CN108314709A - Recombinant glycosylated albumen P39 and its preparation method and application - Google Patents

Abstract

A kind of recombinant glycosylated albumen P39 and its preparation method and application, is related to recombinant protein field.The present invention includes：P39 genes are cloned, PCR fragment is connected with cloning vector pMD 19T (Simple), be connected target fragment with carrier for expression of eukaryon pPICZ α A after sequencing and digestion verification are correct structure recombinant expression plasmid pPICZ α A P39；Electrotransformation converts plasmid pPICZ α A P39 to Pichia pastoris GS115 competent cell, and through induced expression, affinitive layer purification, it is destination protein that molecular sieve, which is verified after further isolating and purifying through western blot, and optimal conditions of fermentation is：PH6, phosphate concn 0.02M, methanol induction concentration 1%, induction 72h, a concentration of 5mg/ml of recombinant protein P39 after purification.The present invention is that brucella epidemic disease immunoprophylaxis lays the foundation.

Description

Recombinant glycosylated albumen P39 and its preparation method and application

Technical field

The present invention relates to recombinant protein technical fields, and in particular to a kind of recombinant glycosylated albumen P39 and preparation method thereof And application.

Background technology

Brucellosis (abbreviation cloth disease) is a kind of infectious disease of Zoonosis caused by brucella, all over the world All it is widely current, especially in developing country, currently, effective commercialization vaccine for man is there is no, and animal vaccine is mostly weak There is malicious seedling certain toxicity and pathogenicity, hardly possible to be distinguished with natural infection, and therefore, it is that control is dynamic to study effective vaccine The main method of object cloth disease.Currently, existing effective brucella vaccine type has killed vaccine, attenuated live vaccine etc., attenuated live Immune effect of vaccine is preferable.Existing poultry attenuated live vaccine has S19, Rev.1, RB51, S2 and M5 etc., and people is main with live vaccine There is 19-B and 104M etc..Domestic and foreign current brucella vaccine is usually 70~80% to the protection of livestock, therefore, single to rely on Vaccine inoculation is come to eliminate brucellosis be unpractiaca.Ox opens brucellosis with RB51 rough types mutation strain vaccine and prevents The frontier controlled, effectively improves the control effect of brucellosis, but in the short term will in vivo in be eliminated.Epidemic disease Seedling is using relatively broad, and in many countries and regions by the lamb at 3-6 monthly age of Rev-1 vaccine immunities, they think Rev-1 pairs Sheep has 100% protectiveness, the area of this inoculated vaccine, and people suffers from cloth disease incidence and substantially reduces, but this vaccine has people Infectivity needs to take animal doctor personnel stringent safeguard measure.And attenuated live vaccine restores there are virulence and recessive sense Dye etc. is dangerous outer, and can be interfered to current widely used serological diagnostic method so that infection animal cannot be distinguished With immune animal, certain interference and difficulty are caused to the thorough purification elimination of cloth disease.S19 attenuated strains are immune to ox and sheep Effect is good and effect stability, but poor to the immune efficacy of sheep, invalid to pig.In addition, S19 attenuated vaccines have virulence reversion existing As the miscarriage of pregnancy ox can be caused.Rev.1 and RB51 is mutation strain vaccine, and application is relatively broad, once but mutant strain Vaccine virulence is returned by force, and consequence will be hardly imaginable.M5 bacterial strains are widely used in the prevention of Brucella melitensis, but the vaccine can also draw The miscarriage of pregnant female is played, and is unfavorable for antidiastole.

Currently, researcher has begun using genetic engineering and molecular biology method to known immunogenicity egg The white research and development for carrying out gene engineering vaccine.Research find some protective antigens molecules have BCSP31, OMP25, L7/L12, P39, CuZn SOD and heat shock protein etc..New generation vaccine mainly has DNA vaccination, recombinant protein vaccine and Anti-idiotype Antibody Vaccine Deng.The development of DNA vaccination, which obtains, has achieved some achievements, but since its immune protective is not high, there is presently no DNA vaccinations can Preferably to protect attack of the animal from pathogenic strain.With the completion of ox kind and brucella melitensis genome sequencing, section Scholars are more and more thorough to the function understanding of the various genes of brucella, develop gene delection Attenuate vaccine fusion protein and recombinate epidemic disease The novel brucella recombinant vaccine such as seedling and protein modified vaccine becomes the hot spot that they study.

Invention content

The object of the present invention is to provide a kind of recombinant glycosylated albumen P39 and its preparation method and application, are system research It lays the foundation as the effect of a kind of novel vaccine and immunologic adjuvant in brucella epidemic disease immunization therapy.

The present invention is that technical scheme applied to solve the technical problem is as follows：

The preparation method of recombinant glycosylated albumen P39 of the present invention a kind of, includes the following steps：

Step 1: the structure of carrier for expression of eukaryon pPICZ α A-P39

P39 genes are cloned, the segment after PCR is connected with cloning vector pMD-19T (Simple), through sequencing and After digestion verification is correct, by the target fragment obtained after double digestion and the carrier for expression of eukaryon pPICZ α A phases equally through double digestion Even structure recombinant expression plasmid pPICZ α A-P39；

Step 2: expression, purifying and fermentation of the P39 albumen in Pichia pastoris GS115

The carrier for expression of eukaryon pPICZ α A-P39 built are converted to Pichia pastoris GS115 using electrotransformation and are experienced In state cell, the induced expression of albumen is carried out using methanol, destination protein is purified using affinity chromatography method, and utilizes Molecular sieve further isolates and purifies destination protein, and is verified to the albumen of purifying using western blot, passes through optimization Experiment of single factor obtains optimal conditions of fermentation：PH value is 6, phosphate concn 0.02M, methanol induction are a concentration of 1%, induction 72h, a concentration of 5mg/ml of recombinant protein P39 after purification.

As preferred embodiment, in step 2, carrier for expression of eukaryon pPICZ α A-P39 are converted to Pichia pastoris Detailed process in GS115 competent cells is as follows：

1, the extraction of recombinant expression plasmid pPICZ α A-P39

The single bacterium colony of picking culture is inoculated in 5mL LB liquid mediums, 37 DEG C, after 200rpm shake cultures are stayed overnight Expand overnight incubation：

(1) 40ml bacterium solutions are collected to be put into 50ml centrifuge tubes, 10000rpm, centrifuges 3min and is discarded after more collections several times Clearly, it is upside down on filter paper；

(2) 10mlP1 solution is added into the 50ml centrifuge tubes for have precipitation, while 50ul TIANRED reagents are added, utilizes Liquid-transfering gun blows and beats mixing or with oscillator mixing；

(3) 10mlP2 solution is added into centrifuge tube, mild spins upside down 6-8 times, makes cellular lysate, is placed at room temperature for 5min；

(4) 10mlP4 solution is added into centrifuge tube, mild spins upside down 6-8 times, until white dispersion wadding occurs in solution Shape precipitates, and is placed at room temperature for 10min, and 10000rpm centrifuges 5-10min；

(5) complete soln is carefully poured into filter, filters, provides 50ml centrifuge tubes for oneself；

(6) the 5M Nacl solution of the isopropanol and 1/2 isopropanol volume of 0.35 times of filtrate volume is added into filtrate, on Reverse mixing down；

(7) under the conditions of 4 DEG C, 10000rpm centrifuges 30min, outwells supernatant, is upside down on filter paper and is absorbed water；

(8) ethyl alcohol that 6ml 70% is added into pipe fully rinses precipitation, and 4 DEG C, 10000rpm centrifuges 10min, outwells Clearly, it is upside down on filter paper；

(9) the 7th step is repeated；

(10) centrifuge tube opening is placed on indoor 10-20min, it is molten that 1-1.5mlTB buffer are added after so that ethyl alcohol is volatilized Solution, Nano Drop detect plasmid concentration；

2, the linearisation of recombinant expression plasmid pPICZ α A-P39

The recombinant expression plasmid pPICZ α A-P39 of extraction are linearized using SalI, reaction system is as follows：Plasmid 20 μ l, 5 μ l of SalI restriction enzymes, 5 bufferH μ l, ddH₂20 μ l of O, totally 20 samples；37 DEG C of water-bath 3h；5 μ l are taken to carry out Nucleic acid electrophoresis detects whether digestion is complete, and non-digested plasmid compares；

3, the plasmid of alcohol precipitation method recycling linearisation

It draws in the 500ul plasmids to the centrifuge tube of 1.5ml after digestion, isometric phenol, chloroform, isoamyl alcohol is added, It uniformly mixes, under the conditions of 4 DEG C, 12000rpm centrifuges 10min；Upper layer supernatant is taken, the absolute ethyl alcohol and 1/10 of 2 times of volumes is added The 5M Nacl solution of volume, static 2h under the conditions of -20 DEG C；Under the conditions of 4 DEG C, 13000rpm centrifuges 10min, discards supernatant；With 75% ethyl alcohol washing precipitation, 12000rpm centrifuge 5min；Naturally dry is volatilized completely to ethyl alcohol, and 10ul TB buffer are returned Molten, precipitation is linearization plasmid, and Nano Drop detect nucleic acid concentration；

4, electrotransformation Pichia pastoris GS115 competent cell

Plasmids of the 10ul after SalI is linearized is taken to be added the Pichia pastoris GS115 competent cell of 100ul, after mixing It being added in the electric shock cup of 0.1cm precoolings, ice bath 5min, is 1.8kV in voltage, carrying out electricity under conditions of the time 4-5ms that shocks by electricity turns, 1M sorbierite 1ml are added after turning in electricity, transfer them in sterilized 2ml centrifuge tubes, 30 DEG C, are incubated under conditions of 250rpm 2h；200ul is taken to carry out coated plate, the zeocin resistant panels of various concentration.

As preferred embodiment, in step 2, the detailed process of the induced expression of recombinant protein P39 is as follows：

1, the single bacterium colony grown in zeocin resistant panels is chosen into YPD fluid nutrient mediums and is cultivated；1ml is taken to stay overnight Culture 8000rpm centrifuges 5min, discards supernatant liquid；Into precipitation be added 500ul PBS solution fully suspend, 8000rpm from Heart 5min, discards supernatant liquid；It is suspended and is precipitated with the TE buffer of 100ul, boiling water bath 10min；Freeze with -80 DEG C under the conditions of 30min；Boiling water bath 10min again, 8000rpm centrifugation 5min collect supernatant and take 1ul as pcr template, carry out PCR amplification, PCR Condition is：94℃3min；94 DEG C of 30s, 60 DEG C, 72 DEG C of 90s (35 cycles)；72℃10min.

