CN105176897A - Recombinant engineering strain expressing GnRH/M2 fusion polypeptide and construction and application thereof - Google Patents
Recombinant engineering strain expressing GnRH/M2 fusion polypeptide and construction and application thereof Download PDFInfo
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- CN105176897A CN105176897A CN201510458317.4A CN201510458317A CN105176897A CN 105176897 A CN105176897 A CN 105176897A CN 201510458317 A CN201510458317 A CN 201510458317A CN 105176897 A CN105176897 A CN 105176897A
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Abstract
The invention discloses a recombinant Escherichia coli engineering strain and construction and application thereof. The Escherichia coli strain is BL21/pEDGM2, CCTCC M 2015369. The Escherichia coli engineering strain carries recombinant plasmids capable of expressing ansB-C-GnRH3-hinge-MVP-M2 fusion polypeptide. ansB-C-GnRH3-hinge-MVP and M2 gene segments are colonized through a PCR (polymerase chain reaction) process separately, inserted into non-carrier plasmids pET28a sequentially and converted to Escherichia coli, so that the recombinant Escherichia coli engineering strain is enabled to express recombinant protein, and the ansB-C-GnRH3-hinge-MVP-M2 fusion polypeptide is obtained through separation and purification and by removing a fusion partner. The recombinant Escherichia coli engineering strain and the fusion polypeptide expressed by the strain can be applied to anti-tumor treatment.
Description
Technical field
In genetically engineered field, the invention discloses a kind of take intestinal bacteria as the recombinant bacterial strain of carrier, relates to structure and the application thereof of engineering strain simultaneously.
Background technology
Gonadotropin releasing hormone (GnRH) is a kind of peptide hormone be made up of ten amino-acid residues, and its structure is pGlu
1-His
2-Trp
3-Ser
4-Tyr
5-Gly
6-Leu
7-Arg
8-Pro
9-Gly
10-NH
2.GnRH is found in hypothalamic neuroendocrine cell the earliest, and it discharges in a pulsed fashion, with suprapituitary GnRH receptor-specific combine, stimulate luteotropic hormone (LH) and the synthesis of follicular stimulating hormone (FSH) and release.LH and FSH promotes that sexual gland discharges specific hormones again, and as women stimulates ovarian secretion estradiol and progesterone, the male sex stimulates testicular secretion testosterone and prostaglandin(PG), and also plays a role to mammary gland, adrenal cortex, Tiroidina and bone growth.Except hypothalamic neuroendocrine cell secretion GnRH, many malignant tumor tissues such as mammary cancer, prostate cancer, liver cancer, melanoma etc. also can this hormones of autocrine.GnRH is a kind of small haptens, and immunogenicity is very weak, it can be used as separately vaccine to be difficult to cause immunne response.
Research shows, specificity epitope can be caused to suppress when GnRH being connected to immune animal on some macromolecular carriers.Thus this laboratory utilizes auxiliary T epi-position to improve the immunogenicity of GnRH.In Fusion protein of measles virus sequence, the 288th amino acids is a typical t cell epitope (SEIKGVIVRLEGVAK is hereafter all called for short MVP) to the 302nd amino acids fragment, can be used in vaccine for man.Abroad have been reported, being connected with haptens with MVP forms T1249, can induce for haptenic antibody response, and can not produce significantly for the antibody of this t cell epitope.In addition, the immunogenicity of the self-peptide sequences albumen that research shows containing multiple copied linearly connected can be greatly improved, and thus, the nucleotide fragments of coding three sections of end to end GnRH is cloned into efficient expression vector by this laboratory.Also there are some researches show, haptenic dimerization physical efficiency produces very strong immunogenicity.Human IgG
1hinge area (hingeregion) oligopeptide fragments 226-232/226 '-232 ' (CPPCPAP/CPPCPAP) is the pivot of natural immunity albumen.In native protein, the rigidity double-stranded circular structure of this proline rich spontaneously can be folded into a double focusing proline(Pro)-II α spiral, in the sterie configuration of the hinge region polypeptide 225-232/225 '-232 ' of synthetic and native protein, the configuration of hinge fragment is completely the same, and further research also finds at IgG
1when the one or both ends of hinge area connect the peptide sequence of non-IgG1, still can form double focusing proline(Pro)-II α spiral.Therefore, we add three sections of GnRH fragments repeated at hingeregion fragment N-terminal, connect the T epi-position of Measles virus, wish that GnRH can form dimer by this, strengthen immunogenicity at its C-terminal.
