CN101591622A - Melittin gene fission yeast engineering bacteria and construction process thereof and application - Google Patents
Melittin gene fission yeast engineering bacteria and construction process thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of Melittin gene fission yeast engineering bacteria and construction process thereof and application, this project bacterium is the fission yeast that is transformed by the Melittin gene fission yeast recombinant expression vector; Its construction process comprises structure Melittin gene fission yeast recombinant expression vector, transforms fission yeast totally 2 steps with the Melittin gene fission yeast recombinant expression vector; Use separation and purification totally 2 steps that method that this project bacterium produces mellitin comprises expression, the mellitin of mellitin; Easy to operation, mellitin expression level height, be easy to separation and purification, active strong, with short production cycle, cost is low, remedied the deficiency of existing mellitin preparation method and prokaryotic expression system and other yeast expression system, can reduce native conformation and the activity that mellitin guarantees mellitin in to the host cell lethality, solve that mellitin extracts purification difficult, synthetic cost is crossed problems such as height, thereby provide strong instrument for the fundamental research and the production application of mellitin.
Description
Technical field
The present invention relates to a kind of genetic engineering bacterium, particularly Melittin gene fission yeast engineering bacteria also relates to the preparation method of this project bacterium and uses the method that this project bacterium produces mellitin.
Background technology
Antibacterial peptide claims antimicrobial peptide or peptide antibiotic again, make a general reference to be present in and have the external microbe of resisting infringement in the organism, remove the small molecule polypeptide of vivo mutations cell, being distributed widely in bacterium, virus and the various animal and plant body, is the important component part of biological innate immune defence system.For microbiotic, antibacterial peptide have has a broad antifungal spectrum, effective concentration low, be difficult for making the microorganisms resistance, can transform easily and advantage such as modification.
Mellitin (Melittin) is a kind of of antibacterial peptide, has broad-spectrum bactericidal action, the growth and breeding that can suppress more than 20 kind of Gram-positive and negative bacteria, can resist the drug-fast streptococcus aureus of penicillin, can also strengthen the antibacterial efficacy of sulfamido and penicillin medicine, multiple fungi, virus are also had toxicity, be expected to become efficient, the microbiotic of new generation of low toxicity and the candidate of agricultural chemicals.
Mellitin can obtain from bee venom in separation and purification, also can direct chemical synthesize, but existing separating and purifying technology is difficult to and will sensitivity response is arranged in the bee venom and remove fully with the approaching Phospholipase A2 of mellitin molecular weight, and direct chemical synthetic cost is higher, and the clinical application of mellitin is severely limited.The acquisition mellitin that develops into of genetic engineering technique provides a new approach, obtained certain progress in recent years, but still have many weak points, the big more options coli expression system of heterogenous expression as mellitin, though intestinal bacteria have plurality of advantages as the expression system of genetically engineered drug first-selection, but its translation post-treatment is modified the system imperfection, and the expression product activity is low; Also there is research to express mellitin with the Pichia anomala expression system, though pichia spp possesses certain post transcriptional modificaiton function, the too late far away fission yeast of its biological similarity with higher eucaryote.
Fission yeast (Schizosaccharomyces pombe) is a kind of unicellular eukaryote, has with the extremely similar characteristic of higher eucaryote: as the splicing of regulation of Cell Cycle, chromosomal 26S Proteasome Structure and Function, RNA, proteinic glycosylation, phosphorylation and acylations function etc.Compare with pichia spp with yeast saccharomyces cerevisiae, fission yeast can better be expressed eukaryotic protein, and keeps these proteic native conformation and functions.Its genom sequence in 2002 and obtaining of each gene function more help studying the expression of foreign protein in fission yeast.Fission yeast draws attention recently as the research of efficient heterologous gene expression system, and present domestic research report is less.
Summary of the invention
In view of this, one of purpose of the present invention is to provide a kind of Melittin gene fission yeast engineering bacteria, can when the reduction mellitin is to the host cell lethality, guarantee the native conformation and the activity of mellitin, solve that mellitin extracts purification difficult, synthetic cost is crossed problems such as height, thereby provide strong instrument for the fundamental research and the production application of mellitin.
For reaching this purpose, the invention provides a kind of Melittin gene fission yeast engineering bacteria, this project bacterium is the fission yeast that is transformed by the Melittin gene fission yeast recombinant expression vector.
Further, described Melittin gene fission yeast recombinant expression vector contains the Melittin gene of nucleotide sequence shown in SEQ IDNo.1, the mellitin of its encoding amino acid sequence shown in SEQ ID No.2;
Further, the fission yeast expression vector of described Melittin gene fission yeast recombinant expression vector employing is pESP-2;
Further, described fission yeast is schizosaccharomyces pombe SP-Q01.