2, the extraction of Yeast genome

Biomass is detected using spectrophotometer or plating method, when OD600 values are 1.0, i.e. 1-2 × 10⁷cells/ Ml extracts pastoris genomic dna；

3, picking is grown in the positive single bacterium colony on Zeocin resistance YPD tablets, is inoculated in the BMGY fluid nutrient mediums of 5ml In, it is cultivated in 50ml triangular flasks, 28 DEG C, 250r/min shaking table culture 16-20h, until OD600 values are about 2.0-4.0, ferment at this time Female bacterium is in exponential phase, and the methanol for being added 1% carries out induced expression；The GS115 bacterium of electrotransformation empty carrier pPICZ α A simultaneously Strain is used as negative control, culture to collect sample, 10000r/min after a certain period of time, and 4 DEG C of centrifugation 2min take supernatant to carry out SDS- PAGE analyzes the expression of albumen, is as a result shown in the purpose that the size of successful expression in Pichia pastoris GS115 is about 43kDa Albumen.

As preferred embodiment, the specific extraction process of Yeast genome is as follows：

(1) yeast cells is taken, most 5 × 10⁷Cells, 12000rpm centrifuge 1min, absorb supernatant；

(2) yeast cell wall is abolished：600 μ l sorbierite buffer are added into thalline, 50U Lyticase are added, fill Divide mixing；30 DEG C of processing 30min；4000rpm centrifuges 10min, abandons supernatant, collects precipitation；

(3) 200 μ l buffer solutions GA are added into precipitation, precipitation is resuspended, mix well, if necessary to remove RNA, 4 μ are added L, 100mg/mlRNaseA solution vibrates 15sec, is placed at room temperature for 5min；

(4) 20 μ l Proteinase K solution, mixing is added；

(5) 220 μ l buffer solution GB are added, fully reverse mixing, 70 DEG C of placement 10min are centrifuged to remove cap wall Droplet；

(6) add 220 μ l absolute ethyl alcohols, fully reverse mixing, it may appear that flocculent deposit is centrifuged to remove the water of cap wall Pearl；

(7) previous step acquired solution and flocculent deposit being all added in an adsorption column CB3,12000rpm centrifuges 30sec, Waste liquid is outwelled, adsorption column CB3 is put into collecting pipe；

(8) 500 μ l buffer solutions GD, 12000rpm centrifugation 30sec are added into adsorption column CB3, waste liquid are outwelled, by adsorption column CB3 is put into collecting pipe；

(9) 600 μ l rinsing liquids PW, 12000rpm centrifugation 30sec are added into adsorption column CB3, waste liquid are outwelled, by adsorption column CB3 is put into collecting pipe；

(10) repetitive operation step (9)；

(11) adsorption column CB3 is put back in collecting pipe, 12000rpm centrifuges 2min, outwells waste liquid, adsorption column CB3 is placed in It is placed at room temperature for several minutes, to dry rinsing liquid remaining in sorbing material；

(12) adsorption column CB3 is transferred in centrifuge tube, it is slow that 50-200 μ l elutions are vacantly added dropwise to the intermediate position of adsorbed film Fliud flushing TE, is placed at room temperature for 2-5min, and 12000rpm centrifuges 2min, solution is collected into centrifuge tube.

As preferred embodiment, in step 2, the detailed process of purifying and the identification of recombinant protein P39 is as follows：

1, ammonium sulfate precipitation recombinant protein

It collects in 400ml bacterium solutions to 500ml centrifuge tubes, 1000rpm centrifugation 10min, collection supernatant, under the conditions of 4 DEG C, 70% ammonium sulfate precipitation destination protein, pulverulence is ground to by ammonium sulfate, is stirred to dissolve with glass bar when being added, 4 DEG C overnight, precipitation is collected by centrifugation again；10ml, 10mMPBS back dissolving precipitate, be used in combination the bag filters of 3000 molecular weight to back dissolving liquid into Row desalting processing；

2, Ni columns affinitive layer purification recombinant protein；

3, as a result Sephacryl S-100 purification of recombinant proteins P39 is shown in successful expression in Pichia pastoris GS115 Size is the recombinant protein P39 of 43kDa；

4, identify that the destination protein is recombinant protein P39 through immunoblot experiment.

Recombinant glycosylated albumen P39 prepared by the present invention is pure using HPLC Reverse High Performance Liquid Phase Method detection recombinant protein Degree, the results show that the purity of recombinant protein P39 after purification is 93%.

Applications of the recombinant glycosylated albumen P39 in preparing brucella vaccine and immunologic adjuvant prepared by the present invention.

Applications of the recombinant glycosylated albumen P39 in preparing immunologic adjuvant prepared by the present invention.

Recombinant glycosylated albumen P39 prepared by the present invention is preparing the application in having Immunogenic agents.

The beneficial effects of the invention are as follows：

P39 albumen is the Brucella melitensis immunoprotection candidate antigens albumen filtered out by immunoproteomics, can To induce the powerful specific immune response of body.Present invention secreting, expressing P39 albumen in Pichia pastoris, and in eukaryotic system It is interior it is carried out it is glycosylation modified, pass through glycosylation improve protein immunogenic.

Brucella can be decomposed, while can discharge some cells after entering host cell by host immune system Matter binding protein, these protein molecules can continue to play immunostimulation and dissemination, effectively induce body to generate special Property immune response, the encoding gene of P39 albumen is highly conserved in multiple and different biological species type Brucella sps, with its preparation it is novel Cloth disease vaccine may form cross protection in the Brucella sp of not isotype.P39 can also be expressed in prokaryotic system, but There is no modification system after the protein translation of eukaryotic system in prokaryotic system, established using Pichia pastoris as expressive host Stable rP39 Pichia pastoris high efficient expression systems, by optimization of fermentation conditions, expression quantity is up to 5mg/ml.With prokaryotic expression system System is compared, and pichia yeast expression system has following features：(1) expression system is highly stable, and the recombinant vector after linearisation can The problem of stable is integrated into the chromosome of yeast, and there is no plasmid loss；(2) efficient secreting, expressing approach, recombination Protein expression afterwards is started expression recombinant protein by the stringent control of alcohol oxidase promoter (AOX1) under methanol induction, and Recombinant protein can be secreted into solubility expression in culture medium by presence signal peptide α-factor on expression vector, be easy to collect and Purifying；(3) expression yield is high, and fermentation condition is easily controllable, and shake flask scale expression quantity of the rP39 in Pichia pastoris is up to 5mg/ ml；(4) contaminated with endotoxins problem is not present, escherichia expression system has certain contaminated with endotoxins, and finishes red Yeast is without this problem；(5) there are posttranslational modification systems, can modify recombinant protein, thus it is possible to vary protein molecular Property.

Description of the drawings

Fig. 1 is the PCR amplification electrophoresis result for recombinating cloned plasmids pMD-19T-P39.M：DL2000 DNA molecular amount standards； 1：PMD-19T-P39PCR products.

Fig. 2 is the double digestion electrophoresis result for recombinating cloned plasmids pMD-19T-P39.M：DL5000 DNA molecular amount standards； 1：PMD-19T-P39 double digestion products.

Fig. 3 is the PCR amplification electrophoresis result of recombinant expression plasmid pPICZ α A-P39.M：2000Marker；1-2：P39 bases Cause.

Fig. 4 is the double digestion electrophoresis result of recombinant expression plasmid pPICZ α A-P39.M：2000Marker；1：Recombinate table Up to plasmid pPICZ α A-P39；2：EcoRI the and NotI double digestions of recombinant expression plasmid pPICZ α A-P39.

Fig. 5 is the fermentation condition optimization schematic diagram of recombinant protein P39.Fig. 5 A are that different pH value express recombinant protein P39 Influence；Fig. 5 B are the influence that different potassium phosphate concentration express recombinant protein P39；Fig. 5 C are various concentration methanol induction pair The influence of recombinant protein P39；Fig. 5 D are the influence that different induction times express recombinant protein P39.

Fig. 6 is that SDS-PAGE analyzes expressions of results of the recombinant protein P39 in Pichia pastoris.M is albumen average molecular matter Amount standard；1-4 is recombinant protein P39 after purification.

The western blot identifications that Fig. 7 is recombinant protein P39.M is albumen relative molecular mass standard；1-2 is Western blot specific bands.

Fig. 8 is high-efficient liquid phase chromatograms of the recombinant protein P39 of purifying at Detection wavelength 280nm.

Fig. 9 is that PAS dyes PNGaseF glycosidases enzymolysis eukaryon P39 albumen.

Figure 10 is that nuclear magnetic resonance spectroscopy detects recombinant protein P39 level of glycosylation.

Figure 11 is that mtt assay detects mice spleen lymphocytes proliferation situation.

Figure 12 is that macrophage swallows FITC fluorescent marker protein situations.A-D be respectively eukaryon modification P39 albumen 1h, 2h, 4h, 6h cell swallow situation.

Figure 13 is that ELASA detects splenocyte supernatant cell factor IL-2 expressions.

Figure 14 is that ELASA detects splenocyte supernatant cell factor IL-4 expressions.

Figure 15 is that ELASA detects splenocyte supernatant cell factor IL-10 expressions.

Figure 16 is that ELASA detects splenocyte supernatant cell factor IFN-γ expression.

Figure 17 is that ELASA detects IgG antibody changes of contents level in different time mice serum.

Specific implementation mode

The present invention clones brucella dominant antigen albumen P39 genes, and builds carrier for expression of eukaryon pPICZ α A-P39, Zeocin resistance screening high copy number transformant, electrotransformation convert Pichia pastoris GS115 competent cell, final concentration For the expression of 1% methanol induction recombinant protein P39, destination protein is purified by affinity chromatography method, and utilizes molecule Sieve is further purified, and liquid chromatography detects its purity, and BCA methods detect albumen concentration, western blot Western blots pair Destination protein is identified.Multiple factors optimize the optimum condition of P39 secreting, expressings in Pichia pastoris；Pass through macrophage pair The phagocytosis of recombinant glycosylated albumen P39 and the tentatively more recombinant glycosylated egg of the influence to mice spleen lymphocytes proliferation White P39 and the immunogenicity that albumen is not glycosylated；Macrophage Raw264.7 is acted on after FITC fluorescent marker recombinant proteins；With not It is determined by the amount of phagocytosis albumen with time observation fluorescence intensity；Recombinant glycosylated albumen P391:1 mixing Freund's adjuvant is immune Balb/c mouse surroundings, mtt assay measure the spleen lymphocyte proliferation of immune rear 4th week, measure recombinant glycosylated albumen P39 thorns It is horizontal to swash immunized mice spleen small cell cytokine secretion, indirect EILSA methods detect second week to the 8th week serum IgG antibody water of mouse It is flat；Western Blot detect cell surface TLR2, TLR4 expression of receptor situation.

Invention is further described in detail with reference to embodiments.

The clone of 1 p39 genes of embodiment and the structure of carrier for expression of eukaryon pPICZ α A-P39

The present embodiment clones p39 genes, and the segment after PCR is connected with cloning vector pMD-19T (Simple), After being sequenced and digestion verification is correct, by the target fragment obtained after double digestion and the equally carrier for expression of eukaryon through double digestion PPICZ α A, which are connected, builds recombinant expression plasmid pPICZ α A-P39.

