CN100340656C - High-efficiency expression in Pichia yeast, fermentation and purification of human gamma-interferon - Google Patents

Abstract

The present invention designs and artificially synthesizes a people gamma-interferon gene sequence which is especially suitable for being expressed in pichia pastoris. A carrier pPIC 3.5K-ifn for expressing human gamma-interferon protein in a cell is constructed, SMD1163 Pichia pastoris with a proteinase defect is adopted as parasitifer pastoris, a multi-copy clone is sieved by a G418 resistance concentration incremental method, and then a high expression clone strain Pichia pastoris SMD 1163 (pPIC 3.5k-ifn, his 4 pep 4 delta prbl)-181 is obtained. The pastoris is cultivated for 48 hours in a cultivation medium without methanol at 28 to 30 DEG C and then is cultivated for 36 to 48 hours in an induced cultivation medium with 1 % of methanol, and the expression quantity of gamma-interferon can reach 25% of the quantity of total protein of the pastoris body; about 1 g of a gamma-interferon primary product per litre is contained. Human gamma-interferon protein of which the purity is more than 97.5 % (HPLC) is obtained by EAE-Sepharose gel adsorption, Sephacryl S-200 molecular sieve chromatography, DEAE-Sepharose anion exchange chromatography, and CM-Sepharose cation exchange chromatography.

Description

Human gama-interferon efficiently expressing in Pichia yeast, fermentation and purifying

Technical field

The invention belongs to gene engineering technology field, more particularly relate to a kind of with the synthetic human gama-interferon of DNA recombinant technology, contain the recombinant vectors of this gene, with this carrier host transformed and the fermentation and the purification process that utilize transformant generation human gama-interferon.

Background technology

Gamma-interferon (IFN-γ) is that wheelock found in nineteen sixty-five, claims the immunologic pattern Interferon, rabbit again.It has physiological actions such as antiviral, antitumor, immunomodulatory, has a wide range of applications clinically.Natural human IFN-γ stimulates the T lymphocyte to produce by mitogen, intracellular toxin or exotic antigen, and the very low and purification difficult of content is difficult to meet clinical needs.The people IFN-γ that uses clinically adopts the DNA recombinant technology to produce at present.

Recombinant human gamma-interferon prior art is done the host bacterium by intestinal bacteria (Escherichia coli.) and is expressed with prokaryotic expression system.The Liu Xin of Shanghai Inst. of Biochemistry, Chinese Academy of Sciences wall etc. 1994 with synthetic human gama-interferon cDNA the expression in escherichia coli human gama-interferon (see Chinese science (B collects), 24 (10), 1076-1084,1994; With Chinese patent 94112091).The Hou Yun of Virology Inst., Chinese Academy of Preventive Medical Science moral and Shanghai Biological Products Inst., Ministry of Public Health also adopt the hybrid plasmid pBV220 transfection Escherichia coli DH5 α strain that has the human, set up into expression engineering bacteria (viral journal, 4,97,1988).The human gama-interferon that above-mentioned two expression strains are expressed all is to exist with the inclusion body form at first, and it is water insoluble promptly to express product, and human gama-interferon albumen is the non-natural folded conformation, need through complex steps purification process such as renaturing inclusion bodies (viral journal, 13,134-139,1997; The microorganism journal, 33 (4), 248-254,1993; The biotechnology journal, 14 (2), 203-207,1998).Because the limitation of prokaryotic expression system and the complicacy of renaturing inclusion bodies and purification process, cause the human gama-interferon C end heterogeneity disappearance of expression sometimes, lose 13,15,18 amino-acid residues respectively, make the human gama-interferon primary structure of gene engineering expression and natural human gama-interferon primary structure that very big difference (biotechnology journal be arranged, 10 (1), 50-55,1994).For overcoming the deficiency of prior art existence, it is that the host efficiently expresses human gama-interferon and ferments and the novel technical method of purifying with pichia spp (Pichiapastoris) that design is sought a kind of, has certain theoretical significance and important application.

Summary of the invention

The objective of the invention is to seek that a kind of human efficiently expresses in Pichia yeast (P.pastoris), the novel technical method of fermentation and purifying, to overcome the deficiency that prior art exists.Human gama-interferon suitability for industrialized production bacterial strain on the domestic and international market all adopts intestinal bacteria as host expresses at present.The present invention adopts the suitability for industrialized production bacterial strain of methanol evoked yeast as human gama-interferon.Because be interior expression of born of the same parents of eucaryon yeast cell, so the gamma-interferon of natural generation is in full accord in proteic primary structure of human gama-interferon that expression obtains and secondary structure and the human body, the gamma-interferon of expression is water-soluble fully, does not contain intracellular toxin.

