CN103435701B - Pig antibacterial peptide cystatin11 fusion protein, and encoding gene and application thereof - Google Patents
Pig antibacterial peptide cystatin11 fusion protein, and encoding gene and application thereof Download PDFInfo
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- CN103435701B CN103435701B CN201310312961.1A CN201310312961A CN103435701B CN 103435701 B CN103435701 B CN 103435701B CN 201310312961 A CN201310312961 A CN 201310312961A CN 103435701 B CN103435701 B CN 103435701B
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- cystatin11
- antibacterial peptide
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Abstract
The invention belongs to the field of genetic engineering of biotechnology pharmacy industrials, and specifically relates to a pig antibacterial peptide cystatin11 fusion protein, and an encoding gene and an application thereof. The advantages of antibacterial peptides and pichiapastoris are well utilized by the pig antibacterial peptide cystatin11 fusion protein, and the encoding gene of the pig antibacterial peptide cystatin11 fusion protein is realized in efficient expression in pichiapastoris, so that the production cost of the antibacterial peptide is reduced. The fusion protein is applicable to preparation of medicaments for inhibiting bacteria especially such as escherichia coli and staphylococcus, so that a novel green safe and no-residual antibacterial medicament is developed, and therefore the pig antibacterial peptide cystatin11 fusion protein has extremely high economic value and social benefit.
Description
Technical field
The invention belongs to genetically engineered field in biological-pharmacy, specifically boar antibacterial peptide cystainll fusion rotein and encoding gene and an application.
Background technology
Along with the continuous expansion of cultivation scale, bacillary and viral infectious has become the serious key factor that restricts China's aquaculture development, and has caused great financial loss.Although being widely used of chemicals in the prevention and control of disease, bring into play important effect, its high residue in vivo and the appearance of pathogenic micro-organism multi-drug resistant have brought serious threat to the health of animals and humans.Therefore, the antimicrobial agents of development of new seems particularly urgent.Antibacterial peptide is almost present in all organisms as the important composition composition of body innate immunity, but can kill bacterium, fungi, virus, parasite and cancer cells, and effect rapidly, is not subject to the resistance mutation that microorganism has produced to affect, be difficult for making microorganism to occur resistance mutation.Along with deepening continuously of research, antibacterial peptide has more and more demonstrated its unique application in association areas such as medical science, veterinary science and has been worth, and antibacterial peptide is regarded as desirable Substitutes For Antibiotic for a long time.But antibacterial peptide natural yield poorly and synthesize expensive be the problem that antimicrobial peptide medicaments development must solve.
Pichia spp (Pichia pastoris) expression system is a kind of novel exogenous protein expression system growing up the phase at the beginning of the eighties in last century, it both had prokaryotic expression system operation simple and easy, be easy to that cultivation, fast growth, expression amount are high, low cost and other advantages, also there is the features such as posttranslational modification to foreign protein that prokaryotic expression system does not have, as glycosylation, protein phosphorylation etc.It has also avoided the defects such as yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) secernment efficiency is poor, expression strain is stable not, the easy loss of expression plasmid simultaneously.In addition, due to it aspect protein expression, also have advantages of following other expression system incomparable, make this system become one of current most study, most widely used eukaryotic expression system: (1) its entrained aox promotor is a kind of strong promotor, be subject to the strict induction regulating controlling of methyl alcohol and express, so can strictly regulate and control the expression of foreign protein; (2) can carry out to expressing protein a series of posttranslational modifications such as glycosylation, protein phosphorylation, folding, signal sequence processing, lipid acidylate, thereby make the albumen of its expression there is biological activity; (3) expression amount is high, and up to now, existing more than 300 kind of heterologous protein obtained high efficient expression in this expression system, and as TTFC can reach 12g/L, and the expression amount of gelatin has even reached 14.8g/L; (4) expression product of foreign gene both can be present in born of the same parents, can secrete to extracellular by the signal peptide of its distinctive signal peptide α-factor or native protein itself again, and meanwhile, the albumen of this system self secretion is considerably less, and this has simplified purge process greatly; (5) be integrated into the recon expression system of foreign gene very stable; Have bibliographical information by homologous recombination be integrated into the recon expression system of foreign gene can cultured continuously 50 generations but have no the phenomenon that foreign gene is lost; (6) degree of glycosylation is low, the mean length that is added to every side chain of foreign protein in Pichia pastoris is 8-14 mannose residue, than the average 50-150 mannose residue of every side chain of yeast saccharomyces cerevisiae, want much shorter, simultaneously, because can not resembling yeast saccharomyces cerevisiae, Pichia pastoris forms α-1 at core polysaccharide end, 3 end seminoses, make its protein antigenicity well below the latter, thereby are more suitable in therepic use; (7) this expression system is due to low to nutritional requirement, and Media Components cheap and simple can be carried out the fermentation culture of high-density high yield, is convenient to suitability for industrialized production.The efficient expression system that utilizes Pichia pastoris to build can significantly improve the output of antibacterial peptide, thereby reduces the production cost of antibacterial peptide, and the suitability for industrialized production of antibacterial peptide is had broad application prospects.