Main agents are as follows：

Amp storing liquids (100mg/mL)：It takes 1g ammonia benzyls to be dissolved into 7mL deionized waters, is settled to 10mL, filtered with 0.22 μm Membrane filtration is distributed into 1mL and often manages, and is stored in -20 DEG C of refrigerators.

LB liquid medium：Weigh peptone 10g, NaCl 10g, yeast powder 5g, distilled water 1L be added, adjust pH to 7.2,121 DEG C of sterilizing 20min.

Solid LB media：Weigh peptone 10g, NaCl 10g, yeast powder 5g, distilled water 1L be added, adjust pH to 7.2,121 DEG C of sterilizing 20min.

50×TAE：Weigh 242g Tris and NaEDTA2H₂O 37.2g are added 800ml distilled water and are dissolved, added The acetic acid for entering 57.1ml is sufficiently stirred and is settled to 1L, room temperature preservation.

One, the structure of recombinant clone plasmid pMD-19T-P39

1, the preparation of bacillus coli DH 5 alpha competent cell

(1) E.coli DH5 α original strains are taken out from -80 DEG C of refrigerators, in scribing line culture and picking on LB solid plates Single bacterium colony is inoculated in 5mL LB liquid mediums, 37 DEG C, 150rpm, shake culture 12h, by the obtained bacterium solution of culture by According to 1:1000 ratio inoculation with 100mL LB liquid mediums in, 37 DEG C, 150rpm, shake culture to OD600 be 0.4~ 0.5。

(2) bacterium solution that culture obtains is collected into 50mL centrifuge tubes, ice bath handles 30min, and 4 DEG C, 6000rpm is centrifuged 8min。

(3) it discards supernatant, with the 0.1mol/L CaCl being pre-chilled in ice bath in advance₂The thalline that solution suspension is collected, 4 DEG C, 6000rpm centrifuges 8min, which is repeated once.

(4) it discards supernatant, the CaCl of 4ml precoolings is added₂The thalline that solution suspension is collected, after mixing in each centrifuge tube plus Enter the sterile glycerol that 1.6mL is pre-chilled in advance, mixing is dispensed with 1.5mL EP pipes, and often 100 μ L of pipe, deposit in -80 DEG C of ice It is spare in case.

2, the connection of target fragment and cloning vector pMD-19T (Simple)

According to the p39 gene orders recorded in GenBank, using Primer Premier5.0 software Design primers, by upper Hai Shenggong Co., Ltds synthesize, and primer sequence is as follows：

Sense primer-P1：5'-gaattc(EcoRl)ATGGCACCTGTTGCCAATGC-3'

Downstream primer-P2：5'-gcggccg(Notl)TTTTGCGGCTTCAACCGCC-3'

P39 genes are cloned by template of brucella M16 genomes using round pcr, PCR reaction conditions are：94 DEG C pre- It is denaturalized 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 40s, 72 DEG C extend 10min eventually, and PCR reaction systems are as follows It is shown, the detection of 1% agarose gel electrophoresis.Using brucella 16M genomes as template, P1, P2 are that primer progress PCR amplification obtains To the band of about 1206bp sizes, as shown in Figure 1.

The segment of PCR is connect with cloning vector pMD-19T (Simple), 4 DEG C of constant-temperature incubations are stayed overnight, and recombination gram is built into Grand plasmid pMD-19T-P39, coupled reaction system are as shown in the table.

3, the conversion of recombinant clone plasmid pMD-19T-P39

Competent cell DH5 α are taken out from -80 DEG C of refrigerators, and recombinant clone plasmid pMD-19T- is added after ice bath recovery P39 10 μ L connection products pipettor is added in 50 μ L competent cell DH5 α, ice bath 30min, 42 DEG C of water-bath thermal shock 45s, Ice bath 5min (should during not be aggressively shaken EP pipe) immediately later.200 μ L non-resistant LB liquid mediums are added thereto, Shake culture 1h, condition are 37 DEG C, shaking table shaken cultivation under the conditions of 200rpm.Cultured bacterium solution is taken out 100 μ L uniformly to apply It is distributed in the LB solid mediums containing Amp resistances, the constant temperature incubation 10-12h in 37 DEG C of incubators, until it is opposite to obtain size It is uniform, the more smooth white colony in edge.

4, the extraction of recombinant clone plasmid pMD-19T-P39

The sterile lancet choicest single bacterium colony on plate of making even, which is gripped, with tweezers is placed in the LB Liquid Cultures that 5mL contains Amp resistances In base, 37 DEG C in shaking table, 200rpm shaken cultivations are stayed overnight.Steps are as follows for plasmid extraction：

(1) bacterium solution being incubated overnight is collected, 13000rpm centrifuges 1min, discards supernatant.

(2) SolutionI of 250 μ L of often pipe addition, blows and beats mixing thalline.

(3) SolutionII of 250 μ L of often pipe addition, covers EP pipes, gently overturns for several times, until liquid in pipe becomes phase To clarification, this step is no more than 5min.

(4) SolutionIII of 350 μ L of often pipe addition, covers EP pipes, gently overturns 7-8 times, 13000rpm centrifugations 10min。

(5) supernatant obtained after centrifugation is collected, is drawn in supernatant to pillar, 12000rpm centrifuges 1min, discards lower liquid.

(6) to Buffer W1,12000rpm the centrifugation 1min that 500 μ L are added in pipe are prepared, lower liquid is discarded.

(7) to Buffer W2,12000rpm the centrifugation 1min that 700 μ L are added in pipe are prepared, lower liquid is discarded.

(8) it is primary to repeat step (7).

(9) pipe 12000rpm skies will be prepared from 1min, discard lower liquid.

(10) to the Eluent solution that 40 μ L are added in pipe is prepared, room temperature stands 1min, and 12000rpm centrifuges 1min, will carry The plasmid taken is placed in -20 DEG C of refrigerators and saves backup.

5, the identification of recombinant clone plasmid pMD-19T-P39

(1) PCR is identified

PCR identifications are carried out by template of the different recombinant clone plasmids of extraction, PCR reaction conditions are：94 DEG C of pre-degenerations 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 40s, 72 DEG C extend 10min, PCR reaction systems such as following table institute eventually Show, the detection of 1% agarose gel electrophoresis.

Using different recombinant clone plasmids as template, P1, P2 are the band that primer PCR expands to obtain about 1206bp sizes, knot Fruit sees Fig. 1.

(2) digestion is identified

Digestion identification is carried out to the recombinant clone plasmid pMD-19T-P39 of extraction with restriction enzyme EcoRI and NotI, Double digestion reaction system is as shown in the table, 37 DEG C of metal bath 2h, the detection of 1% agarose gel electrophoresis.

With restriction enzyme EcoRI, NotI double digestion recombinant clone plasmid, a treaty 2500bp and a treaty are obtained The band of 1206bp sizes, as shown in Fig. 2, two results all meet expection.

(3) sequencing identification

PCR and the correct plasmid of digestion qualification result are sent to Shanghai Sheng Gong Co., Ltds and be sequenced, sequencing is tied Fruit is compared with NCBI original series.

PCR will be passed through and the correct recombinant clone plasmid of double digestion qualification result is sequenced, after sequencing result is translated It is as a result completely the same with the amino acid alignment of the p39 genes recorded in GENBANK, illustrate recombinant clone plasmid pMD- 19T-P39 is built successfully.

Two, the structure of recombinant expression plasmid pPICZ α A-P39

1, the connection of target fragment and carrier pPICZ α A

It is bis- through EcoRI and NotI to select sequencing result correct recombinant clone plasmid pMD-19T-P39 and carrier pPICZ α A Digestion, double digestion system is as shown in the table, the detection of 1% agarose gel electrophoresis.

The Ago-Gel containing target gene is cut under bale cutting instrument to be placed in centrifuge tube, is recycled and is tried using DNA gel Agent box is recycled, and operating procedure is as follows：

(1) calculated for gel weight, using this weight as the volume (example of a gel：100mg=100 μ L).

(2) thereto be added three times gel volume Buffer DE-A, centrifuge tube is placed in 75 DEG C of water-baths heat it is molten Glue, should overturn a centrifuge tube per 2-3min in the process, observe colloidal sol situation at any time until glue dissolves completely.

(3) the Buffer DE-B of 0.5 Buffer DE-A volume are added into centrifuge tube, mixing is blown and beaten with pipettor.

(4) liquid in step (3) centrifuge tube is transferred to and is prepared in pipe, 12000rpm centrifuges 1min, discards lower liquid.

(5) to Buffer W1,12000rpm the centrifugation 30s that 500 μ L are added in pipe are prepared, lower liquid is discarded.

(6) to Buffer W2,12000rpm the centrifugation 1min that 700 μ L are added in pipe are prepared, lower liquid is discarded.

(7) it is primary to repeat step (6).

(8) pipe 12000rpm skies will be prepared from 1min, discard lower liquid.

(9) to the Eluent solution that 30 μ L are added in pipe is prepared, room temperature stands 1min, and 12000rpm centrifuges 1min, will return The segment of receipts is placed in -20 DEG C of refrigerators and saves backup.

(10) target gene that gel recycles is attached with pPICZ α A carriers with T4DNA ligases, connection is anti- Answer system as follows, 4 DEG C are incubated overnight connection.

2, the preparation of bacillus coli DH 5 alpha competent cell

DH5 α bacterial strains are taken out from -80 DEG C of refrigerators, simultaneously picking single bacterium colony, competent cell preparation method are same for scribing line culture On.

3, the conversion of recombinant expression plasmid pPICZ α A-P39

Connection product is converted into E.coliDH5 α competent cells, the solid LB cultures containing Amp resistances are coated on In base, conversion of the method with recombinant clone plasmid pMD-19T-P39.

4, the extraction of recombinant expression plasmid pPICZ α A-P39

The sterile lancet choicest single bacterium colony on plate of making even, which is gripped, with tweezers is placed in the LB Liquid Cultures that 5mL contains Amp resistances In base, 37 DEG C in shaking table, 200rpm shaken cultivations are stayed overnight.Plasmid extraction method is the same as recombinant clone plasmid pMD-19T-P39's Extraction.

5, the identification of recombinant expression plasmid pPICZ α A-P39

(1) PCR is identified

PCR identifications are carried out by template of the different recombinant plasmids of extraction, method is the same as recombinant clone plasmid pMD-19T-P39's PCR is identified.

Using different recombinant expression plasmids as template, P1, P2 are the band that primer PCR expands to obtain about 1206bp sizes, knot Fruit sees Fig. 3.

(2) digestion is identified

The correct recombinant plasmid of PCR qualification results is subjected to digestion identification, method with restriction enzyme EcoRI and NotI Digestion with recombinant clone plasmid pMD-19T-P39 is identified.

With restriction enzyme EcoRI, NotI double digestion recombinant expression plasmid, a treaty 2500bp and a treaty are obtained As a result the band of 1206bp sizes is shown in that Fig. 4, two results meet expection.