407-426 fragment (M) in the mHSP70 of heat-shock protein family strengthens peptide section as the non-specific immunity that HSP70 is main, by raising costimulatory molecules and the MHC molecule on DC surface, promote Th1 type irritant reaction, enhance the cytotoxicity of killer cell, thus mediate strong cellullar immunologic response; Strengthen the DC that mHSP70 or CD40L induce by being combined with CD40 ripe simultaneously, thus absorb efficiently, processing treatment and present antigen, the cytotoxic T lymphocyte of inducing specific generates.This laboratory early-stage Study finds that M is through tandem sequence repeats twice (M
2) after can strengthen to DC effect of stimulation, there is good anti-tumor activity.
But restructuring GnRH polypeptide only has more than 50 amino acid, if directly very low in escherichia coli expression efficiency, because its mRNA and expression product are all easily degraded.Therefore the method for amalgamation and expression can be taked to address this problem, select the existing expression system PED in this laboratory.In this system, fusion partners ansB-C is connected by aminoacid sequence Asp-Pro with object bioactive peptide, and wherein, Asp-Pro is unique acid hydrolysis site.In acid condition, the peptide bond between Asp-Pro disconnects fusion rotein, thus discharges desired polypeptides.
In order to strengthen GnRH further
3the anti-tumor activity of-hinge-MVP polypeptide, the present invention constructs GnRH/M
2gene recombination bacterium, DC vaccine is obtained with APC co culture system in vitro after expression and purification fusion rotein, feed back in body and APC can be impelled to have higher loading and process antigenic capacity, the immunne response of body to tumour cell can be improved, reach the object of identification and killing off tumor cells, strengthen antitumor action further.
Summary of the invention
The present invention discloses a kind of recombinant escherichia coli strain PEDGM
2.
An object of the present invention is to provide the construction process of recombinant bacterium.
Another object of the present invention is the purposes disclosing this recombinant protein.
In first of the present invention, provide a kind of recombination bacillus coli engineering strain.This colibacillus engineering strain called after BL21/pEDGM2, bacterial strain has submitted China typical culture collection center preservation to, preservation address: China. Wuhan. Wuhan University, preservation date on June 14th, 2015, deposit number is CCTCCNO:M2015369, Classification And Nomenclature: Escherichia.coliBL21/pEDGM
2.This bacterial strain carries can express ansB-C-GnRH
3-hinge-MVP-M
2the recombinant plasmid of fusion rotein.
In second of the present invention, provide recombinant escherichia coli strain pET-28a-ansB-C-GnRH
3-hinge-MVP-M
2structure and the purification process of expressed recombinant protein, details are as follows for its technological line:
1. recombinant plasmid pET-28a-ansB-C-GnRH
3-hinge-MVP-M
2and the structure of corresponding engineering bacteria
PEDGD (the pET-28a-ansB-C-GnRH built with this laboratory
3-hinge-MVP-NRLLLTG) be template, design upstream primer P1 and downstream primer P2, NcoI restriction enzyme site is contained in upstream, and NheI site is introduced in downstream.By the ansB-C-GnRH of amplification
3-hinge-MVP sequence is connected with empty pET28a plasmid, obtains recombinant plasmid pET-28a-ansB-C-GnRH
3-hinge-MVP.
