sequence alignment analysis KanMX fragment sequence is identical with the KanMX sequence on the pUG6 plasmid.
KanMX is scaled off from T carrier enzyme with BamH I, cut glue and reclaim the back, cut glue and reclaim the fragment connection that obtains, connect liquid Transformed E .coli TOP10 bacterial strain, coating LB (100 μ g/mL penbritin) flat board with Bgl II single endonuclease digestion pYB-1.The some transformants of picking utilize Pst I to carry out enzyme and cut checking after carrying out the preparation of a small amount of plasmid.If the KanMX forward inserts the BamH I site of pYB-1, then, Pst I enzyme will obtain three electrophoretic bands after cutting, and size is respectively 276bp, 1200bp and 6720bp.The transformant of choosing the forward insertion is with its called after pYB-6 (pYIP-6).PYIP-6 is and makes up the multi-copy integration expression vector that finishes.Its sequence is shown in SEQ ID NO.1.
The preparation method of antibacterial peptide cecropin fodder additives, step is following:
(I) structure of cecA1 expression vector: pUC57-cecA1 and carrier pYIP-6 that embodiment 1 is made cut with Not I enzyme; Cutting glue reclaims corresponding fragment and connects; Connect liquid Transformed E .coli TOP10 bacterial strain, coating LB (100 μ g/mL penbritin) flat board.
The some transformants of picking shake after bacterium spends the night, and are template with bacterium liquid, carry out bacterium liquid PCR, and primer sequence is following:
P1：CAGATCATCAAGGAAGTA，SEQ?ID?NO.15
P2：CCTTACCTTCCAATAATTCC?SEQ?ID?NO.16
Reaction conditions is: 95 ℃ of 2min; 94 ℃ of 30sec, 54 ℃ of 30sec, 72 ℃ of 30sec, cycle repeats 34 times; 72 ℃ of 10min.
Obtain about 323bp size fragment (Fig. 2), promptly the cecA1 of 203bp adds on the carrier distance of about 120bp between P1 and P2 primer binding site.This conforms to expection, explains that cecA1 is with the successful connection of pYIP-6 carrier.To insert segmental several transformant and send to order-checking, and analyze sequencing result and will oppositely insert the segmental transformant called after of cecA1 pYIP-cecA1.
(II) expression vector transformed saccharomyces cerevisiae ABXL-1D and the screening that contains the multi-copy gene transformant: extract the pYIP-cecA1 plasmid, cut, cut glue recovery linearization plasmid with Hpa I enzyme.The linearization plasmid that recovery is obtained joins 80 μ L yeast saccharomyces cerevisiae ABXL-1D electricity transformed competence colibacillus cell, and mixture is added in the 0.1cm electric shock cup, chooses the yeast shock parameters and shocks by electricity.After having shocked by electricity, coating contains the YEPD flat board of 200 μ g/mL G418, is inverted for 30 ℃ and cultivates 2 days.The transformant that obtains through gradient concentration G418 plate screening is the transformant that contains high copy number.
(III) the bacterium liquid PCR check of reorganization cecropin saccharomyces cerevisiae engineered yeast: the select transformant that contains high copy number of step (II) and blank bacterium utilized freeze-boil-method of freezing prepares template; Utilization is carried out the PCR checking according to the cecA1 designed primer, and primer sequence is following:
Sense： ATGAATTTCTATAATATTTT SEQ?ID?NO.17
Antisense：TTAACCTCTTGCTGTAGCAG SEQ?ID?NO.18
Reaction conditions is: 94 ℃ of 2min, 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 30sec, cycle repeats 34 times, 72 ℃ of 10min.Get 10 μ L PCR reaction product and carry out the agarose gel electrophoresis detection.The fragment that transformant PCR obtains is approximately 204bp; Meet isotype the hardening of primer and close the distance between the zone; And the blank bacterium PCR that does not transform through carrier does not obtain band (Fig. 3); Explain in the transformant genome that screening obtains and contain cecA1, pYIP-cecA1 has been integrated in the S.cerevisiae ABXL-1D genome.
(IV) Tricine SDS-PAGE detects express recombinant cecA1 situation in the yeast saccharomyces cerevisiae born of the same parents: the inoculation transformant is in the 50mLYEPD liquid nutrient medium; Behind 30 ℃ of 200rpm shaking culture 48h; Get 1mL bacterium liquid centrifugal after, after 1mL PBS washed twice, behind 200 μ L PBS dissolution precipitations; After adding 2 * sample-loading buffer mixing, boiling water bath 10min.Get appearance SDS-PAGE (Fig. 4) on the 20 μ L treatment solutions.Can be known that by Fig. 5 transformant is expressed a kind of not expressed proteins in blank bacterium, size is about 4.6kD, conforms to the Cecropins molecular weight, Cecropins successful expression in the yeast saccharomyces cerevisiae transformant is described, but expression amount is lower.
Claims (4)
1. the preparation method of an antibacterial peptide cecropin fodder additives, step is following:
(1) with antibacterial peptide cecropin gene
CecA1Fragment and multi-copy integration expression vector are cut with Not I enzyme respectively, after connecting with dna ligase then, and Transformed E .coli TOP10 bacterial strain, coating LB is dull and stereotyped, utilizes the Not I to carry out enzyme and cuts checking, through analyzing order-checking, chooses reverse insertion
CecA1Segmental transformant called after pYIP-
CecA1
(2) pYIP-that makes of extraction step (1)
CecA1Plasmid, Hpa I digested plasmid, reclaim big fragment after, join 80 μ L yeast electricity transformed competence colibacillus cell, after the mixture electric shock,, obtain to express the yeast cell of cecropin through the dull and stereotyped screening of YEPD that contains G418;
(3) yeast cell of the expression cecropin that makes of culturing step (2) through dry, makes antibacterial peptide cecropin fodder additives;
The nucleotide sequence of the multi-copy integration expression vector in the said step (1) is shown in SEQ ID NO.1.
2. preparation method as claimed in claim 1 is characterized in that, the electric shock time that electricity transforms in the said step (2) is 3.5～4.0 ms.
3. preparation method as claimed in claim 1 is characterized in that, the culture condition in the above-mentioned steps (3): in the YEPD of no selective pressure substratum, cultivated 2～3 days for 28～30 ℃.
4. preparation method as claimed in claim 1 is characterized in that, the yeast in the above-mentioned steps (2) is the wild-type yeast saccharomyces cerevisiae ABXL-1D with flocculence, and bacterial classification is available from U.S. ATCC company.
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| CN102304541A (en) * | 2011-08-17 | 2012-01-04 | 中国药科大学 | Saccharomyces cerevisiae expression carrier containing constitutive promoter pPGK and G418 resistance gene and construction method thereof |
| CN102578417B (en) * | 2012-02-27 | 2013-09-11 | 贵阳台农种养殖有限公司 | Piglet feed and additive thereof |
| CN104381419A (en) * | 2014-11-20 | 2015-03-04 | 洞头县水产科学技术研究所 | Biological preservative and preserving method thereof |
| CN114106133A (en) * | 2021-12-01 | 2022-03-01 | 郑州人民医院(郑州人民医院医疗管理中心) | Gene editing housefly antibacterial peptide Sarcotoxin-1B nucleotide sequence and method for preparing antibacterial peptide |
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