(3) sequencing identification

PCR and the correct plasmid of digestion qualification result are sent to Shanghai Sheng Gong Co., Ltds and be sequenced, sequencing is tied Fruit is compared with NCBI original series.

PCR will be passed through and the correct recombinant expression plasmid of double digestion qualification result is sequenced, after sequencing result is translated It is as a result completely the same with NCBI sequence alignments, illustrate that recombinant expression plasmid pPICZ α A-P39 are built successfully.

Expression, purifying and fermentation condition optimization of the 2 P39 albumen of embodiment in Pichia pastoris GS115

The present embodiment is converted the carrier for expression of eukaryon pPICZ α A-P39 built to Pichia pastoris using electrotransformation In GS115 competent cells, the induced expression of albumen is carried out using methanol, and destination protein is carried out using the method for affinity chromatography Purifying, and destination protein is further isolated and purified using molecular sieve, and the albumen of purifying is tested using western blot Card.The yield of albumen is further increased by optimizing experiment of single factor, obtaining optimal conditions of fermentation is：PH value is that 6, phosphate is dense Degree can obtain optimum experimental condition when being 0.02M, methanol induction a concentration of 1%, induction 72h, be optimized with this condition Fermentation condition, recombinant protein concentration after purification is up to 5mg/ml.

Main agents are as follows：

(1)10×YNB：It weighs 13.4gYNB to be dissolved in 100ml water, is heated to YNB and is completely dissolved, 0.22 μm of filter membrane mistake Bacterium is filtered out, is preserved under the conditions of 4 DEG C.

(2) 500 × biotins：It weighing 10mg biotins and is dissolved in 50ml, 0.22 μm of membrane filtration degerming deposits in 4 DEG C, It can use 1 year.

(3) 10% glycerine：Mix the distilled water of 10ml glycerine and 90ml, 121 DEG C of high pressure steam sterilizations.

(4) 10% methanol：Mix the distilled water of 10ml methanol and 90ml, 0.22 μm of membrane filtration degerming.

Kaliumphosphate buffer (1M)：

(5) 50% (W/V) glucose：Addition 100ml distilled water in 50g glucose, 0.22 μm of membrane filtration degerming, -20 It is preserved under the conditions of DEG C.

(6) YPD fluid nutrient mediums：2g peptones are weighed, yeast extract 1g is settled to 96ml, and 121 DEG C of high steams go out The glucose of 4ml 50% is added in bacterium, and 4 DEG C spare.

(7) YPD solid mediums：Weigh 2g peptones, yeast extract 1g, agar 1.5g are settled to 96ml, 121 DEG C The glucose of 4ml 50% is added in high pressure steam sterilization, and 4 DEG C spare.

(8) BMGY culture mediums：10g yeast extracts are dissolved, 20g albumen is in 700ml water, 121 DEG C of high pressure steam sterilizations 20min is cooled to room temperature, the kaliumphosphate buffer of addition 100ml 1M, 100ml 500 × biotins of 10 × YNB, 2ml, 10% glycerine of 100ml, deposits in 4 DEG C.

(9) YPD-zeocin tablets：2g peptones are weighed, yeast extract 1g is settled to 96ml, 121 DEG C of high steams The glucose of 4ml 50% is added in sterilizing, and 4 DEG C spare.

(10)Binding buffer：Weigh Na₂HPO₄·12H₂O 3.041g, imidazoles 0.544g, NaCl 11.688g, Add 150ml water, adjust pH to 7.4, is settled to 400ml, 0.22 μm of membrane filtration, 4 DEG C of preservations.

(11)Elution buffer：Weigh Na₂HPO₄·12H₂O 3.041g, imidazoles 13.616g, NaCl 11.688g, Add 150ml water, adjust pH to 7.4, is settled to 400ml, 0.22 μm of membrane filtration, 4 DEG C of preservations.

(12)Stripping buffer：Weigh Na₂HPO₄·12H₂O1.5306g, EDTA 3.722g add 150ml water, PH to 7.4 is adjusted, 200ml, 0.22 μm of membrane filtration, 4 DEG C of preservations are settled to.

(13) 10% (W/V) SDS：1g SDS are added 8mL deionized waters, are fully settled to 10mL after dissolving, preservation waits for With.

(14) 5 × Tris- glycine running buffers：Glycine 75.2g, Tris 12.08g, 10%SDS 40mL, adds Deionized water is put in 4 DEG C of refrigerators and preserves to 800mL.

(15) 1M Tris-HCl (pH6.8) buffer solution：2.1g Tris are weighed, 70mL deionized waters are added, after to be dissolved With 1M HCl tune PH to 6.8, it is settled to l00mL after mixing, 121 DEG C, 20min high pressure sterilizations are placed in room temperature preservation.

(16) 1.5M Tris-HCl (pH8.8) buffer solution：It weighs 36.34g Tris to be put in beaker, 150mL is added and goes Ionized water is adjusted to 8.8 with 1M HCl after to be dissolved, is settled to 200mL after mixing, 121 DEG C, 20min high pressure sterilizations are placed in room Temperature preserves.

(17) 10% ammonium persulfates (W/V)：20mg ammonium persulfates are added 200 μ L deionized waters and mix well, matching while using.

(18) 2 × sample-loading buffers：Bromophenol blue 0.lmg, 10%SDS 4mL, This-HC1 (pH6.8) buffer solution 2mL, 10% glycerine 1mL, uses ddH₂O is diluted to 10mL.

(19) coomassie brilliant blue R_250 dyeing liquor：2g Coomassie brilliant blues, 500mL isopropanols, 1300mL deionized waters, 200mL glacial acetic acid, filter paper filters after mixing.

(20) destainer：Absolute ethyl alcohol 300ml, glacial acetic acid 100ml, is settled to 1L.

(21) film transfer buffer solution：Deionized water 800mL is added, fully in Tris5.8g, glycine 2.9g, SDS 0.38g 20% methanol 200mL is added in dissolving, instant to match.

(22)10×PBS：Weigh NaCl 80g, KCl 2g, Na₂HPO₄35.8g KH₂PO₄2.7g is dissolved in 800ml water In, pH to 8.0 is adjusted, 1L is settled to.

(23)PBST(pH8.0)：1mL Tween-20 are added in 1000mL, 0.01mol/LPBS.

(24) sorbierite buffer：1.2M sorbierites are prepared with 0.1M sodium phosphate buffers (pH7.4).

(25) preparation of 0.1M sodium phosphate buffers (pH7.4)：77.4ml、0.1mol/L Na₂HPO₄+22.6ml、 0.1mol/L NaH₂PO₄。

(26) mobile phase A：100% methanol.

(27) Mobile phase B：Pure water containing 0.1%TFA.

One, recombinant expression plasmid pPICZ α A-P39 electrotransformations Pichia pastoris GS115 competent cell

1, a large amount of extractions of recombinant expression plasmid pPICZ α A-P39

The single bacterium colony of picking culture is inoculated in 5mL LB liquid mediums, 37 DEG C, after 200rpm shake cultures are stayed overnight Expand overnight incubation.A large amount of extraction steps of plasmid are as follows：

(1) 40ml bacterium solutions are collected to be put into 50ml centrifuge tubes, 10000rpm, centrifuges 3min and is discarded after more collections several times Clearly, it is upside down on clean filter paper；

(2) 10mlP1 solution is added into the 50ml centrifuge tubes for have precipitation, while 50ul TIANRED reagents are added, utilizes Liquid-transfering gun blows and beats mixing or with oscillator mixing；

(3) 10mlP2 solution is added into centrifuge tube, mild immediately spins upside down 6-8 times, so that thalline is fully cracked, room Temperature places 5min；

(4) 10mlP4 solution is added into centrifuge tube, mild immediately spins upside down 6-8 times, until white point occurs in solution Flocculent deposit is dissipated, 10min is placed at room temperature for, 10000rpm centrifuges 5-10min, the time can be appropriately extended to 20-30min；

(5) complete soln is carefully poured into filter, slowly pushes filtering, provides the clean centrifuge tubes of 50ml for oneself；

(6) the 5M Nacl solution of the isopropanol and 1/2 isopropanol volume of 0.35 times of filtrate volume is added into filtrate, on Lower overturn mixes well；

(7) under the conditions of 4 DEG C, 10000rpm centrifuges 30min, gently outwells supernatant, is upside down on filter paper and is absorbed water；

(8) ethyl alcohol that 6ml 70% is added into pipe fully rinses precipitation, and 4 DEG C, 10000rpm centrifuges 10min, gently falls Fall supernatant, is upside down on filter paper；

(9) the 7th step is repeated；

(10) centrifuge tube opening is placed on indoor 10-20min, 1-1.5mlTB buffer is added after so that ethyl alcohol is fully volatilized Fully dissolving, Nano Drop detect plasmid concentration.

2, the linearisation of recombinant expression plasmid pPICZ α A-P39

The recombinant expression plasmid pPICZ α A-P39 of extraction are linearized using SalI, reaction system is as follows：Plasmid 20 μ l, 5 μ l of SalI restriction enzymes, 5 bufferH μ l, ddH₂20 μ l of O, totally 20 samples.37 DEG C of water-bath 3h.5 μ l are taken to carry out Nucleic acid electrophoresis detects whether digestion is complete, and non-digested plasmid compares.

3, the plasmid of alcohol precipitation method recycling linearisation

It draws in the 500ul plasmids to the centrifuge tube of 1.5ml after digestion, isometric phenol, chloroform, isoamyl alcohol is added, It uniformly mixes, under the conditions of 4 DEG C, 12000rpm centrifuges 10min；Upper layer supernatant is taken, the absolute ethyl alcohol and 1/10 of 2 times of volumes is added The 5M Nacl solution of volume, static 2h under the conditions of -20 DEG C；Under the conditions of 4 DEG C, 13000rpm centrifuges 10min, discards supernatant；With 75% ethyl alcohol washing precipitation, 12000rpm centrifuge 5min；Naturally dry is volatilized completely to ethyl alcohol, and 10ul TB buffer are returned Molten, precipitation is linearization plasmid, and Nano Drop detect nucleic acid concentration.

4, the preparation of Pichia pastoris GS115 competent cell

GS115 bacterial strains are taken out from -80 DEG C of refrigerators, simultaneously picking single bacterium colony, 28 DEG C of overnight incubations take 1ml for scribing line culture In GS115 overnight cultures to 1.5ml centrifuge tubes, OD values are 0.6-0.8 or so；4 DEG C, 10000rpm centrifuges 10min, discards The sterile water of clear liquid, precipitation precooling washes twice, and centrifuges, and retains precipitation；The treatment fluid of 1ml, room temperature is added in each centrifuge tube Lower placement 20min；10000rpm is centrifuged, and is discarded supernatant, and 1ml sorbierites are added, and is centrifuged 10min, is discarded supernatant liquid；Use sorbierite Precipitation is washed twice, and it is 100ul, as Pichia pastoris GS115 competent cell finally to retain to volume.