Two of purpose of the present invention is to provide the construction process of described Melittin gene fission yeast engineering bacteria, and is easy to operation, and expression product is easy to separation and purification.
For reaching this purpose, the invention provides the construction process of described Melittin gene fission yeast engineering bacteria, may further comprise the steps:
A, the multiple clone site place that Melittin gene is inserted the fission yeast expression vector make up the Melittin gene fission yeast recombinant expression vector;
B, the Melittin gene fission yeast recombinant expression vector that makes up with step a transform fission yeast, promptly get Melittin gene fission yeast engineering bacteria.
Further, described step a prepares the Melittin gene of nucleotide sequence shown in SEQ ID No.1 earlier, and the gained Melittin gene is cloned into the pMD18-T carrier, makes up Melittin gene recombinant vectors pMD18-T-Melittin; After again the Melittin gene recombinant vectors pMD18-T-Melittin that makes up being used restriction enzyme BamHI single endonuclease digestion, be connected with the fission yeast expression vector pESP-2 that handles through BamHI single endonuclease digestion and alkaline phosphatase dephosphorylation, make up Melittin gene fission yeast recombinant expression vector pESP-2-Melittin;
Further, described step b adopts the Lithium Acetate chemical transformation to transform schizosaccharomyces pombe SP-Q01 with the Melittin gene fission yeast recombinant expression vector pESP-2-Melittin that step a makes up, with leucine auxotroph substratum screening positive transformant, promptly get Melittin gene fission yeast engineering bacteria SP-Q01/pESP-2-Melittin.
Three of purpose of the present invention is to provide uses the method that described Melittin gene fission yeast engineering bacteria is produced mellitin, easy to operation, mellitin expression level height, is easy to separation and purification, active strong, and with short production cycle, cost is low.
For reaching this purpose, the invention provides and use the method that described Melittin gene fission yeast engineering bacteria is produced mellitin, may further comprise the steps:
A, cultivation Melittin gene fission yeast engineering bacteria also make it express mellitin, collect culture;
B, the culture that step a is collected carry out separation and purification, promptly get mellitin.
Further, described step a is earlier with containing the YES liquid nutrient medium enlarged culturing of VitB1 to OD with Melittin gene fission yeast engineering bacteria
600Be 1.0, after washed cell is removed residual YES liquid nutrient medium and VitB1, with the EMM liquid nutrient medium inducing culture that does not contain VitB1 18 hours, collect culture again;
Further, described step b gets the culture that step a collects, centrifugal removal supernatant liquor, smudge cells, the centrifugal removal cell debris of ultra-high speed, supernatant liquor get the fusion rotein GST-Melittin of glutathione-S-transferase and mellitin again through the separation and purification of GST affinity chromatography, GST-Melittin with enteropeptidase (Ek) excision GST, is promptly got mellitin.
Beneficial effect of the present invention is: the invention provides a kind of Melittin gene fission yeast engineering bacteria and construction process thereof and application, easy to operation, mellitin expression level height, be easy to separation and purification, active strong, with short production cycle, cost is low, remedied the deficiency of existing mellitin preparation method and prokaryotic expression system and other yeast expression system, can when the reduction mellitin is to the host cell lethality, guarantee the native conformation and the activity of mellitin, solve mellitin and extract purification difficult, synthetic cost is crossed problems such as height, thereby provides strong instrument for the fundamental research and the production application of mellitin.
Description of drawings
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:
Fig. 1 is a schematic flow sheet of the present invention;
Fig. 2 is the structure synoptic diagram of Melittin gene;
Fig. 3 is the agarose gel electrophoresis figure of Melittin gene PCR product;
Fig. 4 is the structure synoptic diagram of Melittin gene fission yeast recombinant expression vector pESP-2-Melittin;
Fig. 5 is the agarose gel electrophoresis figure of Melittin gene fission yeast engineering bacteria PCR product;
Fig. 6 is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) figure of GST-Melittin;
Fig. 7 is protein immunoblot (Western blot) figure of GST-Melittin;
Fig. 8 cuts the SDS-PAGE figure of product for the enteropeptidase enzyme of GST-Melittin.
Embodiment
Below with reference to accompanying drawings, the preferred embodiments of the present invention are described in detail, schematic flow sheet of the present invention as shown in Figure 1.