With the pET28a-hVEGF121I-M that this laboratory builds
2for template, design upstream and downstream primer V1 and V2, two ends introduce NheI site and HindIII site respectively.PCR reaction amplification M
2sequence.By pET-28a-ansB-C-GnRH
3the M of-hinge-MVP plasmid and acquisition
2sequence NheI and HindIII restriction enzyme carry out double digestion, then add DNA ligase and obtain recombinant plasmid pET-28a-ansB-C-GnRH
3-hinge-MVP-M
2.By recombinant plasmid transformed to E.coliBL21 competent cell, kantlex screening positive transformant, verifies with primer P1 and V2.
Through DNA sequencing analysis display, M
2fragment is correctly inserted into pET-28a-ansB-C-GnRH
3in-hinge-MVP (PEDG) recombinant plasmid, and be positioned at pET-28a-ansB-C-GnRH
33 ' end of-hinge-MVP gene fragment.So far two sections of goal gene fragments are inserted in pET-28a according to correct order, and form the recombination fragment of fusion rotein, pET-28a-ansB-C-GnRH
3-hinge-MVP-M
2expression vector establishment completes and sequencing analysis is correct.
2. the abduction delivering of fusion rotein and separation and purification
Picking engineering bacteria list bacterium colony from flat board, is seeded in the LB substratum containing 50 μ g/mL kantlex and shakes overnight incubation, transfers in fresh LB nutrient solution by 1%, 37 DEG C are cultured to logarithmic phase, add lactose and carry out abduction delivering, 37 DEG C are continued to cultivate, centrifugally after 5h collect bacterial sediment.
Collect thalline, abundant cracking thalline, the centrifugal 10min of 12000r/min, abandons supernatant, retains precipitation.Precipitation is through washing, and obtain pure inclusion body, inclusion body crude product adds 8mol/L urea and dissolves completely to inclusion body.Inclusion body solution is carried out dehydrated alcohol fractionation precipitation, collecting precipitation; The albumen of alcohol settling is added the HCl of 60mM, put 48 DEG C of water-bath 72h.
Protein solution after water-bath is crossed anion-exchange column DEAE-cellulose be further purified, with the chromatography buffer gradient elution containing NaCl, collect and penetrate peak and elution peak, combining data detection target protein peak component, freeze-drying after distilled water enough hemodialysis is preserved.
Accompanying drawing explanation
Fig. 1 construction of recombinant plasmid principle schematic.
Fig. 2 PCR obtains 1.5% agarose gel electrophoresis detected result of GnRH recombination.
Lane1:DNAMarker, Lane2:PCR product.
PET28a-ansB-C-GnRH in Figure 31 .5% sepharose
3the PCR checking of-hinge-MVP recombinant plasmid
Lane1:DNAMarker, Lane2:PCR product.
Fig. 4 pET-28a-ansB-C-GnRH
3the gene forward sequencer map of-hinge-MVP recombinant plasmid.
Fig. 5 PCR obtains 1.5% agarose gel electrophoresis detected result of M2 gene.
Lane1:DNAMarker, Lane2:PCR product.
The restriction enzyme checking electrophorogram of recombinant plasmid in Figure 60 .8% sepharose
Lane1:DNAMarker, Lane2: the recombinant plasmid after HindIII single endonuclease digestion, Lane3: the recombinant plasmid after NheI and HindIII double digestion, Lane4: the recombinant plasmid cut without enzyme.
PET-28a-ansB-C-GnRH in Figure 71 .5% sepharose
3-hinge-MVP-M
2the PCR checking of recombinant plasmid
Lane1:PCR product, Lane2:DNAMarker.
Fig. 8 pET-28a-ansB-C-GnRH
3-hinge-MVP-M
2the gene forward sequencer map of recombinant plasmid.
Figure 91 2%SDS-PAGE electrophoresis detection recombinant bacterium is through the induction curves of lactose-induced expressed fusion protein.
Lane1:ProteinMarker, Lane2: the whole bacterial protein sample before induction, Lane3-10: the induction whole bacterial protein sample of latter 1-8 hour each hour.
Figure 101 2%SDS-PAGE electrophoresis detection ultrasonic degradation thalline result.