5, electrotransformation Pichia pastoris GS115 competent cell

Plasmids of the 10ul after SalI is linearized is taken to be added the Pichia pastoris GS115 competent cell of 100ul, after mixing It being added in the electric shock cup of 0.1cm precoolings, ice bath 5min, is 1.8kV in voltage, carrying out electricity under conditions of the time 4-5ms that shocks by electricity turns, 1M sorbierite 1ml are added after turning in electricity, transfer them in sterilized 2ml centrifuge tubes, 30 DEG C, are incubated under conditions of 250rpm 2h；200ul is taken to carry out coated plate, the zeocin resistant panels of various concentration.

Two, the screening of positive colony

1, the single bacterium colony grown in zeocin resistant panels is chosen into YPD fluid nutrient mediums and is cultivated；1ml is taken to stay overnight Culture 8000rpm centrifuges 5min, discards supernatant liquid；Into precipitation be added 500ul PBS solution fully suspend, 8000rpm from Heart 5min, discards supernatant liquid；It is suspended and is precipitated with the TE buffer of 100ul, boiling water bath 10min；Freeze with -80 DEG C under the conditions of 30min；Boiling water bath 10min again, 8000rpm centrifugation 5min collect supernatant and take 1ul as pcr template, carry out PCR amplification, PCR Condition is：94℃3min；94 DEG C of 30s, 60 DEG C, 72 DEG C of 90s (35 cycles)；72℃10min.

2, the extraction of Yeast genome

Biomass is detected using spectrophotometer or plating method, when OD600 values are 1.0, i.e. 1-2 × 10⁷cells/ Ml, for extracting cerevisiae dna.Specific extraction process is as follows：

(1) yeast cells is taken (to be no more than 5 × 10⁷Cells), 12000rpm (~13400 × g) centrifuges 1min, to the greatest extent Amount absorbs supernatant (bacterial sediment can be collected into a centrifuge tube by centrifuging several times when bacterium solution is more)；

(2) yeast cell wall is abolished：600 μ l sorbierite buffer are added into thalline, about 50U is added Lyticase is mixed well；30 DEG C of processing 30min；4000rpm (~1500 × g) centrifuges 10min, abandons supernatant, collects precipitation；

(3) 200 μ l buffer solutions GA are added into precipitation, precipitation is resuspended, mix well.If necessary to remove RNA, 4 can be added μ l RNaseA (100mg/ml) solution vibrates 15sec, is placed at room temperature for 5min；

(4) 20 μ l Proteinase K solution, mixing is added；

(5) 220 μ l buffer solution GB are added, fully reverse mixing, 70 DEG C of placement 10min, limpid, the brief centrifugation of solution strain To remove the droplet of cap wall；

(6) add 220 μ l absolute ethyl alcohols, fully reverse mixing, at this time it is possible that flocculent deposit, brief centrifugation is to remove The droplet of cap wall；

(7) previous step acquired solution and flocculent deposit being all added in an adsorption column CB3,12000rpm centrifuges 30sec, Waste liquid is outwelled, adsorption column CB3 is put into collecting pipe；

(8) 500 μ l buffer solutions GD, 12000rpm centrifugation 30sec are added into adsorption column CB3, waste liquid are outwelled, by adsorption column CB3 is put into collecting pipe；

(9) 600 μ l rinsing liquids PW, 12000rpm centrifugation 30sec are added into adsorption column CB3, waste liquid are outwelled, by adsorption column CB3 is put into collecting pipe；

(10) repetitive operation step (9)；

(11) adsorption column CB3 is put back in collecting pipe, 12000rpm centrifuges 2min, outwells waste liquid.Adsorption column CB3 is placed in It is placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in sorbing material；

(12) adsorption column CB3 is transferred in a clean centrifuge tube, 50- is vacantly added dropwise to the intermediate position of adsorbed film 200 μ l elution buffer TE, are placed at room temperature for 2-5min, and 12000rpm centrifuges 2min, solution is collected into centrifuge tube.

Three, the induced expression of recombinant protein P39

Picking is grown in the positive single bacterium colony on Zeocin resistance YPD tablets, is inoculated in the BMGY fluid nutrient mediums of 5ml In, it is cultivated in 50ml triangular flasks, 28 DEG C, 250r/min shaking table culture 16-20h, until OD600 values are about 2.0-4.0, ferment at this time Female bacterium is in exponential phase, and the methanol for being added 1% carries out induced expression.The GS115 bacterium of electrotransformation empty carrier pPICZ α A simultaneously Strain is used as negative control, collects sample, 10000r/min after cultivating a period of time, 4 DEG C of centrifugation 2min take supernatant to carry out SDS- PAGE analyzes the expression of albumen.As a result it is shown in the purpose that the size of successful expression in Pichia pastoris GS115 is about 43kDa Albumen.

Four, the fermentation condition optimization of recombinant protein P39

1, influence of the pH value to recombinant protein P39 expression quantity

Positive restructuring bacterium is taken to be respectively connected to the 200mlBMGY that pH value is respectively 3.0,5.0,6.0,8.0 with 1% inoculum concentration In culture medium, salinity 0.05M, 28 DEG C of shaking table cultures, until when OD values are 0.6-0.8,1% methanol induction 72h.

As a result albumen yield first increases with the raising of pH and reduces afterwards it can be seen from Fig. 5-A, when most 6 pH, albumen Yield highest, experiment show that recombinant protein expression condition under the conditions of acid or weakly alkaline is preferable, so, best fermentation PH should be between 6-7, and highest rate is 3.86mg/ml.

2, influence of the salinity to recombinant protein P39 expression quantity

It is respectively 0.02M, 0.05M, 0.1M, 0.2M to take positive restructuring bacterium to be respectively connected to salinity with 1% inoculum concentration BMGY culture mediums are cultivated, pH6.0, when OD values are 0.6-0.8,1% methanol induction 72h.

As a result by Fig. 5-B as it can be seen that with potassium phosphate concentration raising albumen yield reduce, potassium phosphate concentration be 0.02M when, Albumen yield highest, excessively high salinity may inhibit the expression of albumen, so, best potassium phosphate concentration is 0.02- Between 0.05M, highest yield is 3.64mg/ml.

3, influence of the methanol concentration to recombinant protein P39 expression quantity

Take positive restructuring bacterium in 1% inoculum concentration BMGY culture mediums, 28 DEG C of shaking table cultures, salinity 0.05M, pH6.0, When OD values are 0.6-0.8, respectively with the methanol induction 72h of 0.5%, 1%, 2%, 3% various concentration.

As a result by Fig. 5-C as it can be seen that must take the lead in reducing after increasing with the increase albumen of methanol induction concentration, in methanol concentration When 1%, the methanol of albumen yield highest, low concentration and high concentration all adversely affects the expression of albumen, so, best first Alcohol-induced a concentration of 1%, highest rate is 4.96mg/ml.

4, influence of the induction time to recombinant protein P39 expression quantity

Take positive restructuring bacterium in 1% inoculum concentration BMGY culture mediums, 28 DEG C of shaking table cultures, salinity 0.05M, pH6.0, OD values be 0.6-0.8 when, with the methanol of 1% concentration induce respectively for 24 hours, 48h, 72h, 96h.

As a result by Fig. 5-D as it can be seen that with induction time increase, albumen yield occur first increase after downward trend, it is long The induction of time is unfavorable for the expression of destination protein, and experiment shows under conditions of inducing 72h that prolonged induction may cause Protein degradation, so, 72h albumen yield highests, highest rate is 5.05mg/ml.

5, SDS-PAGE polyacrylamide gel electrophoresises testing goal albumen, PAGE gel are formulated as follows shown in table.

By optimization of fermentation conditions, obtain recombinant protein P39 in pH6.0, potassium phosphate concentration 0.02M, methanol induction concentration 1%, under conditions of inducing 72h, yield reaches as high as 5mg/ml.

Five, the purifying and identification of recombinant protein

1, ammonium sulfate precipitation recombinant protein

It collects in 400ml bacterium solutions to 500ml centrifuge tubes, 1000rpm centrifugation 10min, collection supernatant, under the conditions of 4 DEG C, 70% ammonium sulfate precipitation destination protein, pulverulence is ground to by ammonium sulfate, is stirred to dissolve with glass bar when being added, 4 DEG C overnight, precipitation is collected by centrifugation again；10ml, 10mMPBS back dissolving precipitate, be used in combination the bag filters of 3000 molecular weight to back dissolving liquid into Row desalting processing.

2, Ni columns affinitive layer purification recombinant protein

(1) ddH of 5ml is first used₂O washs pillar；

(2) the Elution buffer of 5ml elute pillar；

(3) the Binding buffer of 5ml elute pillar；

(4) ddH of 5ml₂O washs pillar；

(5) the Binding buffer of 10ml elute pillar；

(6) 10ml recombinant proteins sample crosses pillar, and flow control is in 1ml/min；

(7) the Binding buffer of 20ml elute pillar；

(8) the Elution buffer of 5ml elute destination protein, packing to 1.5ml centrifuge tubes；

(9) SDS-PAGE electrophoresis detections each component.

3, Sephacryl S-100 purification of recombinant proteins P39

The supernatant gathered is subjected to vacuum freeze-drying, phosphate solution (PBS) the dialysis 48h, 0.22 μ of 10mM, pH7.4 M sterilizing filters filter, and purify column purification using Sephacryl S-100 protein chromatographics, 20mM Nacl elute albumen, often manage 3ml, Nanodrop detect light absorption value at 280nm, and SDS-PAGE identifies each chromatography ingredient.The results are shown in Figure 6, in complete red ferment The recombinant protein P39 that successful expression size is about 43kDa in female GS115.

4, recombinant protein P39 immunoblot experiments (westen blot) are identified

(1) transferring film：After SDS-PAGE, nitrocellulose filter (pvdf membrane) and filter paper are cut out according to gel same size 10, pvdf membrane is impregnated into 10s in methyl alcohol, the pvdf membrane handled well, filter paper are placed in transferring film buffer solution and impregnate 30min； It is installed by following sequence：Cathode-filter paper (5)-gel-pvdf membrane-filter paper (5)-anode, at the same ensure filter paper, Pvdf membrane, gel alignment, bubble is lightly driven away with glass bar；Power on, with constant pressure 100V, electric current is not higher than 250mA's Condition transfers 50min.

(2) it closes：1g skimmed milk powers, which are dissolved in 20mL PBST, is made confining liquid, and the pvdf membrane transferred is placed in closing In liquid, room temperature gently shakes 1h on shaking table and is closed to it, is during which washed 3 times, each 2-3min with PBST, and closing is completed After discard confining liquid.

(3) with an anti-binding：The mouse monoclonal antibody of anti-histidine tag is added, antibody is diluted with confining liquid, and work is dense Degree is 1:500,4 DEG C of overnight incubations, after primary antibody is incubated, with PBST by Membrane cleaning 3 times, about 5min every time.