One, the structure of Melittin gene fission yeast engineering bacteria
The construction process of Melittin gene fission yeast engineering bacteria may further comprise the steps:
A, structure Melittin gene fission yeast recombinant expression vector
The clone of a1, Melittin gene
Reasonably combined according to the preference of yeast and e. coli codon and codon; design mellitin coding gene sequence under the prerequisite that does not change the mellitin aminoacid sequence; for improving the specificity pairing between primer; during design partial password has been carried out same sense mutation; 5 ' end at the mellitin coding gene sequence adds protection base, restriction enzyme digestion sites and Ek recognition site sequence again; add restriction enzyme digestion sites and protection base at 3 ' end, design Melittin gene sequence is as follows:
5 '-aaggatcc
GgatccAa-3 ' (SEQ ID No.1); Wherein, single underscore partly is the BamHI restriction enzyme site, and italicized item is the Ek restriction enzyme site, and black matrix partly is the mellitin encoding gene, and double underline partly is the XbaI enzyme cutting site;
According to the Melittin gene sequence, design following PCR primer and entrust Shanghai Ying Jun Bioisystech Co., Ltd to synthesize:
P1：5’-aaggatccgatgatgatgataaaggaattggagcagttctgaaggtattaaccac-3’(SEQ?ID?No.3)；
P2：5’-ttaatccaacttatgagggcgggcaatcctgtggttaataccttcagaac-3’(SEQ?ID?No.4)；
P3：5’-ttggatcctctagattactactgttgcctcttacgtttaatccaacttatgagggc-3’(SEQ?ID?No.5)；
Adopt the gene fusion strategy of overlapping extension to prepare Melittin gene, make up synoptic diagram as shown in Figure 2, comprise two-wheeled PCR altogether: first round PCR passes through terminal complementary pairing primer each other by P1 and P2, extends to make the Melittin gene fragment; Reaction system is: sterilized water 18 μ L, 10 * PCR reaction buffer, 2.5 μ L, concentration are the MgCl of 25mM
22.5 μ L, concentration are that the dNTP 0.5 μ L of 10mM, P1 that concentration is 20 μ M and each 1 μ L of P2, concentration are Taq archaeal dna polymerase (Takara company) the 0.2 μ L of 5U/ μ L; Second to take turns PCR be to be template with first round PCR product, and P1 and P3 are the upstream and downstream primer, make the Melittin gene total length, and reaction system is: sterilized water 17 μ L, 10 * PCR reaction buffer, 2.5 μ L, MgCl
22.5 μ L, dNTP 0.5 μ L, P1 1 μ L, P3 1 μ L, template 1 μ L, Taq archaeal dna polymerase 0.2 μ L; The reaction conditions of two-wheeled PCR is identical: at first, 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute; Secondly, 94 ℃ of sex change 30 seconds, 58 ℃ of annealing 30 seconds, 72 ℃ were extended totally 30 circulations 30 seconds; At last, 72 ℃ were extended 10 minutes.
Second takes turns the PCR product, and to adopt mass percent concentration be that 1% agarose gel electrophoresis is identified, the result as shown in Figure 3,500,400,300,250,200,150,100 wherein the M swimming lane is dna molecular amount standard (molecular weight is followed successively by from big to small:, 50bp), and 1 and 2 swimming lanes are second to take turns the PCR product; A special DNA band all appears in 1 and 2 swimming lanes at the about 120bp of molecular weight place, conform to goal gene expection size; Identify that correct second takes turns the PCR product and adopt DNA product purification test kit (TIANGEN company) to cut glue to reclaim purifying, promptly get Melittin gene; Melittin gene is connected under T4DNA ligase enzyme (Takara company) effect with carrier pMD18-T (Takara company), connect product and be transformed into e. coli jm109 (Takara company) competent cell, usefulness contains the penbritin that concentration is 100mg/L, 5-bromo-4-chloro-3-indoles-α-D-galactoside (X-gal) and the concentration that concentration is 40mg/L is the LB plate culture medium screening positive clone bacterium of the isopropylthiogalactoside (IPTG) of 10mmol/L; Picking positive colony list bacterium colony is inoculated in the LB liquid nutrient medium that contains penbritin, after 37 ℃ of jolting overnight incubation of temperature, extracts recombinant plasmid in a small amount, and verify the positive colony bacterium with the PCR method: with P1 and P3 is upstream and downstream primer amplification Melittin gene; Gained positive colony bacterium entrusts Shanghai Ying Jun Bioisystech Co., Ltd to adopt the Sanger dideoxy method to carry out dna sequencing, the result is presented at and is inserted with among the pMD18-T and the on all four dna fragmentation of nucleotide sequence shown in the SEQ ID No.1, shows that Melittin gene recombinant vectors pMD18-T-Melittin successfully constructs.