But some antibacterial peptide can not be expressed well in Pichia pastoris, therefore, need badly provide a kind of Pichia of meeting pastoris express Preference and can be in Pichia pastoris expression system the antimicrobial peptide protein of high efficient expression.
Summary of the invention
The present invention aim to provide a kind of Pichia of meeting pastoris express Preference and can be in Pichia pastoris expression system the antimicrobial peptide protein of high efficient expression, specifically boar antibacterial peptide cystain11 fusion rotein and encoding gene and an application.
The present invention is achieved by the following technical solutions: pig antibacterial peptide cystain11 fusion rotein, its aminoacid sequence is as shown in SEQ IDNO:2.
The application of described fusion rotein in preparing anti-bacteria medicine.Described bacterium is intestinal bacteria and staphylococcus.
The encoding gene of described fusion rotein, its base sequence is as shown in SEQ ID NO:1.
The encoding gene of fusion rotein of the present invention is comprised of the cDNA sequence in the cDNA sequence of coding pig cystain11 mature peptide, 6 Histidine cDNA sequences and restriction enzyme Xho I and EcoR I site.
In the present invention, the preparation method's of the encoding gene of pig antibacterial peptide cystain11 fusion rotein step is:
The structure of expression vector: the restriction enzyme site that is designed with restriction enzyme in the gene order of the cystatin11 of synthetic, the gene order of cystatin11 and expression plasmid pwPICZalpha, with linking together after same digestion with restriction enzyme, are detected and are screened the recombinant plasmid that contains cystatin11 gene order by double digestion;
The structure of yeast expression system: use restriction enzyme linearizing recombinant expression plasmid, the method transforming by electricity, the gene order of cystatin11 is incorporated in the genome of pichia spp, thalline suspension after transforming is coated on the YPD flat board that contains Zeocin, cultivate 3-4d for 30 ℃, 6 single bacterium colonies of picking, carry out low dose of abduction delivering, and SDS-PAGE detects and expresses supernatant confirmation positive colony;
The purifying of recombinant protein and active detection: select positive colony bacterium colony and carry out heavy dose of abduction delivering, express supernatant and adopt ProteinPure Ni-NTA resin and strong cation-exchanging resin purifying, by SDS-PAGE and Western blot, detect the recombinant protein of purifying; By Bactericidal test, detect the anti-microbial activity of recombinant protein.
Pig antibacterial peptide cystain11 fusion rotein of the present invention has utilized the advantage of antibacterial peptide and Pichia pastoris well, its encoding gene can be in Pichia pastoris high efficient expression, reduced the production cost of antibacterial peptide, application by this fusion rotein in preparing anti-bacteria medicine, particularly intestinal bacteria and staphylococcus, develop the antibacterial newtype drug of a kind of green, safety, noresidue, there is very high economic worth and huge social benefit.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE electrophorogram of ProteinPure Ni-NTA resin purification restructuring cystatin11.
Fig. 2 is the SDS-PAGE electrophorogram of strong cation-exchanging resin purification of Recombinant cystatin11.