Lane1:ProteinMarker, Lane2: the whole bacterial protein sample before induction, Lane3: 5h whole bacterial protein sample after induction, Lane4: cellular lysate supernatant, Lane5: cellular lysate precipitates.
Figure 111 2%SDS-PAGE electrophoresis detection ethanol precipitation fusion rotein result.
Lane1: before alcohol settling, Lane2: the albumen precipitation adding 0.5 times of volume dehydrated alcohol, Lane3: the albumen precipitation adding 1 times of volume dehydrated alcohol, Lane4: the albumen precipitation adding 2 times of volume dehydrated alcohols.
Figure 121 2%tricine-SDS-PAGE electrophoresis detection acid hydrolysis target protein result
Lane1:ProteinMarker, Lane1: the fusion rotein before acid hydrolysis, Lane3 ~ 8: 12h, 24h, 36h, 48h, 60h, 72h sampling after acid hydrolysis.
Figure 131 2%tricine-SDS-PAGE electrophoresis detection DEAE52 anion-exchange chromatography is to target protein purification result.
Lane1:ProteinMarker, Lane2: the protein sample before loading, Lane3: penetrate protein sample during peak-to-peak top, Lane4: protein sample during elution peak summit.
Embodiment
Material
(1) bacterial strain and plasmid
Carrier pET-28a-ansB-C-GnRH
3-hinge-MVP-NRLLLTG, EscherichiacoliBL21 (DE3); PET-28a-hVEGF121I-M
2, EscherichiacoliBL21 (DE3), pET-28a-ansB-C-GnRH
3-hinge-MVP-NRLLLTG and pET-28a-hVEGF121I-M
2plasmid is this laboratory and preserves.
(2) enzyme and reagent
Molecular cloning toolenzyme is TaKaRa Products; Plasmid extraction test kit is Shanghai Jierui Biology Engineering Co., Ltd's product; PCR glue reclaims the product that test kit is Sheng Gong bio tech ltd, Shanghai.
(3) substratum
LB substratum, fill a prescription the document SambrookJ that sees reference, FristshEF, ManiatisT.MolecularCloning; ALaboratoryManual2nded.NY:ColdSpringHarborLaboratoryPres s, 1989.
The recovery of plasmid extraction, PCR reaction, endonuclease digestion, DNA fragmentation in method mentioned by this specification sheets, connection and transformation of E. coli, these are all the conventional practices of genetically engineered research field, specifically see SambrookJ, FristshEF, ManiatisT.MolecularCloning; ALaboratoryManual2nded.NY:ColdSpringHarborLaboratoryPres s, 1989, pp.16-340.
Embodiment 1 carries goal gene ansB-C-GnRH
3the structure of the plasmid vector of-hinge-MVP
The extraction of 1.1 template plasmid PEDGD and empty plasmid pET28a
PEDGD (the pET-28a-ansB-C-GnRH that this laboratory is built
3-hinge-MVP-NRLLLTG) be inoculated into LB substratum, 37 DEG C of incubated overnight, plasmid extraction adopts plasmid extraction kit (Shanghai Jierui Biology Engineering Co., Ltd).
The extracting method of empty plasmid pET-28a is the same.
1.2ansB-C-GnRH
3the amplification of-hinge-MVP gene fragment
Be template with PEDGD, utilize primer-design software Oligo to design two couples of primers P1, P2 respectively:
P1：5’-CATGCCATGGATACGCCATTCGATG-3’
P2：5’-CGGCTAGCAGCAACACCCTCCAGA-3’
Primer is synthesized by JaRa company.NcoI restriction enzyme site is contained in upstream, and NheI site is introduced in downstream.Adopt round pcr from pET-28a-ansB-C-GnRH
3in-hinge-MVP-NRLLLTG recombinant plasmid, clone obtains ansB-C-GnRH
3-hinge-MVP sequence.
PCR response procedures:
PCR reaction conditions:
PCR reaction is carried out on eppendorfPCR amplification instrument.94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 62 DEG C of annealing 45s, 72 DEG C extend 1min, totally 35 circulations; 72 DEG C extend 5min.