(4) with two anti-bindings：The mountain sheep anti-mouse igg of horseradish peroxidase label is added, antibody is diluted with confining liquid, is worked A concentration of 1:5000, it is incubated at room temperature 2h, after secondary antibody is incubated, with PBST by Membrane cleaning 3 times, about 5min every time.

(5) it develops the color：Substrate o-phenylene diamine (DAB) reaction solution is added on film, is protected from light and gently shakes a few minutes, observe at any time Purpose band shows situation, after purpose band appearance, deionized water is added immediately and terminates and reacts and scans preservation.

(6) identify that the destination protein is recombinant protein P39 through western blot Western blots.

Six, the reversed efficient liquid phases of HPLC detect recombinant protein purity

The protein sample of recombination is carried out using the C18 chromatographic columns of the reversed high performance liquid chromatograph of Japanese Shimadzu and Agilent Analysis, operation are as follows：

(1) preparation of samples：Recombination P39 protein samples are filtered with 0.22 μm of filter, mobile phase is：(A) contain The pure water and (B) 100% methanol of 0.05% trifluoroacetic acid (TFA), mobile phase ultrasound 1h after 0.22 μm filters are molten to remove Minute bubbles in liquid.

(2) instrument uses：It opens high performance liquid chromatograph and preheats 10min, be exhausted 3 times, this experiment uses Agilent Zorbax 300SB-C18 (250mm × 4.6mm, 5 μm) reverse-phase chromatographic column, 85% methanol aqueous solution are used first as mobile phase For line of flow as baseline, isobase starts hand sampling when balancing, and sample size is 20 μ l.

(3) flow velocity 0.8ml/min, 35 DEG C of column temperature are set, and 45min carries out gradient elution to sample.

(4) Detection wavelength is 280nm, draws and calculates purity of protein.

(5) after sample detection pillar 1h is cleaned with 100% methanol.

(6) reversed-phase high performance liquid chromatography figure is computed egg the results show that the purity of recombinant protein P39 after purification is higher Bai Chundu is up to 93%.

Seven, BCA detects recombinant protein concentration

According to PierceTM BCAProteinAssay Kit standard curve standard items dilution process, enzyme is used after reaction It marks instrument and measures light absorption value of the albumen at 562nm.

Recombinant plasmid is transferred in Pichia pastoris GS115 competent cell by this experiment by electrotransformation, and utilizes methanol Induced expression is verified by SDS-PAGE, and Western Blot analyses confirmation (as shown in fig. 7, there is specific band, size About 43kDa), brucella rP39 albumen success secreting, expressing in yeast, and by optimization of fermentation conditions, expression quantity is up to 5mg/ml.The purity of albumen after purification is detected up to 93% by efficient liquid phase.

3 brucella rP39 protein glycosylations of embodiment are identified

It is glycoprotein, the composite junction that glycoprotein is made of oligonucleotide chain and polypeptide chain that majority, which has the albumen of clinical application, Structure.There is more abundant posttranslational modification system in Pichia pastoris, as the formation of disulfide bond, protein folding, glycosylation are repaiied Decorations, phosphorylation modification, the processing of signal peptide sequence and acyl.Yeast glycosylation modification is complex, to the outer of secretion Source protein can also carry out glycosylation modified.According to the difference of connection type on sugar chain and gal4 amino acid residue, can be divided into N- is glycosylated to be glycosylated with O-.N- glycosylations refer on albumen that there are one section of conserved sequence Asn-X-Thr/Ser, sugar chain can be with day Winter amide residues are connected to form glycosylation modified.O- glycosylations refer to that oligosaccharides is connected with the hydroxyl of lysine or proline.It utilizes Yeast expression system production glycosylation albumen has been applied in practice production, and this mode of production is not only at low cost, operation letter It is single, and yield high-purity is good, it is safe.The degree of glycosylation to compare with saccharomyces cerevisiae, Pichia pastoris degree of glycosylation compared with It is low, generally 8-50 mannose, the appropriate glycosylation modified stability and immunogenicity that can improve albumen.The sugar of yeast Base approach can also make it generate the glycosylation modified form beneficial to people by artificial reconstructed method.

The present embodiment digests brucella rP39 albumen using PNGaseF glycosidases, and PAS methods are dyed, knot Close the analysis that nuclear magnetic resonance spectroscopy carries out the P39 albumen after recombination glycosylation modified level.

One, main agents：

1, Shiff reagents：It weighs 0.5g basic fuchsins to be added in the distilled water that 100ml boils, constantly shake, then boil 15min is cooled to 50 DEG C and is filtered with filter paper, the HCl of 10ml1M is added until being completely dissolved, and is uniformly mixed, is cooled to 25 DEG C, 0.5g Sodium Metabisulfites are added, bottle stopper is stoppered after fully shaking, it is colourless or faint yellow to wait for that solution is presented by the static 48h of room temperature When, 0.5g activated carbons are added and remove color, is sealed up for safekeeping after filter paper filtering and in brown reagent bottle, blackens paper bag outside and wrap up in, and refrigerated in 4 It is spare in DEG C environment.

2,10% (W/V) SDS：1g SDS are added 8mL deionized waters, are fully settled to 10mL after dissolving, preserve for use.

3,5 × Tris- glycine running buffers：Glycine 75.2g, Tris 12.08g, 10%SDS 40mL, adds Ionized water is put in 4 DEG C of refrigerators and preserves to 800mL.

4,1mol/LTris-HCl (pH6.8) buffer solution：2.1g Tris are weighed, 70mL deionized waters are added, after to be dissolved With 1M HCl tune PH to 6.8, it is settled to l00mL after mixing, 121 DEG C, 20min high pressure sterilizations are placed in room temperature preservation.

5,1.5mo1/LTris-HCl (pH8.8) buffer solution：It weighs 36.34g Tris to be put in beaker, 150mL is added and goes Ionized water is adjusted to 8.8 with 1M HCl after to be dissolved, is settled to 200mL after mixing, 121 DEG C, 20min high pressure sterilizations are placed in room Temperature preserves.

6,10% ammonium persulfate (W/V)：20mg ammonium persulfates are added 200 μ L deionized waters and mix well, matching while using.

7,2 × sample-loading buffer：Bromophenol blue 0.lmg, 10%SDS 4mL, This-HC1 (pH6.8) buffer solution 2mL, 10% Glycerine 1mL is diluted to 10mL with ddH2O.

8, coomassie brilliant blue R_250 dyeing liquor：2g Coomassie brilliant blues, 500mL isopropanols, 1300mL deionized waters, 200mL Glacial acetic acid, filter paper filters after mixing.

9, destainer：Absolute ethyl alcohol 300ml, glacial acetic acid 100ml, is settled to 1L.

10, fixer：50ml absolute methanols, are settled to 100ml.

11, oxidation solution：It is mixed with the mixed liquor of 1% periodic acid and 3% acetic acid.

12, rinsing liquid：It is final concentration of to its to hydrochloric acid is added in the mixed liquor of the periodic acid for being mixed with 1% and 3% acetic acid 10mmol/L。

13, developing solution：100ml water dissolutions are added in 0.5g Sodium Metabisulfites.

Two, PNGaseF glycosidases enzymolysis recombinant protein P39

1, in the system of 10 μ l, the recombinant protein P39 of 1-20 μ g is added, 1 μ l 10 × glycoprotein denaturation buffers are added buffer。

2, under conditions of boiling for 100 DEG C, 10min makes recombinant protein P39 be denaturalized.

3,2 μ 10 × Glycobuffer of l buffer solutions, 2 μ l 10%NP-40,1 μ l PNGaseF glycosidases is added, adds water It is 20 μ l to total volume.

4, it is incubated 1h under the conditions of 37 DEG C.

5, SDS-PAGE analyses are carried out after PNGaseF glycosidases enzymolysis recombinant protein P39, the results are shown in Figure 8, before enzymolysis Recombinant protein molecular weight afterwards does not occur significantly to change.

Three, the PAS dyeing of recombinant protein running gel

1, after electrophoresis, gel is placed in 50% ethyl alcohol and fixes 30min.

2, gel is rinsed 3 times with distilled water, is put into the mixed solution for being mixed with 1% sodium metaperiodate and 3% acetic acid and is aoxidized 30min。

3, gel distilled water is rinsed 3 times, is put into the mixing for being mixed with 0.1% Sodium Metabisulfite and 10mmol/L hydrochloric acid It is rinsed 2 times in solution, each 10min.

4, gel is put into Schiff and is protected from light dyeing 1h.

5, gel is put into the mixed solution for being mixed with 1% Sodium Metabisulfite and 10mmol/L hydrochloric acid and is protected from light rinsing 1h.

6,0.5% Sodium Metabisulfite solution of gel is rinsed 3 times, each 20min, observes the purplish red vitta of appearance Band.

The recombinant protein P39 samples after recombinant protein sample and PNGaseF glycosidases enzymolysis are contaminated using PAS dyeing Color, the results are shown in Figure 9, and albumen is dyed to pink, illustrates to carry sugar chain ingredient on two kinds of albumen.

Four, nuclear magnetic resonance spectroscopy detects recombinant protein P39 level of glycosylation

Recombinant protein P39 to Yeast expression and the recombinant protein P39 digested using PNGase glycosidases are utilized respectively core Magnetic resonance hydrogen spectrum has carried out glycosylation modified analysis, the results showed that, as shown in Figure 10, recombinant protein P39 collection of illustrative plates is in 3.6- Occur oligosaccharides H signal at 3.8ppm, illustrates that there are Sugar signals on recombinant protein P39 after purification, the P39 albumen after recombination exists Occur in Pichia pastoris glycosylation modified.The recombinant protein P39 sugar after PNGase glycosidase enzymolysis protein N- sugar chains is utilized simultaneously Signal does not disappear, and recombinant protein P39 has potential glycosylation modified site, but Sugar signal still has after enzymolysis, illustrates weight The glycoforms of histone P39 are not theoretically sugar-modified types of N-.

The Analysis of Immunogenicity of 4 recombinant glycosylated albumen P39 of embodiment

The present embodiment carries out Evaluation of Immunogenicity to the recombinant glycoprotein of Pichia pastoris secreting, expressing after purification, compares recombination Rear sugar-modified P39 albumen and the immunogenicity for not carrying out glycosylation modified protokaryon P39 albumen.Analyze recombinant glycosylated albumen P39 is on the reactive influence of animal immune, using mice spleen lymphocytes proliferation index as judgment criteria.After detection is 8 weeks immune The serum IgG antibody levels variation of mouse is measured, mice serum antibody change is observed, is swallowed in conjunction with macrophage Experimental analysis protein immunogenic.

1, experimental animal

8 week old female Balb/c mouse.