A2, Melittin gene are connected with the fission yeast expression vector
Adopt Melittin gene recombinant vectors pMD 18-T-Melittin and fission yeast expression vector pESP-2 to make up Melittin gene fission yeast recombinant expression vector pESP-2-Melittin, make up synoptic diagram as shown in Figure 4.Fission yeast expression vector pESP-2 comprises with the lower section: (1) intestinal bacteria replication initiation sequence C olE1 and penbritin (ampicilin) resistant gene, and it allows to carry out duplicating with microbiotic of carrier when escherichia coli cloning and selects; (2) fission yeast autonomously replicating sequence ARS1 can be provided in and makes carrier duplicate needed replication origin automatically in the fission yeast; (3) selectable marker gene LEU2-d can be used as the selection that transforms expression structure in fission yeast and demarcates thing; (4) expression cassette, comprise in the fission yeast strict nmt1 promotor (Pnmt1) and terminator (Tnmt1), glutathione-S-transferase (GST) full length sequence, zymoplasm (Thrombin) recognition site and the multiple clone site (MCS) that is subjected to the regulation and control of VitB1 concentration, can be in fission yeast the fusion rotein of abduction delivering band GST label.Concrete operations are as follows:
Melittin gene recombinant vectors pMD18-T-Melittin is carried out single endonuclease digestion with BamHI (Takara company), adopt DNA product purification test kit to cut glue and reclaim the purifying Melittin gene, again with through BamHI single endonuclease digestion and calf intestine alkaline phosphatase (CIAP, Takara company) the fission yeast expression vector pESP-2 (Invitrogen company) of dephosphorylation processing connects under the effect of T4DNA ligase enzyme, connect product and be transformed into escherichia coli jm109 competent cell, with containing the penbritin that concentration is 100mg/L, concentration is the LB plate culture medium screening positive clone bacterium of the X-gal of 40mg/L and the IPTG that concentration is 10mmol/L; Picking positive colony list bacterium colony, be inoculated in the LB liquid nutrient medium that contains penbritin, after 37 ℃ of jolting overnight incubation of temperature, extract recombinant plasmid in a small amount, verify the positive colony bacterium with the PCR method: the nucleotide sequence according to pESP-2 multiple clone site two ends designs following PCR primer (can clone the fragment of about 260bp to the pESP-2 empty carrier): Yf:5 '-gtacttgaaatccagcaagtatatagc-3 ' (SEQ ID No.6); Yr:5 '-caaaatcgtaatatgcagcttgaatgggcttcc-3 ' (SEQ ID No.7); With Yf and Yr is upstream and downstream primer amplification recombinant plasmid, with P1 and P3 is upstream and downstream primer amplification Melittin gene, reaction system is: each 10pmol of upstream and downstream primer, dNTP 200 μ mol, bacterium liquid 1 μ L, 10 * PCR reaction buffer, 2.5 μ L, Taq archaeal dna polymerase 1U, and it is 25 μ L that sterilized water is supplemented to cumulative volume; Reaction conditions is: at first, and 95 ℃ of pre-sex change 5 minutes; Secondly, 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended totally 35 circulations 1 minute; At last, 72 ℃ were extended 5 minutes; Gained positive colony bacterium entrusts Shanghai Ying Jun Bioisystech Co., Ltd to adopt the Sanger dideoxy method to carry out dna sequencing, the result is presented at and is inserted with among the pESP-2 and the on all four dna fragmentation of nucleotide sequence shown in the SEQID No.1, shows that Melittin gene fission yeast recombinant expression vector pESP-2-Melittin successfully constructs.