1.5% agarose gel electrophoresis qualification PCR primer (see accompanying drawing 2), pillar location is consistent with the 572bp of expection.
1.3ansB-C-GnRH
3-hinge-MVP gene is connected to plasmid pET-28a.
PCR primer through cut glue reclaim obtain gene fragment and pET28a plasmid respectively through NcoI and NheI double digestion, double digestion system sees the following form:
37 DEG C of enzymes cut 3h.Digestion products is used PCR primer Purification Kit respectively.
By ansB-C-GnRH
3-hinge-MVP gene fragment is connected with pET-28a carrier segments according to the form below system:
DNA ligase 4 DEG C of connections are spent the night, and are converted into E.coliBL21 competent cell.Carry out as follows: the enzyme drawing 5 μ L in super clean bench connects product and joins in 40 μ L competence engineering bacterias, ice bath 30min after concussion mixing gently, thermal shock 110s in 42 DEG C of water-baths again, take out ice bath 5min immediately, in the bacterium liquid in conversion, now add the fresh LB liquid nutrient medium of 900 μ L, mixing is placed in 37 DEG C of shaking tables, 50r/min, 45min is cultivated in recovery, take out EP pipe collected by centrifugation thalline, 8000r/min, centrifugal 1min, abandon 800 μ L supernatants, join in the LB solid medium flat board containing 50mg/mL kalamycin resistance after remaining bacterium liquid bullet is even, use spreading rod even spread, flat-plate inverted is placed in overnight incubation in 37 DEG C of incubators, the positive bacterium colony of picking, through qualification (see next step), called after PEDG.
The qualification of 1.4 plasmid PEDG:
Use plasmid extraction kit, from positive bacterium colony, extract plasmid PEDG, carry out PCR checking, with recombinant plasmid PEDG for masterplate, P1 and P2 is that primer carries out PCR, can obtain the nucleic acid bands (see accompanying drawing 3) that size is 572bp, conforms to expection size.
Positive colony is delivered to the Jin Sirui company that checks order to check order, sequencing result entirely true after software comparison (see accompanying drawing 4).So far recombinant plasmid pET-28a-ansB-C-GnRH
3-hinge-MVP has built.
Embodiment 2 carries goal gene ansB-C-GnRH
3-hinge-MVP-M
2the structure of plasmid vector
2.1 template plasmid pET-28a-hVEGF121I-M
2with the extraction of vector plasmid PEDG
By the pET-28a-hVEGF121I-M that this laboratory builds
2be inoculated into LB substratum, 37 DEG C of incubated overnight, plasmid extraction adopts plasmid extraction kit (Shanghai Jierui Biology Engineering Co., Ltd).
The extracting method of vector plasmid PEDG is the same.
2.2PCR increases M
2gene fragment
With pET-28a-hVEGF121I-M
2for template, design upstream and downstream primer V1 and V2:
V1：5’-CGGCTAGCTCTAGCCAGCCTTCC-3’
V2：5’-CCCAAGCTTACTTGTTGTGAGCG-3’
Upstream and downstream primer introduces NheI site and HindIII site respectively.Primer is synthesized by JaRa company.By PCR reaction from pET-28a-hVEGF121I-M
2increase in plasmid M
2sequence.
PCR response procedures is as follows with embodiment 1, PCR reaction conditions:
94 DEG C of denaturation 5min; 94 DEG C of sex change 45s, 60 DEG C of annealing 30s, 72 DEG C extend 30s, totally 35 circulations; 72 DEG C extend 5min.
1.5% agarose gel electrophoresis qualification PCR primer (see accompanying drawing 5), pillar location is consistent with the 150bp of expection.
2.3M
2gene is connected to recombinant plasmid PEDG
By pET-28a-ansB-C-GnRH
3the M that-hinge-MVP recombinant plasmid and amplification obtain
2sequence NheI and HindIII enzyme carry out double digestion, and double digestion system sees the following form:
37 DEG C of enzymes cut 3h.Digestion products is used PCR primer Purification Kit respectively.
By M
2gene fragment is connected with PEDG carrier segments according to the form below system:
DNA ligase 4 DEG C of connections are spent the night, and are converted into E.coliBL21 competent cell.Method for transformation is with embodiment 1.The positive bacterium colony of picking, through qualification (see next step), called after PEDGM
2.