Fig. 3 is the Westernblot analysis chart of restructuring cystatin11.
Fig. 4 is the fungistatic effect figure of restructuring cystatin11, in figure: (A) intestinal bacteria; (B) staphylococcus; (1) penbritin; (2) blank; (3) PBS; (4) restructuring cystatin11.
Embodiment
The following stated, is only that the present invention is further narrated, and is not the present invention to be done to the restriction of other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not depart from, any simple modification, equivalent variations and the remodeling above embodiment done according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.
1, agents useful for same and preparation thereof
(1) LB (Luria-Bertani) liquid nutrient medium:
By peptone, yeast powder, NaCl and ddH
2o mixes, and adds NaOH and regulates pH to 7.0, is settled to 1000mL, and autoclaving (121 ℃, 1.034 * 10
5pa) 20min, 4 ℃ of preservations.
(2) LB (Luria-Bertani) solid medium:
At LB liquid nutrient medium, press every 1000mL ddH
2o adds 15g agar powder, autoclaving, 4 ℃ of preservations.
(3) preparation method of each stock solution:
(4) YPD substratum:
Yeast extract and peptone is dissolved in 900mL ddH
2in O, (adding 20g agar YPD plate processed) autoclaving, then add 100mL10 * glucose, 4 ℃ save backup.
(5) YPG substratum:
Yeast extract and peptone is dissolved in 900mL ddH
2in O, autoclaving, then add 100mL10 * glycerine, 4 ℃ save backup.
(6) BMMYC substratum:
Yeast extract, peptone and ddH
2o mixes, and autoclaving, is cooled to room temperature, adds 100mL1M dipotassium hydrogen phosphate damping fluid, and pH=7.0,100mL10 * YNB, 2mL500 * vitamin H, 100mL10 * methyl alcohol, 100mL10 * acid hydrolysis casein mix, 4 ℃ of preservations.
(7) damping fluid
2, the optimization of antibacterial peptide cystatin11 gene order and acquisition
Pig cystatin11 (CST11) gene order (GeneID:100302695) and the Pichia yeast codon preference that according to NCBI, log in, gene order to CST11 is optimized, for the ease of purifying, at C end, add 6 Histidines (6 * His tag), at 5 ' and 3 ' end of sequence, introduce Xho I and two restriction enzyme sites of EcoR I.CST11 gene order after optimization is synthesized by Invitrogen company.
3, the structure of expression vector
Utilize two restriction enzymes of Xho I and EcoR I to carry out double digestion to CST11 and plasmid pwPICZalpha after synthetic, the product after enzyme is cut carries out agarose electrophoresis and adopts glue to reclaim test kit reclaiming DNA fragmentation and connecting.Be specially: in aseptic Eppendorf pipe, add the goal gene fragment of 7 μ L double digestions, the plasmid of 1 μ L double digestion, 1 μ L T4 ligase enzyme, 1 μ L ligase enzyme damping fluid, mixes, and 4 ℃ connect 12h; 10 μ L are connected to product and join in 90 μ L competence intestinal bacteria TOP10, mix, 42 ℃ of heat shock 90s transform, add 400 μ L LB liquid nutrient mediums, 37 ℃ of shaking culture 1h, are coated on bacterium liquid on the LB solid medium of Zeocin resistance 37 ℃ of overnight incubation.From the flat board transforming, 6 colony inoculations of picking are to containing in the 5mL LB liquid nutrient medium of Zeocin, and 37 ℃ of incubated overnight, extract recombinant plasmid with plasmid extraction kit.With Xho I and EcoR I, recombinant plasmid is carried out to double digestion evaluation, checking gene fragment is correctly inserted, this recombinant plasmid called after pwPICZalpha-CST11.