The production that makes up among the present invention bacterial classification [Pichiapastoris SMD1163 (pPIC3.5K-ifn of the Pichia yeast of expressing human gamma-interferon, his4 pep4 Δ prb1)-181] preserving number is CGMCCNO.0758, date saved: on 07 03rd, 2002, address: BeiJing ZhongGuanCun China Committee for Culture Collection of Microorganisms common micro-organisms center, postcode 100080.

When the design human, the 3rd base of some amino acid whose triplet codon changed, be the codon that methanol yeast is liked.Can not change the proteinic amino-acid sequence of human gama-interferon like this, but can improve the expression efficiency of gamma-interferon in methanol yeast.The codon-bias statistics of Pichia pastoris has had relevant report, according to this codon bias cartogram a small amount of rare password that exists in the known array being carried out point mutation to realize the high expression level of gene, is a kind of method of effective realization high expression level.But when there being more rare password during the complete synthesis gene of needs, only adopt the preference password may make synthetic gene phenomenons such as the secondary structure of G+C% imbalance, non-expectation and aminoacyl-tRNA be crowded occur, net result may can not get higher expression amount on the contrary.We design synthetic IFN-γ gene in accordance with the following methods to reject the rare codon in the gene in the present invention:

1. by online data retrieval, from Http: //www.kazusa.or.jp/codonThe place obtains the codon usage frequency table of P.pastoris.This table has been added up 39 sub-service conditions of proteinic amino acid code (table slightly) among the P.pastoris altogether.

2. the P.pastoris codon usage frequency table of above acquisition is suitably revised the back as parameter input CODOP biosoftware, and, natural human gamma-interferon gene order is redesigned based on human gama-interferon aminoacid sequence (Fig. 1).Codon significant figure (Effective number ofcodons wherein, Nc) threshold value setting is 0.7, and is worth with this and judges rare password (annotate: the major function of CODOP biosoftware is automatically rare password to be replaced in proportion at random the same amino acid whose non-rare password of coding).

3. adopt Biosuite VectorNTI biosoftware to make the restriction mapping of primary design gene, and inner Sac I, PshA I, Sal I, Bgl II, BamH I, Avr II, Xho I, the Xba I restriction enzyme site that may exist of manual removal, with convenient follow-up genetic manipulation.

4. according to the tumor-necrosis factor glycoproteins of (G+C) %, gene inside with whether there is mRNA splicing site (Fig. 3) etc., combining cipher uses table, the further manual gene order of revising design.

5. draw 24 primer sequence (see figure 2)s of required synthetic according to the complete synthesis scheme of determining of PCR method, the modification by gene order makes the fusing point of all primers all more than 50 ℃.

6. add suitable restriction enzyme site as required to make things convenient for genetic manipulation at the gene order two ends of design.

7. to the gamma-interferon gene machine check as calculated of design, gets rid of unnecessary secondary structure, especially will get rid of the 5 ' secondary structure of holding, in order to avoid influence the interpretative function of mRNA.

IFN-γ gene (G+C) % that obtains according to above principle redesign is 40.37; Inner potential mRNA split-join model of gene and tumor-necrosis factor glycoproteins are removed; Codon uses and obtains reasonable balance; The secondary structure of having got rid of the interpretative function that influences mRNA has especially been got rid of the 5 ' secondary structure of holding.Fusing point usefulness [4 * C+G+2 * A+T] calculating of 4 primer (see figure 2)s of required Synthetic 2 all between 52-60 ℃, adopts [81.5+0.41 * (G+C/ (A+C+G+T) * 100)-675/ (A+C+G+T)] algorithm all between 60-70 ℃.

The method that reference dna shuffles, we have finally obtained dna fragmentation (Fig. 4) about 500bp by a step PCR assembling and amplification.Because having adopted pfu polymeric enzyme reaction, the dna fragmentation that obtains is the flush end product, can directly the PCR product that obtains be packed in the pUC18 cloning vector of Sma I enzymolysis.After the blue hickie screening of X-Gal/IPTG, clone of picking delivers the order-checking of match hundred victory companies, and the IFN-gamma gene sequences of result and Design Theory is in full accord.