2.3 plasmid PEDGM
2qualification:
Use plasmid extraction kit, from positive bacterium colony, extract plasmid PEDGM
2single endonuclease digestion is carried out and NheI and HindIII carries out double digestion with HindIII, 0.8% agarose gel electrophoresis detects, little compared with after HindIII single endonuclease digestion of the molecular weight of result display recombinant plasmid after NheI and HindIII double digestion, and produce two nucleic acid bands after double digestion, the nucleic acid bands between 500bp and 750bp conforms to (see accompanying drawing 6) with the 712bp molecular weight of goal gene.Identifying with PCR further, take recombinant plasmid as masterplate, carries out PCR with primer P1 and V2, can obtain the amplified band (see accompanying drawing 7) that size is 712bp, conforms to expection size.
By positive colony through the display of DNA sequencing analytical results, M
2fragment correctly inserts pET-28a-ansB-C-GnRH
3in-hinge-MVP (PEDG) plasmid (see accompanying drawing 8).So far two sections of goal gene fragments are inserted in pET-28a according to correct order, recombinant plasmid pET-28a-ansB-C-GnRH
3-hinge-MVP-M
2successfully construct, and called after PEDGM
2.
Embodiment 3ansB-C-GnRH
3the expression of-hinge-MVP-M2 peptide gene in intestinal bacteria and cultivation
Recombinant expressed bacterium is with 1: 100 inoculum size, 37 DEG C of shaken overnight in LB liquid medium, 1: 100 switching shaking flask bulk culture again, adding final concentration after cultivating 4h is the lactose-induced expression of 7mM, collects thalline, keep sample and carry out SDS-PAGE analysis (see accompanying drawing 9) after induction 5h.Fusion rotein 5h after induction reaches stable maximum expression amount.
The soluble analysis of embodiment 4 fusion rotein
Recombinant expressed bacterium is inoculated in LB liquid nutrient medium with 1: 100 inoculum size, 37 DEG C of incubated overnight; Incubated overnight liquid is transferred into LB substratum, 37 DEG C of enlarged culturing, chooses optimal culture conditions and obtains fermentation thalli.The centrifugal 15min of 8000r/min, supernatant and precipitation all keep sample.In the thalline collected, add the cellular lysate liquid (10mL/g thalline) prepared, after fully stirring, carry out ultrasonic degradation operation, with abundant cracking thalline; Cracking to solution not viscous time take out lysate, the centrifugal 15min of 12000r/min, collect supernatant and precipitation all keeps sample mark.SDS-PAGE electrophoresis result display target protein is mainly present in cellular lysate precipitation (see accompanying drawing 10), therefore can judge that fusion rotein is expressed with inclusion bodies.
The initial gross separation purifying of embodiment 5 fusion rotein
Inclusion body precipitation is respectively washed once by the buffer A and distilled water that contain 0.5%Triton100 (v/v) and 2% Sodium desoxycholate respectively, obtain pure inclusion body, every gram of inclusion body crude product adds in the 8mol/L urea of 12.5ml, 4 DEG C of stirrings are spent the night, dissolve completely to inclusion body, the centrifugal 10min of 12000rpm.Add the dehydrated alcohol of the precooling of 0.5 times of volume by the volume of inclusion body solution, limit edged stirs, mixing ,-20 DEG C of centrifugal 20min of standing 1h, 10000rpm; Repeat the dehydrated alcohol (accompanying drawing 11) that above-mentioned steps respectively adds the precooling of 1 times and 2 times volume.The albumen of second time alcohol settling is added 100mL60mmol/LHCl by every 6g fusion rotein dissolve, put 48 DEG C of water-baths 72 hours (accompanying drawing 12).Slowly adding the NaOH solution of 1mol/L after completion of the reaction while stirring, is about 2.5 to pH value, now has foreign protein to precipitate and generates.4 DEG C, the centrifugal 30min of 10000rpm, collects supernatant.