B, usefulness Melittin gene fission yeast recombinant expression vector transform fission yeast
Adopt the Lithium Acetate chemical transformation to transform schizosaccharomyces pombe SP-Q01, with leucine auxotroph substratum screening positive transformant with Melittin gene fission yeast recombinant expression vector pESP-2-Melittin; Schizosaccharomyces pombe SP-Q01 comprises the LEU1 gene of a variation, the 3-propyl group desaturase of LEU1 genes encoding leucine biosynthesizing indispensability, LEU1 genovariation causes the leucine auxotrophy, and Melittin gene fission yeast recombinant expression vector pESP-2-Melittin contains selectable marker gene LEU2-d, can realize the complementation of leucine auxotrophy, so have only the SP-Q01 that transforms pESP-2-Melittin on leucine auxotroph substratum, to grow.Concrete operations are as follows:
Select the single bacterium colony of 3 schizosaccharomyces pombe SP-Q01 (Invitrogen company), be inoculated among the YPD liquid nutrient medium 10mL, 28 ℃ of temperature, overnight incubation under the condition of jolting speed 200rpm, get bacterium liquid 2mL, be inoculated among the fresh YPD liquid nutrient medium 50mL, under similarity condition, be cultured to OD600 about 0.5, centrifugal 5 minutes of rotating speed 3000rpm, abandoning supernatant, (be made up of following component: concentration is 10mmol/L with 1 * TE damping fluid, pH is 8.0 Tris-HCl, concentration is the EDTA of 1mmol/L) the 6mL re-suspended cell, centrifugal 5 minutes of rotating speed 3000rpm, abandoning supernatant, with 1 * TE/LiAC (Lithium Acetate LiAC is made the solution that concentration is 0.1mol/L with the dissolving of 1 * TE damping fluid), 0.5～1.0mL re-suspended cell, make fission yeast SP-Q01 competent cell; Get fission yeast SP-Q01 competent cell 100 μ L, add the salmon sperm dna solution 10 μ L that Melittin gene fission yeast recombinant expression vector pESP-2-Melittin solution 5 μ L and concentration are 10 μ g/ μ L (salmon sperm dna solution boils earlier and put on ice cooling rapidly in 2 minutes again) before adding, abundant mixing, adding PEG/LiAC/TE (is 50% PEG-40008mL with mass percent concentration, concentration be the LiAC 1mL of 1mol/L and 10 * TE damping fluid 1mL mixing promptly) 600 μ L, abundant mixing, 30 ℃ of temperature, cultivated 30～50 minutes under the condition of jolting speed 200rpm, add dimethyl sulfoxide (DMSO) 70 μ L, put upside down abundant mixing gently, 42 ℃ of water-baths of temperature 15～20 minutes, put cooled on ice again 2 minutes, centrifugal 5～10 seconds of rotating speed 13000rpm, abandoning supernatant, with 1 * TE damping fluid, 150 μ L re-suspended cells, centrifugal 5～10 seconds of rotating speed 13000rpm, abandoning supernatant, with 1 * TE damping fluid, 150 μ L re-suspended cells, draw 50 μ L, be seeded to contain on the EMM plate culture medium of VitB1 that concentration is 25 μ mol/L and screen positive transformant, use the Parafilm seal plate, temperature is inverted for 28 ℃ and was cultivated 4～5 days; Picking positive transformant list bacterium colony, be seeded to contain carry out on the EMM plate culture medium of VitB1 that concentration is 25 μ mol/L streak culture, extract recombinant plasmid in a small amount, verify positive transformant with the PCR method: with Yf and Yr is upstream and downstream primer amplification recombinant plasmid, the result as shown in Figure 5,5000,3000,2000,1000,750,500,250 wherein the M swimming lane is dna molecular amount standard (molecular weight is followed successively by from big to small:, 100bp), and 1～11 swimming lane is the PCR product; A special DNA band all appears in 1～11 swimming lane at the about 380bp of molecular weight place, conform to the segmental expection size of purpose, shows that Melittin gene fission yeast engineering bacteria SP-Q01/pESP-2-Melittin successfully constructs.
Two, the application of Melittin gene fission yeast engineering bacteria
The natural bee phallotoxins is expressed with the mellitin precursor forms in the honeybee body, so mellitin does not have toxicity to honeybee.The present invention adds GST label and EK recognition sequence at the N of mellitin end, make the formal representation of mellitin with fusion rotein, the native conformation and the activity of mellitin both can when the reduction mellitin is to the host cell lethality, have been guaranteed, can utilize the GST label to carry out the separation and purification of fusion rotein easily again, fusion rotein behind the purifying can obtain mellitin with EK excision GST label.
The method of using Melittin gene fission yeast engineering bacteria production mellitin may further comprise the steps:
The abduction delivering of a, GST-Melittin
Be subjected to the characteristics of the strict regulation and control of VitB1 content in the substratum according to promotor Pnmt1 expression activity, Melittin gene fission yeast engineering bacteria is at first cultivated with the YES substratum that contains VitB1, when suppressing exogenous protein expression, make engineering bacteria fast, raised growth, collect the engineering bacteria and the thorough washing of enlarged culturing, remove residual YES liquid nutrient medium and VitB1; Cultivate with the EMM liquid nutrient medium that does not contain VitB1 again, remove the check effect of VitB1 promotor Pnmt1, thus the expression of inducing GST-Melittin.Concrete operations are as follows:
Melittin gene fission yeast engineering bacteria SP-Q01/pESP-2-Melittin is seeded to contains among the YES liquid nutrient medium 10mL that mass percent concentration is 5% VitB1,28 ℃ of temperature, cultivated 16 hours under the condition of jolting speed 200rpm, get bacterium liquid 3mL, be seeded to fresh containing among the YES liquid nutrient medium 100mL that mass percent concentration is 5% VitB1, under similarity condition, be cultured to OD600 about 1.0, centrifugal 5 minutes of rotating speed 5000rpm, abandoning supernatant, with sterilized water 100mL re-suspended cell, centrifugal 5 minutes of rotating speed 5000rpm, abandoning supernatant, with sterilized water 100mL re-suspended cell, centrifugal 5 minutes of rotating speed 5000rpm, abandoning supernatant is with the EMM liquid nutrient medium 200mL re-suspended cell that does not contain VitB1, be transferred in the aseptic 1000mL triangular flask, 28 ℃ of temperature, difference abduction delivering 12 under the condition of jolting speed 200rpm, 18 and 22 hours, collect culture, standby.