4, the expression of recombinant protein
(1) low-dose induction is expressed
Utilize restriction enzyme Sac I that 5-10 μ g recombinant plasmid pwPICZalpha-CST11 is carried out to linearizing, adopt PCR product purification test kit to reclaim linearizing pwPICZalpha-CST11, join in 90 μ L competence pichia spp, mix, after ice bath 5min, electric shock transforms, add after 1mL sorbyl alcohol 30 ℃ to place 2h, bacterium liquid is coated on the YPD solid medium of Zeocin resistance, cultivate 3-4d for 30 ℃.From the flat board transforming, 6 colony inoculations of picking are to containing in the 5mL YPD liquid nutrient medium of Zeocin, 30 ℃ of 250rpm cultivate 24h, 3500rpm is centrifugal, after abandoning supernatant, add 5mL YPG liquid nutrient medium, 30 ℃ of 250rpm cultivate 24h, the centrifugal supernatant that discards of 3500rpm, add 2mL BMMYC liquid nutrient medium, 27 ℃ of 225rpm cultivate 48h, every 12h, supplement a methyl alcohol, make methanol concentration maintain 0.5%.The centrifugal rear collection supernatant liquor of 3500rpm, SDS-PAGE analyzes supernatant liquor.
(2) heavy dose of abduction delivering
Positive bacterium colony of picking is inoculated in YPD liquid nutrient medium, 30 ℃ of 250rpm cultivate 24h as seed culture fluid, getting 13mL seed culture fluid is inoculated in 250mLYPD liquid nutrient medium, 30 ℃ of 250rpm cultivate 24h, 3500rpm is centrifugal, after abandoning supernatant, add 250mL YPG liquid nutrient medium, 30 ℃ of 250rpm cultivate 24h, the centrifugal supernatant that discards of 3500rpm, add 125mL BMMYC liquid nutrient medium, 27 ℃ of 225rpm cultivate 48h, every 12h, supplement a methyl alcohol, make methanol concentration maintain 0.5%.The centrifugal rear collection supernatant liquor of 3500rpm, SDS-PAGE analyzes supernatant liquor.
5, the purifying of recombinant protein
(1) ProteinPure Ni-NTA resin purification
In the supernatant liquor of abduction delivering, add 50mM sodium phosphate, 0.3M sodium-chlor, 10mM imidazoles and 10mM pH8.0Tris-HCl, filter with the filter of 0.45 μ m.Getting 10mL ProteinPure Ni-NTA resin packs in the chromatography column of XK16/20, after installing, pillar is connected with constant flow pump, Buffer I balance pillar with 10 times of volumes, after pillar balance is good, carry out loading, Buffer I with 8 times of volumes after end of the sample rinses pillar, with the BufferII wash-out target protein of 8 times of volumes and collect in 8 different pipes, the flow velocity of whole process is 5mL/min again.With SDS-PAGE, the albumen of purifying is analyzed.SDS-PAGE result in Fig. 1 shows, target protein concentrate on No. 2 of Buffer II and No. 3 pipes in.The BufferII that contains target protein is mixed, packs in the dialysis tubing of 3.5kDa, under 4 ℃ of conditions with containing 20mM Tris-HCl pH8.0,1mM EDTApH8.0, the dialyzate of the 5% glycerine 8h that dialyses, changes dialyzate one time every 4h.
(2) strong cation-exchanging resin purifying
Get in the chromatography column that 10mL strong cation-exchanging resin packs XK16/20 into, after installing, pillar is connected with constant flow pump, BufferIII balance pillar with 10 times of volumes, the sample of having dialysed is carried out to loading, BufferIII with 8 times of volumes after end of the sample rinses pillar, use respectively BufferIV and the BufferV wash-out target protein of 8 times of volumes again, collect in 8 different pipes, the flow velocity of whole process is 5mL/min.With SDS-PAGE, the albumen of purifying is analyzed, target protein concentrates in washings as shown in Figure 2.The washings that contains target protein is mixed, adopt the method for ultrafiltration and concentration to concentrate target protein, then pack in the dialysis tubing of 3.5kDa, under 4 ℃ of conditions, with the PBS dialysis 8h that contains 5% glycerine, every 4h, change PBS one time.By BCA kit measurement target protein concentration, protein concentration is 1mg/mL, illustrates that this protein gene obtains high efficient expression in pichia spp.Utilize anti-His tag antibody to carry out western blot analysis to this albumen, this Western blot analysis chart is confirmed it for albumen to be expressed.