2, reagent

75% alcohol, FITC, it is splenic lymphocytes separating liquid, RPMI-1640 cell culture fluids up to section, it is FBS (BI), dual anti- Corning, EILSA cell factor (IL-2) detection kit, EILSA cell factors (IL-4) detection kit, EILSA cells It is that biotechnology is limited that the factor (IL-10) detection kit, EILSA cell factors (IFN-γ) detection kit, which are purchased from up to section, Company；GoatAnti-Mouse IgG are purchased from the Beijing HRP bio tech ltd Quan Shijin, and tetrazole indigo plant is public purchased from sigma Department

3, the preparation of reagent

(1) coating buffer：The antibody of anti-igg is diluted to its a concentration of 1 μ g/ml (pH7.4) with PBS.

(2) cleaning solution PBST：To addition lmL Tween20 in 1000mL PBS (0.01mol/L, pH7.4).

(3) Incubating Solution：The BSA of 0.1g is added into 100mLPBST, 4 DEG C save backup.

4, method

(1) animal immune and grouping

8 week old Balb/c mouse, it is respectively P39 eukaryons, P39 protokaryons, PBS to be divided into nine groups, every group 5.One is immunized weekly Secondary, every mouse immunizing dose is 50 μ g every time, is immunized 4 times altogether.First immunisation protein solution and isometric Freund's complete adjuvant Mixing, after twice immune protein mixed with isometric incomplete Freund's adjuvant.Third time is immune to take spleen separation spleen leaching after a week Bar cell.

(2) preparation of mouse spleen lymphocyte

The immune mouse after a week of third time, takes off neck and puts to death, and 75% alcohol impregnates 5min, and spleen is taken under aseptic condition.In 50mm 4ml mouse lymphocyte separating liquids are added in culture dish, one layer of 200 mesh nylon screen of lid is lived on spleen sieve with syringe The careful grinding spleen of plug, until being completely dissolved in liquid.It draws splenocyte suspension and is transferred to 15mL centrifuge tubes, and be slowly added into 500 μ l RPMI-1640 culture solutions (contain 5% serum), and room temperature 1200r/min centrifuges 10min.Gentle aspiration 800 at the top of from liquid level μ L liquid is transferred to another 15mL centrifuge tube, and 10mL RPMI-1640 culture solutions are added, and 1000r/min centrifuges 5min, abandons Clearly.4mL erythrocyte cracked liquids are added, gently mixing, is stored at room temperature 5min, and erythroclasis is made to crack, 1000r/min centrifugations 5min abandons supernatant.10mL RPMI-1640 culture solutions are added to rinse, 1000r/min centrifuges 5min, abandons supernatant, is repeated 2 times.Most Cell is resuspended in 2mL RPMI-1640 culture solutions afterwards, is counted with cell counting board, and adjust cell concentration to 5 × 10⁶A/mL, -80 DEG C save backup.

(3) mtt assay detection spleen lymphocyte proliferation experiment

Cell concentration is adjusted to 2 × 10⁶A/ml.100 μ l are taken to be added in 96 well culture plates, 2 × 10⁵A cells/well, every group If 3 holes.It is respectively (10 μ g/ml, 1 μ g/ml, 0.1 μ g/ml, PBS) that final concentration, which is added, and PBS stimulations are negative control group.Blank Control group is the hole that RPMI-1640 culture mediums are added and are incubated.Final concentration of (10 μ g/ml, 1 μ g/ml, 0.1 μ g/ is added in experimental group Ml) various corresponding recombinant protein antigens and protokaryon proteantigen.37 DEG C, 5%CO₂Cell incubator culture 12h.20 μ are added LMTT reagents continue to measure OD values at 490nm wavelength after cultivating 4h.Lymphocytic proliferation rate is calculated according to the OD values of measurement. Formula is as follows.IS=(OD-OD1640)/(ODPBS-OD1640).

As a result as shown in figure 11, recombinant glycosylated P39 pairs of the albumen of spleen lymphocyte proliferation case study is detected by mtt assay The influence of mice spleen lymphocytes proliferation finds that recombinant glycosylated albumen P39 can remarkably promote the proliferation of mouse spleen lymphocyte, The effect of its promoting lymphocyte proliferation is 30 times or so of protokaryon albumen P39, illustrate it is glycosylation modified after recombinant protein P39 its Immunogenicity is apparently higher than unmodified protokaryon albumen P39.

(4) supernatants factors check

With the various protein immunization mouse of dosage, 4th week takes spleen, is separately cultured splenic lymphocytes.Adjust concentration of cell suspension It is 2 × 10⁶A/ml spreads 96 orifice plates, per 100 μ l of hole, corresponding albumen is added to final concentration of 10 μ g/ml, at 37 DEG C, 5%CO₂ Under conditions of cultivate 12 hours.Cells and supernatant is drawn, certain Ploidy testing mouse primary splenic lymphocytes secretion is diluted Cell factor IFN-γ, IL-4IL-2, IL-10.

Use the content for detecting cell factor in spleen cell cultures supernatant for EILSA cytokine detection kits up to section. It is as follows：The cytokine standards liquid diluted is added to standard sample wells in 100 holes μ l/, and dilution is added in 100 holes μ l/ Good sample to sample well, the holes 100l/ is added dilution matter blank well and compares.The antibody of biotin labeling is added in 50 holes μ l/ Working solution (1:100 dilutions), after mixing, cover sealing plate film, 37 DEG C are incubated 90 minutes.300 holes μ l/ of liquid addition in plate is deducted to wash Liquid is washed, liquid in plate is deducted after 1 minute, is repeated 4 times, is patted dry on filter paper every time.Streptavidin- is added in 100 holes μ l/ HRP working solutions.Sealing plate film is covered, 37 DEG C are incubated 30 minutes.Washing such as above-mentioned washing step.TMB is added in 100 holes μ l/, and 37 DEG C are kept away Light is incubated 15 minutes, until shade can debate in hole, terminate liquid is added immediately and terminates reaction.Absorption value is detected at 450nm.Reference Standard curve calculates the concentration of the cells in sample factor, as a result as shown in figures 13-16.

(5) FITC labelled proteins macrophage phagocytosis experiment

1) fluorescent marker protein

FITC is dissolved with 20 μ l dimethyl sulfoxide (DMSO)s first, is diluted with the carbonate buffer solution of PH 9.0 after dissolving, It is diluted to the final concentration of 1mmol/L of FITC；

The FTTC diluted is mixed with recombinant protein P39, by FITC:Recombinant protein P39=10:1 is mixed, Low-temperature dark vibrations reaction 4h after mixing；

It is dialysed with 9.0 carbonate buffer solutions of PH, dialyzate is replaced according to the degree of dialysis, until nothing in dialyzate Free FITC, dialyzate is close to colourless at this time.Sample placement is kept in dark place at low temperature after dialysis.

2) macrophage swallows fluorescent marker protein

It is cultivated after RAW264.7 is recovered, needed for culture to experiment, by cell with 8 × 10⁴The concentration in a/hole is inoculated with It to 24 orifice plates, waits for that cell convergence degree in 24 orifice plates reaches 80% or so, the sugared egg that FITC has been marked is added according to various concentration In vain, the protein sample after label is added according to the concentration of 0.1 μ g/ml, 1 μ g/ml, 10 μ g/ml respectively, each concentration corresponds to respectively 4 phagocytosis times, respectively 2h, 4h, 6h, 8h swallow fluorescence mark at corresponding time point with fluorescence microscope cell respectively The case where remembering albumen.

By carrying out glycosylation modified acquisition recombinant glycosylated albumen P39, a concentration of 5 μ to P39 albumen in Pichia pastoris The recombinant glycosylated albumen P39 of g/ml acts on macrophage, when the phagocytosis time is 4h, it is found that macrophage makees the phagocytosis of albumen With apparent, macrophage is significantly higher than protokaryon albumen P39 to the phagocytosis of recombinant glycosylated albumen P39.

(6) the horizontal variation of Salmonella detection immune serum antibody (IgG)

Animal immune and grouping：8 week old Balb/c mouse, it is respectively eukaryon group, protokaryon group, PBS groups to be divided into 3 groups, every group 6 Only.Interval is immunized once for two weeks, and every mouse immunizing dose is 50 μ g every time, is immunized 3 times altogether.After immune 2nd week, weekly Primary docking blood sampling is carried out, serum is detached, the blood plasma obtained every time first places 2h at ambient temperature, waits for that faint yellow serum is precipitated When, serum is sucked out in 4000rpm low-speed centrifugal 10min, and -80 DEG C save backup.

Using the albumen of purifying as 96 orifice plate of antigen coat (100ul coating buffers, 5 μ g/ml of antigen final concentration, 4 DEG C overnight).It adopts It is washed 5 times with 200 μ l PBST buffer solutions, each 1min is patted dry after clean filter paper each time after washing.It will be immunized with Incubating Solution The mice serum of corresponding antigens presses 1:1000 doubling dilutions are added in 96 orifice plates, and the serum of PBS groups is immunized as negative control, no It is blank control to add the incubation fluid apertures of any serum.3 multiple holes are arranged in the detection of each serum antibody.Per 100 μ l of hole, 37 DEG C incubate Educate 1h.Then 5 times are washed with PBST, ibid washing step.Add 100 μ l 1 per hole:The goat anti-mouse of 1000 diluted ALP labels IgG, 37 DEG C incubation 1h.PBST washes film 5 times, with above-mentioned washing step.Add 100 μ l p-nitrophenyl- per hole Phosphate substrates, often hole adds 50 μ l stop solution solution to terminate reaction to avoid light place 30min. at room temperature, stands 5min measures OD values at 405nm.

As a result as shown in figs. 13-17, found by detecting the IgG situations of change that protein immunization mouse generates, when immune small Recombinant glycosylated albumen P39 can detect the antibody I Gg of higher level in immune mouse the 6th week when mouse 4th week, compare From the point of view of relatively, eukaryotic protein group antibody expression reaches peak value in 5 Zhou Shineng.The stimulation of eukaryotic protein group generates cell factor IL- 10 is horizontal higher than protokaryon protein groups, illustrates that recombinant glycosylated albumen P39 can improve Th1 type cellular immune levels.

Conclusion：Can significantly it be promoted by P39 albumen of the influence discovery to mice spleen lymphocytes proliferation after glycosylation modified Into lymphopoiesis, the mouse antibodies by detecting immune glycosylation albumen are horizontal and cytokine levels find recombination sugar Base albumen P39 can effectively facilitate the level of cellular immunity and humoral immunity, pass through the immune protective efficiency to glycosylating albumen Research finds the sugar-modified immune protective efficiency that can improve P39 albumen, these are all recombinant glycosylated albumen P39 as brucella Vaccine candidate antigen has established experiment basis.