The separation and purification of b, GST-Melittin
Adopt the method separation and purification GST-Melittin of the centrifugal and GST affinity chromatography of ultra-high speed, the cracking culturing cell, the centrifugal removal cell debris of ultra-high speed, supernatant liquor is again through the GST affinity chromatography, thorough washing GST affinity column remove can adsorbable foreign protein after, with reduced glutathion solution GST-Melittin is eluted from chromatography column, promptly get the GST-Melittin of purifying.Concrete operations are as follows:
Get the culture that step a collects, 4 ℃ of temperature, centrifugal under the condition of rotating speed 5000rpm, abandoning supernatant, (be made up of following component: concentration is 50mmol/L with cell pyrolysis liquid, pH is 7.4 Tris-HCl, concentration is the EDTA of 5mmol/L, concentration is the sodium-chlor of 300mmol/L, mass percent concentration is 0.5% IGEPAL, concentration is the Sodium Fluoride of 100mmol/L, concentration is that ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) and the concentration of 1mmol/L is the vanadic acid sodium of 1mmol/L) the 50mL re-suspended cell, under the liquid nitrogen freezing condition, add aluminum oxide and lysis buffer, with mechanical lysis method lysing cell, in collecting cell homogenate on ice, 4 ℃ of temperature, under the condition of rotating speed 10000rpm centrifugal 30 minutes, get supernatant liquor and carry out affinity chromatography with GSTrap FF post (GE company): earlier (be made up of following component: concentration is the sodium-chlor of 140mmol/L with the PBS damping fluid of 3 times of sample volumes, concentration is the Repone K of 2.7mmol/L, concentration is the Sodium phosphate dibasic of 10mmol/L, concentration is the potassium primary phosphate of 1.8mmol/L, pH7.3) with flow velocity 1.0mL/min balance GSTrap FF post, again above-mentioned lysis supernatant liquor is gone up sample with flow velocity 0.2mL/min, PBS damping fluid with 3 times of sample volumes washs GSTrap FF post with flow velocity 1.0mL/min, removal is adsorbed on the foreign protein on the post, (be made up of following component: concentration is the reduced glutathion of 10mmol/L to use the elution buffer of 2 times of sample volumes at last, concentration is the Tris-HCl of 50mmol/L, pH8.0) with flow velocity 0.2mL/min flushing GSTrap FF post, the GST-Melittin that is adsorbed on the post is eluted, collect elutriant, ultrafiltration is centrifugal to be concentrated, promptly get the GST-Melittin of purifying, put temperature-80 ℃ preservation.
The detection of the GST-Melittin of purifying:
(1) SDS-PAGE detects
With the GST-Melittin of purifying with containing the PBS damping fluid 400 μ L dissolving that mass percent concentration is 1% Triton (Triton) X-100, draw 15 μ L, after boiling 5 minutes, add 2 * albumen sample-loading buffer (with deionized water 3.55mL, concentration is 0.5mol/L, pH is 6.8 Tris-HCl 1.25mL, glycerine 2.5mL, concentration is the SDS 2.0mL of 100g/L, concentration be the tetrabromophenol sulfonphthalein 0.2mL of 5g/L and mercaptoethanol 0.5mL mixing promptly) 15 μ L, with mass percent concentration is that 15% separation gel carries out SDS-PAGE, Xylene Brilliant Cyanine G R-250 dyeing; The result as shown in Figure 6, wherein the M swimming lane is pre-dsred protein molecular weight standard (Stained Protein Molecular Weight Marker, Fermentas company), 1 swimming lane is 12 hours purifying GST-Melittin of abduction delivering, 2 swimming lanes are 18 hours purifying GST-Melittin of abduction delivering, and 3 swimming lanes are 22 hours purifying GST-Melittin of abduction delivering; A special protein band all appears in 2 and 3 swimming lanes at the about 29KD of molecular weight place, consistent with the expection molecular weight size of GST-Melittin, and it is few to contain foreign protein, purification effect is good, shows that the abduction delivering time of Melittin gene fission yeast engineering bacteria SP-Q01/pESP-2-Melittin was good with 18 hours.