The pUC18-ifn plasmid that sequence verification is crossed is after amplification, extracting, with Avr II, EcoR I double digestion, agarose gel electrophoresis reclaims the pUC18-ifn gene fragment, then the carrier with the same pPIC 3.5K that reclaims with Avr II, EcoR I double digestion and through agarose gel electrophoresis is connected, and obtains pPIC 3.5K expression vector with ammonia benzyl resistance screening.This pP1C 3.5K-ifn plasmid DNA with Bgl II linearization for enzyme restriction after agarose gel electrophoresis separate and reclaim the fragment that wherein contains human's expression cassette, this expression cassette fragment is used for electric shock transforms.PPIC3.5K empty plasmid with Bgl II linearization for enzyme restriction transforms contrast as electric shock simultaneously.

Electric shock conversion and colony screening method are with reference to Invitrogen pichia spp laboratory manual, and wherein host cell adopts the two scarce bacterial strain Pichia pastoris SMD1163 of proteolytic enzyme.4 RDB flat boards of cell coating after electric shock transforms, host cell Pichia pastoris SMD1163 is a histidine defect, expression vector contains the gene of encoding histidine, utilizes its complementary effect to filter out the Histidine positive colony.Respectively add in 2ml sterile distilled water to the 4 RDB flat board, wash out the Histidine positive colony and merge it, each draws 200 μ l thalline liquid separate application in containing 1,2,4, on the G418YPD flat board of 6mg/ml, selecting the G418 resistance multiple copied clone on G418 concentration 4 and 6mg/ml two flat boards.

Above-mentioned multiple copied clone carries out a small amount of fermentation expression respectively, and its method is as follows: with the strain of sterilization toothpick picking mono-clonal, be inoculated in the test tube that contains an amount of YPD 30 ℃ of concussion overnight incubation.Get an amount of thalline and be seeded in the 50ml centrifuge tube that contains BMGY, cultivate concussion 16 hours for 28-30 ℃; Centrifugal collection thalline.In super clean bench, add a small amount of BMMY again, and change centrifugal lid into 4 layers of sterile gauze.Continue at 28-30 ℃ of concussion and add pure methanol induction after 24 hours; Cultivate again and add an amount of pure methyl alcohol after 24 hours; Continue again to cultivate after 24 hours, centrifugal collection thalline and supernatant, carry out in the SDS-PAGE electrophoresis detection born of the same parents respectively and fermented liquid supernatant in the protein expression situation, found that the specificity that exists in the born of the same parents about 17KD from band, and fermented liquid supernatant does not have almost the specific proteins band to occur.The cell strain that this explanation screening obtains is for expressing in the born of the same parents.

Select on above-mentioned G418 concentration 4 and 6mg/ml two flat boards height copy clone totally 511 strains carry out fermentation expression and centrifugal collection thalline by above-mentioned a small amount of fermentation process respectively; Thalline carries out ultrasonication after with the Zymolase enzymic digestion again, and the direct point sample of supernatant liquor after centrifugal carries out immunoblotting and SDS-PAGE electrophoresis experiment to nitrocellulose membrane, detect expressing protein in the born of the same parents; The interior homogeneous of bacterial strain energy born of the same parents that wherein is numbered pPIC3.5K-ifn-181 is expressed the IFN-γ target protein of 17KD, and expression amount accounts for about 25% of yeast body protein.Preserve the pPIC3.5K-ifn-181 bacterial strain standby.

With the above-mentioned bacterial strain called after Pichia pastoris SMD1163 (pPIC3.5K-ifn that is numbered pPIC3.5K-ifn-181, his4 pep4 Δ prb1)-181, and carry out the bulk fermentation of industrially scalable, its method is as follows: seed liquor is inoculated in 30 ℃ of incubation growth in the YPB substratum.To contain a certain amount of (NH ₄) ₂SO ₄,, KCl, MgSO ₄, CaSO ₄, NaCl solution adds in the fermentor tank autoclaving.A certain amount of glucose, hexamethyl phosphoric acid ester sodium and the micro-mother liquor of adding during to 29 ℃ to be cooled regulated fermented liquid pH to 5 with ammoniacal liquor and phosphoric acid.Seed liquor is seeded in the fermentor tank, under the dissolved oxygen condition of 29-30 ℃ of temperature, 300-800rpm rotating speed and 30%, cultivated 24-36 hour.Add glucose and (NH subsequently ₄) ₂SO ₄Solution and trace element, this stage cultivated 24 hours altogether; Add glucose and pure methyl alcohol at last, this stage cultivated 36-48 hour altogether.Stop to cultivate centrifugal collection thalline.