6, bacteriostatic experiment
Intestinal bacteria and staphylococcus are respectively got 100 μ L and are coated onto in LB solid culture, at flat board, make a call to the hole that 4 diameters are 5mm, in hole, add respectively 50 μ L recombinant C ST11 albumen (1mg/mL), penbritin (100mg/mL), PBS, last 1 hole is as blank, cultivate 12h for 37 ℃, observe and record fungistatic effect, fungistatic effect figure is shown in Fig. 4, the cystatin11 that recombinates as can be seen from Figure can effectively suppress intestinal bacteria and staphylococcic growth, and the cystatin11 that shows to recombinate can carry out further development research as potential antibacterials.
< 110 > Agricultural University Of Shanxi
< 120 > pig antibacterial peptide cystain11 fusion rotein and encoding gene and application
〈160〉2
〈210〉1
〈211〉402
〈212〉DNA
< 213 > artificial sequences
〈220〉
〈223〉
〈222〉(1)…(402)
〈400〉1
CCGCTCGAGA AGAGAGAGGC TGAAGCTTAC CAAACTAAGA AGAAGTCTTT CATTCACGTT 60
CAAGAGTTGC CAGTTTCTGA CCCATTCGTT GTCACTACCT TGGACTTCGT TTCTAAGGAG 120
TTCAACAAGA AGTCTGAGGA CAAGTACAAC TTCAGAATTG TTAGAGTTTT GAAGGCTGTT 180
CAAGTTTTGA CTAACCACTT GGAGTACAGA TTGAACTTGG AGATGCAAAG AACTGTTTGT 240
CCAAAGGCTG AGAGAAACGC TTGTACTTTC CAAGAGGGTG AGTTGTACAA GAAGATTGAC 300
TGTTTCTTCT CTGTTTTCGC TGTTCCATGG TTCGAGAAGT ACAAGATTTT GACTAAGGCT 360
TGTACTGACG GTCACCACCA CCACCACCAC TAAGAATTCC GG 402
〈210〉2
〈211〉121
〈212〉PRT
< 213 > artificial sequences
〈400〉2
Try Gln Thr Lys Lys Lys Ser Phe Iie His Val Gln Glu Leu Pro Val Ser Asp Pro Phe Val Val Thr Thr Leu
25
Asp Phe Val Ser Lys Glu Phe Asn Lys Lys Ser Glu Asp Lys Tyr Asn Phe Arg Iie Val Arg Val Leu
26
Lys Ala
50
Val Gln Val Leu Thr Asn His Leu Glu Tyr Arg Leu Asn Leu Glu Met Gln Arg Thr Val Cys Pro Lys
51
Ala Glu
75
Arg Asn Ala Cys Thr Phe Gln Glu Gly Glu Leu Tyr Lys Lys Ile Asp Cys Phe Phe Ser Val Phe Ala
76
Val Pro
100
Trp Phe Glu Lys Tyr Lys Ile Leu Thr Lys Ala Cys Thr Asp Gly His His His His His His
101 121
Claims (3)
1. pig antibacterial peptide cystain11 fusion rotein, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:2.
2. described in claim 1, fusion rotein suppresses the application in intestinal bacteria and staphylococcus medicine in preparation.
3. the encoding gene of fusion rotein described in claim 1, is characterized in that, its base sequence is as shown in SEQ ID NO:1.
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| CN104017087B (en) * | 2014-06-24 | 2016-03-09 | 科美博瑞科技(北京)有限公司 | One boar derived antimicrobial peptide and preparation method thereof |
| CN104017086B (en) * | 2014-06-24 | 2016-03-09 | 科美博瑞科技(北京)有限公司 | A kind of fusion antibacterial peptide and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102676533A (en) * | 2012-05-16 | 2012-09-19 | ***广州总医院 | Recombinant human cystatin C coding gene and expression method |
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| CN102676533A (en) * | 2012-05-16 | 2012-09-19 | ***广州总医院 | Recombinant human cystatin C coding gene and expression method |
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