(2) Western blot detects
The GST-Melittin of purifying is transferred on the Hybond N nylon membrane (Phamarcia company) through electricity behind the SDS-PAGE, washes film, sealing adds mouse-anti GST tag monoclonal antibody (TIANGEN company), and temperature was hatched 1 hour for 37 ℃, washed film, colour developing; The result as shown in Figure 7, wherein the M swimming lane is a protein molecular weight standard, 1 swimming lane is the GST-Melittin of purifying; A special protein band appears in 1 swimming lane at the about 29KD of molecular weight place, confirm that the gained purifying protein is GST-Melittin.
The detection of mellitin:
(1) SDS-PAGE detects
The GST-Melittin of purifying is carried out enzyme with EK to be cut, getting enzyme cuts product and carries out SDS-PAGE, the result as shown in Figure 8, wherein, 1 swimming lane is lysis supernatant (containing total protein), 66.2,45,35,25.2,18.4 2 swimming lanes are that the EK enzyme of GST-Melittin is cut product, and 3 swimming lanes are the GST-Melittin of purifying, and the M swimming lane is protein molecular weight standard (molecular weight is followed successively by from big to small:, 14.4KD); As seen a special protein band appears in 2 swimming lanes at the about 26KD of molecular weight place, and is consistent with the molecular weight size of GST, shows that GST-Melittin can be excised GST by EK, thereby obtains mellitin.
(2) anti-microbial activity detects
With OD590 is that 0.375 intestinal bacteria are cultivated bacterium liquid 1mL and nutrient agar 25mL mixing, pours in the culture dish, treat culture medium solidifying after, on substratum, punch, the aperture is 0.6cm, pore capacities is 70 μ L; The EK enzyme of GST-Melittin is cut product, and (containing concentration is 1.20 * 10
-2The mellitin of μ g/ μ L) and concentration be that the GST-Melittin of 0.12 μ g/ μ L adds respectively in the hand-hole, (adding concentration is 4.3 * 10 to establish negative control (adding sterilized water) and positive control simultaneously
-5The penbritin of μ g/ μ L), again culture dish was cultivated 24 hours under 37 ℃ of conditions of temperature, measured the size of inhibition zone.It is the inhibition zone of 1.4cm that the EK enzyme of result: GST-Melittin is cut product generation diameter, and it is the inhibition zone of 1.9cm that positive control produces diameter, and GST-Melittin and negative control do not produce obvious inhibition zone; Show that mellitin N end shows anti-microbial activity hardly when having GST label and EK recognition sequence, and show stronger anti-microbial activity after removing GST label and EK recognition sequence.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Sequence table
＜110〉University Of Chongqing
＜120〉Melittin gene fission yeast engineering bacteria and construction process thereof and application
<160>7
<210>1
<211>121
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(24)......(107)
<220>
＜223〉description of artificial sequence: Melittin gene
<400>1
aaggatccga?tgatgatgat?aaa?gga?att?gga?gca?gtt?ctg?aag?gta?tta 50
Gly?Ile?Gly?Ala?Val?Leu?Lys?Val?Leu
1 5
acc?aca?gga?ttg?ccc?gcc?ctc?ata?agt?tgg?att?aaa?cgt?aag?agg?caa 98
Thr?Thr?Gly?Leu?Pro?Ala?Leu?Ile?Ser?Trp?Ile?Lys?Arg?Lys?Arg?Gln
10 15 20 25
cag?tag?taa?tctagaggat?ccaa 121
Gln
<210>2
<211>26
<212>PRT
＜213〉Eastern bee (Apis cerena Fabricius)
<400>2
Gly?Ile?Gly?Ala?Val?Leu?Lys?Val?Leu?Thr?Thr?Gly?Leu?Pro?Ala
1 5 10 15
Leu?Ile?Ser?Trp?Ile?Lys?Arg?Lys?Arg?Gln?Gln
20 25
<210>3
<211>55
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer P1
<400>3
aaggatccga?tgatgatgat?aaaggaattg?gagcagttct?gaaggtatta?accac 55
<210>4
<211>50
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer P2
<400>4
ttaatccaac?ttatgagggc?gggcaatcct?gtggttaata?ccttcagaac 50
<210>5
<211>56
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer P3
<400>5
ttggatcctc?tagattacta?ctgttgcctc?ttacgtttaa?tccaacttat?gagggc 56
<210>6
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer Yf
<400>6
gtacttgaaa?tccagcaagt?atatagc 27
<210>7
<211>33
<212>DNA
＜213〉artificial sequence
<220>
＜223〉description of artificial sequence: primer Yr
<400>7
caaaatcgta?atatgcagct?tgaatgggct?tcc 33
Claims (10)
1, Melittin gene fission yeast engineering bacteria is characterized in that: this project bacterium is the fission yeast that is transformed by the Melittin gene fission yeast recombinant expression vector.