The used synthetic gamma-interferon of barms of the present invention is to utilize type gene A OX1 gene to be regulated and control by the entrained methyl alcohol of this yeast, owing to being subjected to the concentration of methyl alcohol, this gene product influences, control the synthetic of gamma-interferon so can utilize the concentration of methyl alcohol, can be earlier 30 ℃ of grown cultures of carrying out the fs, and then the expression of carrying out subordinate phase in the substratum that contains finite concentration methyl alcohol is cultivated, adopt the training strategy in two stages, obtain efficiently expressing of human gama-interferon easily.The fermentation back is centrifugal, collects thalline; Through adding distil water suspension back centrifugal operation steps, washing thalline secondary.Carrying out ultrasonic bacteria breaking behind the adding pickling glass pearl mixing, the centrifuging and taking supernatant liquor, this supernatant liquor is by Sepharose DEAE anionite-exchange resin, the gamma-interferon albumen of expressing is attracted on the resin, with damping fluid (0.8M NaCl, 0.1M PB pH8) elutes the gamma-interferon crude product that is adsorbed.The gamma-interferon sample that wash-out obtains carries out the molecular sieve gel filtration chromatography through the dialysis desalination with Sephacry1 S-200, with damping fluid (0.1M NH ₄Ac, 0.05%Tween, pH 7.0) as moving phase separate the sample elution profile at 2 peaks, second peak is gamma-interferon sample peak, collects it.The sample of collecting is carried out DEAE Sepharose anion chromatography, and moving phase is 0.1M NH ₄Ac, 2.5% sucrose, the damping fluid of pH7.0.With this understanding, the gamma-interferon sample is not retained, and impurity is adsorbed reservation.Carry out cation-exchange chromatography with CM Sepharose resin again.Moving phase is by A liquid (20mM NH ₄Ac, 5% sucrose is pH8) with B liquid (20mM NH ₄Ac, 5% sucrose, 0.7M NaCl, pH8) form, with ionic strength incremental method wash-out, collect the 3rd sample peak, get the pure product of pure gamma-interferon.The pure product that obtain carry out titration, protein quantification, SDS-PAGE electrophoresis, high pressure liquid chromatographic analysis, residual DNA assay, isoelectric point determination, UV scanning, peptide figure analysis, n terminal amino acid mensuration and the test of immune seal stain, prove that with telling test the product of purifying is human gama-interferon more at last.

Description of drawings

Fig. 1. be adapted at the synthetic human's sequence and the amino acid sequence corresponding thereof that efficiently express in the Pichia yeast (Pichia pastoris).

Fig. 2. 24 Oligonucleolide primers sequences of synthetic.

Fig. 3. the shearing intron of pre-mRNA signal formula in the Pichia yeast (P.pastoris).

1% agarose gel electrophoresis analysis of Fig. 4 .PCR product.

Contain 20ng/ μ L primer mixture (24 Oligonucleolide primers) in the PCR mixture, 5 pfu of unit

The dNTP of archaeal dna polymerase and 300 μ mol/L forms; 1 and 2 for gamma-interferon PCR synthetic

Product; 3 is DL 2000DNA standard molecular weight.

Fig. 5. in Pichia yeast (P.pastoris), express the structure of the expression vector pPIC 3.5K-ifn of gamma-interferon.

Fig. 6. the nitrocellulose membrane immunoblotting assay (screening contains the Pichi strain of multiple copied pPIC3.5K-ifn) of expression product in the gamma-interferon born of the same parents.

The positive contrast of 1-3; 4 and 5 negative contrasts; Remaining is pPIC 3.5K-ifn male SMD1163 strain.

Fig. 7. express gamma-interferon product S DS-PAGE in methanol yeast bacterium Pichia pastoris SMD1163 (pPIC 3.5K-ifn, his4 pep4 Δ prb1)-181 fermentation born of the same parents and analyze.

1-3 is respectively and induces primary fermentation to cultivate broken bacterium sample supernatant after 24,36,48 hours;

4-8 is respectively methanol induction post-fermentation and culture 8,16,24,36, broken bacterium sample supernatant after 48 hours;

9 is standard molecular weight albumen.

Fig. 8. express gamma-interferon product S DS-PAGE in the methanol yeast bacterium Pichia pastoris SMD1163 of different batches (pPIC 3.5K-ifn, his4 pep4 Δ prb1)-181 fermentation born of the same parents and analyze.

1,2 for not inducing contrast; 3-6 is that 1% methanol induction post-fermentation and culture is broken bacterium sample supernatant after 36-48 hour;

7 is the protein standard molecular weight.