Sequence table

<110>Jilin Agriculture University

<120>Recombinant glycosylated albumen P39 and its preparation method and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

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gaattcatgg cacctgttgc caatgc 26

<210> 2

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 2

gcggccgttt tgcggcttca accgcc 26

Claims (10)

1. a kind of preparation method of recombinant glycosylated albumen P39, which is characterized in that include the following steps：

Step 1: the structure of carrier for expression of eukaryon pPICZ α A-P39

P39 genes are cloned, the segment after PCR is connected with cloning vector pMD-19T (Simple), through sequencing and digestion After verification is correct, the target fragment obtained after double digestion is connected structure with the carrier for expression of eukaryon pPICZ α A equally through double digestion Build recombinant expression plasmid pPICZ α A-P39；

Step 2: expression, purifying and fermentation of the P39 albumen in Pichia pastoris GS115

The carrier for expression of eukaryon pPICZ α A-P39 built are converted using electrotransformation thin to Pichia pastoris GS115 competence In born of the same parents, the induced expression of albumen is carried out using methanol, destination protein is purified using affinity chromatography method, and utilize molecule Sieve further isolates and purifies destination protein, and is verified to the albumen of purifying using westernblot, by optimizing single factor test Experiment obtains optimal conditions of fermentation and is：PH value is 6, phosphate concn 0.02M, methanol induction are a concentration of 1%, induction 72h, pure A concentration of 5mg/ml of recombinant protein P39 after change.

2. the preparation method of recombinant glycosylated albumen P39 according to claim 1, which is characterized in that in step 2, eukaryon Expression vector pPICZ α A-P39 convert as follows to the detailed process in Pichia pastoris GS115 competent cell：

1, the extraction of recombinant expression plasmid pPICZ α A-P39

The single bacterium colony of picking culture is inoculated in 5mL LB liquid mediums, 37 DEG C, and 200rpm shake cultures expand after overnight Overnight incubation：

(1) 40ml bacterium solutions are collected to be put into 50ml centrifuge tubes, 10000rpm, centrifuge 3min and discards supernatant after more collections several times, It is upside down on filter paper；

(2) 10mlP1 solution is added into the 50ml centrifuge tubes for have precipitation, while 50ul TIANRED reagents are added, utilizes liquid relief Rifle blows and beats mixing or with oscillator mixing；

(3) 10mlP2 solution is added into centrifuge tube, mild spins upside down 6-8 times, makes cellular lysate, is placed at room temperature for 5min；

(4) 10mlP4 solution is added into centrifuge tube, mild spins upside down 6-8 times, until solution appearance white dispersion is cotton-shaped heavy It forms sediment, is placed at room temperature for 10min, 10000rpm centrifuges 5-10min；

(5) complete soln is carefully poured into filter, filters, provides 50ml centrifuge tubes for oneself；

(6) the 5M Nacl solution of the isopropanol and 1/2 isopropanol volume of 0.35 times of filtrate volume is added into filtrate, runs up and down Mixing；

(7) under the conditions of 4 DEG C, 10000rpm centrifuges 30min, outwells supernatant, is upside down on filter paper and is absorbed water；

(8) ethyl alcohol that 6ml 70% is added into pipe fully rinses precipitation, and 4 DEG C, 10000rpm centrifuges 10min, outwells supernatant, will It is upside down on filter paper；

(9) the 7th step is repeated；

(10) centrifuge tube opening is placed on indoor 10-20min, 1-1.5mlTB buffer dissolvings is added after so that ethyl alcohol is volatilized, Nano Drop detect plasmid concentration；

2, the linearisation of recombinant expression plasmid pPICZ α A-P39

The recombinant expression plasmid pPICZ α A-P39 of extraction are linearized using SalI, reaction system is as follows：20 μ l of plasmid, 5 μ l of SalI restriction enzymes, 5 bufferH μ l, ddH₂20 μ l of O, totally 20 samples；37 DEG C of water-bath 3h；5 μ l are taken to carry out core Whether sour electrophoresis detection digestion is complete, and non-digested plasmid compares；

3, the plasmid of alcohol precipitation method recycling linearisation

It draws in the 500ul plasmids to the centrifuge tube of 1.5ml after digestion, isometric phenol, chloroform, isoamyl alcohol is added, uniformly It mixes, under the conditions of 4 DEG C, 12000rpm centrifuges 10min；Upper layer supernatant is taken, the absolute ethyl alcohol and 1/10 volume of 2 times of volumes is added 5M Nacl solution, static 2h under the conditions of -20 DEG C；Under the conditions of 4 DEG C, 13000rpm centrifuges 10min, discards supernatant；With 75% Ethyl alcohol washing precipitation, 12000rpm centrifuge 5min；Naturally dry is volatilized completely to ethyl alcohol, 10ul TB buffer back dissolvings, is sunk It is linearization plasmid to form sediment, and Nano Drop detect nucleic acid concentration；

4, electrotransformation Pichia pastoris GS115 competent cell

It takes plasmids of the 10ul after SalI is linearized that the Pichia pastoris GS115 competent cell of 100ul is added, is added after mixing In the electric shock cup of 0.1cm precoolings, ice bath 5min is 1.8kV in voltage, and carrying out electricity under conditions of the time 4-5ms that shocks by electricity turns, and electricity turns 1M sorbierite 1ml are added afterwards, transfers them in sterilized 2ml centrifuge tubes, 30 DEG C, 2h is incubated under conditions of 250rpm；It takes 200ul carries out coated plate, the zeocin resistant panels of various concentration.

3. the preparation method of recombinant glycosylated albumen P39 according to claim 2, which is characterized in that in step 2, recombination The detailed process of the induced expression of albumen P39 is as follows：

1, the single bacterium colony grown in zeocin resistant panels is chosen into YPD fluid nutrient mediums and is cultivated；1ml is taken to be incubated overnight Object 8000rpm centrifuges 5min, discards supernatant liquid；The PBS solution that 500ul is added into precipitation fully suspends, 8000rpm centrifugations 5min discards supernatant liquid；It is suspended and is precipitated with the TE buffer of 100ul, boiling water bath 10min；Freeze with -80 DEG C under the conditions of 30min；Boiling water bath 10min again, 8000rpm centrifugation 5min collect supernatant and take 1ul as pcr template, carry out PCR amplification, PCR Condition is：94℃3min；94 DEG C of 30s, 60 DEG C, 72 DEG C of 90s (35 cycles)；72℃10min.

2, the extraction of Yeast genome

Biomass is detected using spectrophotometer or plating method, when OD600 values are 1.0, i.e. 1-2 × 10⁷Cells/ml is carried Take pastoris genomic dna；

3, picking is grown in the positive single bacterium colony on Zeocin resistance YPD tablets, is inoculated in the BMGY fluid nutrient mediums of 5ml, It is cultivated in 50ml triangular flasks, 28 DEG C, 250r/min shaking table culture 16-20h, until OD600 values are about 2.0-4.0, yeast at this time Bacterium is in exponential phase, and the methanol for being added 1% carries out induced expression；The GS115 bacterial strains of electrotransformation empty carrier pPICZ α A simultaneously As negative control, sample, 10000r/min are collected in culture after a certain period of time, and 4 DEG C of centrifugation 2min take supernatant to carry out SDS-PAGE As a result the expression for analyzing albumen is shown in the purpose egg that the size of successful expression in Pichia pastoris GS115 is about 43kDa In vain.

4. the preparation method of recombinant glycosylated albumen P39 according to claim 3, which is characterized in that Yeast genome Specific extraction process is as follows：

(1) yeast cells is taken, most 5 × 10⁷Cells, 12000rpm centrifuge 1min, absorb supernatant；

(2) yeast cell wall is abolished：600 μ l sorbierite buffer are added into thalline, 50U Lyticase are added, it is fully mixed It is even；30 DEG C of processing 30min；4000rpm centrifuges 10min, abandons supernatant, collects precipitation；

(3) 200 μ l buffer solutions GA are added into precipitation, precipitation is resuspended, mix well, if necessary removal RNA, 4 μ l of addition, 100mg/mlRNaseA solution vibrates 15sec, is placed at room temperature for 5min；

(4) 20 μ l Proteinase K solution, mixing is added；

(5) 220 μ l buffer solution GB are added, fully reverse mixing, 70 DEG C of placement 10min are centrifuged to remove the droplet of cap wall；

(6) add 220 μ l absolute ethyl alcohols, fully reverse mixing, it may appear that flocculent deposit is centrifuged to remove the droplet of cap wall；

(7) previous step acquired solution and flocculent deposit are all added in an adsorption column CB3,12000rpm centrifuges 30sec, outwells Adsorption column CB3 is put into collecting pipe by waste liquid；

(8) 500 μ l buffer solutions GD, 12000rpm centrifugation 30sec are added into adsorption column CB3, waste liquid are outwelled, by adsorption column CB3 It is put into collecting pipe；

(9) 600 μ l rinsing liquids PW, 12000rpm centrifugation 30sec are added into adsorption column CB3, waste liquid are outwelled, by adsorption column CB3 It is put into collecting pipe；

(10) repetitive operation step (9)；

(11) adsorption column CB3 is put back in collecting pipe, 12000rpm centrifuges 2min, outwells waste liquid, adsorption column CB3 is placed in room temperature It places several minutes, to dry rinsing liquid remaining in sorbing material；

(12) adsorption column CB3 is transferred in centrifuge tube, 50-200 μ l elution buffers is vacantly added dropwise to the intermediate position of adsorbed film TE, is placed at room temperature for 2-5min, and 12000rpm centrifuges 2min, solution is collected into centrifuge tube.

5. the preparation method of recombinant glycosylated albumen P39 according to claim 1, which is characterized in that in step 2, recombination The detailed process of purifying and the identification of albumen P39 is as follows：

1, ammonium sulfate precipitation recombinant protein

It collects in 400ml bacterium solutions to 500ml centrifuge tubes, 1000rpm centrifugation 10min, collection supernatant, under the conditions of 4 DEG C, 70% sulphur Sour ammonia-sinking shallow lake destination protein, pulverulence is ground to by ammonium sulfate, is stirred to dissolve with glass bar when being added, and 4 DEG C are overnight, Precipitation is collected by centrifugation again；10ml, 10mMPBS back dissolving precipitate, and the bag filter of 3000 molecular weight is used in combination to carry out desalination to back dissolving liquid Processing；

2, Ni columns affinitive layer purification recombinant protein；

3, as a result Sephacryl S-100 purification of recombinant proteins P39 is shown in Pichia pastoris GS115 successful expression size For the recombinant protein P39 of 43kDa；

4, identify that the destination protein is recombinant protein P39 through immunoblot experiment.

6. the preparation method of recombinant glycosylated albumen P39 according to claim 1, which is characterized in that reversed using HPLC High-efficient liquid phase technique detects recombinant protein purity, the results show that the purity of recombinant protein P39 after purification is 93%.

7. the recombinant glycosylated albumen P39 that the preparation method as described in any one of claim 1 to 6 obtains.

8. applications of the recombinant glycosylated albumen P39 in preparing brucella vaccine and immunologic adjuvant as claimed in claim 7.

9. applications of the recombinant glycosylated albumen P39 in preparing immunologic adjuvant as claimed in claim 7.

10. recombinant glycosylated albumen P39 as claimed in claim 7 is preparing the application in having Immunogenic agents.