2, Melittin gene fission yeast engineering bacteria according to claim 1, it is characterized in that: described Melittin gene fission yeast recombinant expression vector contains the Melittin gene of nucleotide sequence shown in SEQ ID No.1, the mellitin of its encoding amino acid sequence shown in SEQ ID No.2.
3, Melittin gene fission yeast engineering bacteria according to claim 2 is characterized in that: the fission yeast expression vector that described Melittin gene fission yeast recombinant expression vector adopts is pESP-2.
4, according to the described Melittin gene fission yeast engineering bacteria of the arbitrary claim of claim 1 to 3, it is characterized in that: described fission yeast is schizosaccharomyces pombe SP-Q01.
5, the construction process of the described Melittin gene fission yeast engineering bacteria of claim 1 is characterized in that: may further comprise the steps:
A, the multiple clone site place that Melittin gene is inserted the fission yeast expression vector make up the Melittin gene fission yeast recombinant expression vector;
B, the Melittin gene fission yeast recombinant expression vector that makes up with step a transform fission yeast, promptly get Melittin gene fission yeast engineering bacteria.
6, the construction process of Melittin gene fission yeast engineering bacteria according to claim 5, it is characterized in that: described step a prepares the Melittin gene of nucleotide sequence shown in SEQ ID No.1 earlier, the gained Melittin gene is cloned into the pMD18-T carrier, makes up Melittin gene recombinant vectors pMD18-T-Melittin; After again the Melittin gene recombinant vectors pMD18-T-Melittin that makes up being used restriction enzyme BamHI single endonuclease digestion, be connected with the fission yeast expression vector pESP-2 that handles through BamHI single endonuclease digestion and alkaline phosphatase dephosphorylation, make up Melittin gene fission yeast recombinant expression vector pESP-2-Melittin.
7, the construction process of Melittin gene fission yeast engineering bacteria according to claim 6, it is characterized in that: described step b adopts the Lithium Acetate chemical transformation to transform schizosaccharomyces pombe SP-Q01 with the Melittin gene fission yeast recombinant expression vector pESP-2-Melittin that step a makes up, with leucine auxotroph substratum screening positive transformant, promptly get Melittin gene fission yeast engineering bacteria SP-Q01/pESP-2-Melittin.
8, application rights requires the method that 1 described Melittin gene fission yeast engineering bacteria is produced mellitin, it is characterized in that: may further comprise the steps:
A, cultivation Melittin gene fission yeast engineering bacteria also make it express mellitin, collect culture;
B, the culture that step a is collected carry out separation and purification, promptly get mellitin.
9, application Melittin gene fission yeast engineering bacteria according to claim 8 is produced the method for mellitin, it is characterized in that: described step a is earlier with containing the YES liquid nutrient medium enlarged culturing of VitB1 to OD with Melittin gene fission yeast engineering bacteria
600Be 1.0, after washed cell is removed residual YES liquid nutrient medium and VitB1, with the EMM liquid nutrient medium inducing culture that does not contain VitB1 18 hours, collect culture again.
10, application Melittin gene fission yeast engineering bacteria according to claim 9 is produced the method for mellitin, it is characterized in that: described step b gets the culture that step a collects, centrifugal removal supernatant liquor, smudge cells, the centrifugal removal cell debris of ultra-high speed, supernatant liquor promptly get the fusion rotein GST-Melittin of glutathione-S-transferase and mellitin again through the separation and purification of GST affinity chromatography, GST-Melittin is excised GST with enteropeptidase, promptly get mellitin.
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| CN108250284A (en) * | 2018-01-24 | 2018-07-06 | 吉林大学 | A kind of method for producing melittin |
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| CN101157898A (en) * | 2007-08-03 | 2008-04-09 | 吉林农业大学 | Schizosaccharomyces pombe engineering strain having cellulase activity and constructing method thereof |
| CN101157897B (en) * | 2007-08-03 | 2011-04-27 | 吉林农业大学 | Schizosaccharomyces pombe engineering strain having soybean MnSOD gene and constructing method thereof |
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