Dialysed three times in 4 DEG C by supernatant ion-exchange damping fluid (pH9.0TrisHCl25mM), with 0.45 μm of membrane filtration before loading, filtrate is crossed anion-exchange column DEAE-cellulose and is further purified.
Embodiment 6DEAE-52 anion-exchange chromatography
The DEAE-Mierocrystalline cellulose DE52 that this experiment adopts is weak-type anionite, DEAE-52 is loaded in chromatography column, chromatography column upper end fluid inlet connects constant flow pump, lower outlet connects nucleic acid-protein detector, utilize ion-exchange damping fluid (pH9.0, 25mMTrisHCl) carry out the balance of chromatography column, carry out the loading operation of protein solution with the speed of 3mL/min after balance, balancing run is repeated after loading, NaCl (0-250mM) is used to carry out gradient elution again, collection penetrates peak and elution peak, carry out SDS-PAGE electrophoretic analysis (see accompanying drawing 13).
Except the above-mentioned fact, the present invention can also have other embodiments, and all employings are equal to the technical scheme of replacement or equivalent transformation, all fall within the protection domain of application claims.
Claims (7)
1. express fusion polypeptide GnRH for one kind
3-hinge-MVP-M
2coli strain, it is characterized in that strain Escherichia coli Escherichia.coliBL21/pEDGM
2, deposit number is CCTCCM2015369.
2. a kind of coli strain BL21 (DE3)/pEDGM according to claim 1
2, it is characterized in that: containing recombinant plasmid pET-28a-ansB-C-GnRH in Bacillus coli cells
3-hinge-MVP-M
2.
3. a coli strain BL21/pEDGM according to claim 2
2in recombinant plasmid pET-28a-ansB-C-GnRH
3-hinge-MVP-M
2, it is characterized in that: containing one section of restructuring GnRH gene, it has the nucleotide sequence shown in SEQIDNO.1.
4. restructuring ansB-C-GnRH according to claim 3
3-hinge-MVP-M
2gene, is characterized in that: the fusion rotein actual molecular weight of this genetic expression is about 25kDa, comprises fusion partners ansB-C, restructuring GnRH
3-hinge-MVP antigenic peptide and M
2assist a ruler in governing a country polypeptide.
5. containing the recombination bacillus coli engineering strain described in any one of Claims 1 to 4.
6., for realizing a preparation method for a kind of colibacillus engineering strain according to claim 1, it comprises the following steps:
A, amplify ansB-C-GnRH by PCR method
3-hinge-MVP gene fragment;
B, use restriction enzyme NcoI and NheI cut ansB-C-GnRH
3-hinge-MVP gene fragment and pET28a empty carrier, connect with DNA ligase, construction recombination plasmid pET-28a-ansB-C-GnRH
3-hinge-MVP (PEDG);
C, amplify M by PCR method
2gene fragment;
D, use restriction enzyme NheI and HindIII cut M
2fragment and recombinant plasmid PEDG, both connect by DNA ligase, construction recombination plasmid pET-28a-ansB-C-GnRH
3-hinge-MVP-M
2(PEDGM
2);
E, by recombinant plasmid PEDGM
2be transformed into intestinal bacteria, cultivate intestinal bacteria and extract plasmid;
F, qualification recombinant plasmid PEDGM
2: by double digestion, PCR method, recombinant plasmid is identified.
7. recombination bacillus coli engineering strain according to claim 5 is in the application of anti-tumor aspect.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106085932A (en) * | 2016-05-19 | 2016-11-09 | 中国药科大学 | A kind of structure of the genetic engineering bacterium of VEGF Yu GnRH fusion protein of recombinating |
| CN107090426A (en) * | 2017-04-25 | 2017-08-25 | 中国药科大学 | Restructuring mGM-CSF and the genetic engineering bacterium of GnRH fusion proteins a kind of structure |
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