Fig. 9. the gamma-interferon sample SDS-PAGE behind the purifying analyzes (silver dyes).

A is the protein standard molecular weight; B, D, F are the gamma-interferon sample that reductive agent Mercapto base Ethanol Treatment is crossed, and C, E, G are the gamma-interferon sample that not reduction is handled.

Figure 10. the gamma-interferon sample molecule flow measurement behind the purifying.

Figure 11. the gamma-interferon sample HPLC purity check behind the purifying.

Figure 12. DNA residue analysis in the gamma-interferon sample behind the purifying.

A, B, E are the gamma-interferon sample; The positive contrast of C; The negative contrast of D.

Figure 13. the gamma-interferon sample isoelectric analysis behind the purifying.

A, B, C are gamma-interferon; D is the standard substance of known iso-electric point.

Figure 14. the gamma-interferon sample ultraviolet absorpting spectrum behind the purifying.

Figure 15. the gamma-interferon sample peptide figure electrophoretic analysis collection of illustrative plates behind the purifying.

A is the standard peptide molecular weight; B-D is a gamma-interferon.

Figure 16. the gamma-interferon sample peptide figure HPLC behind the purifying analyzes collection of illustrative plates.

Figure 17. the gamma-interferon N-terminal analytical sequence behind the purifying.

Figure 18. the gamma-interferon sample immunoblotting assay behind the purifying.

1-3 is all gamma-interferon.

Figure 19. the gamma-interferon sample monoclonal antibody neutralizing effect graph of a relation behind the purifying.

Figure 20. the gamma-interferon sample monoclonal antibody neutralizing effect 96 orifice plate baseline results behind the purifying.

The present invention has following advantages:

1. the gene order (Fig. 1) of the gamma interferon of design is particularly suitable for expressing in the Pichia yeast (P.pastoris), and the carrier of structure (Fig. 5) is integrated in the genome of Pichia yeast, and is very stable, is difficult for losing.

2. adopting the Pichia pastoris SMD1163 of Deficient In Extracellular Proteases be the host, adopts the carrier pPIC 3.5K-ifn of intracellular expression to do to express carrier (Fig. 5), the gamma interferon albumen of great expression is present in the born of the same parents and is difficult for degrading.

3. with the anti-type concentration of G418 incremental method screening multicopy clone, make human gamma-interferon protein expression amount account for 25% (Fig. 8) of total mycoprotein.

4. the fermented and cultured initial stage is adopted the medium culture that does not contain methyl alcohol, and late stage of culture adopts the inducing culture of 1% methyl alcohol to cultivate, and saccharomycetic growth and protein expression are separated effectively, and the gamma interferon expression can maintain higher level, and is difficult for degraded.

5. purification step is succinctly efficient, and the finished product endotoxin content is extremely low, is conducive to clinical use.

Embodiment

By the following example technical characterictic of the present invention is described in detail, but does not limit the present invention.

Embodiment 1: the human gama-interferon production structure of barms

One material and method

(1) experiment material

Pichia spp SMD1163, expression plasmid pPIC3.5K and G418 are Invitrogen company product.Cloning vector pUC18 is that our company preserves.

Restriction enzyme EcoRI, XhoI, XbaI, AvrII, BglII, SmaI and PshAI are TakaRa company product; Plasmid extraction test kit Wizard ^TMPlus Minipreps DNAPurification System and PCR product reclaim test kit Wizard ^TMPlus Minipreps PCRPurification System is available from Promega company: glue reclaims test kit available from Shanghai China Shun biotechnology company limited.

Yeast culture base YPD, BMGY, BMMY, RD, RDH, RDB, RDHB, YPD/G418, MD, MDH, MM and MMH, composition are all with Invitrogen pichia spp laboratory manual.How anti-for making product by oneself the anti-human gama-interferon of rabbit is.

Computer biosoftware CODOP is so kind as to give by Britain Liz doctor Carpenter (National Institutefor Medical Research).

(2) experimental technique

1 conventional genetic manipulation

Routine operation is basic with reference to relevant document.Plasmid extraction and PCR product reclaim and adopt the Promega related kit; Glue reclaims according to Shanghai China Shun product description.

2 IFN-γ gene total synthesis methods

Sequence at pichia pastoris phaff (Pichia pastoris) optimization design is divided 24 primers, entrusts Beijing hundred victory companies that match to synthesize, and 24 primers of synthetic are seen Fig. 2.

Wherein EcoR I, the Xho I, Avr II or the Xba I restriction enzyme site that contain of Oligo 1,12 and 13 is used for clone operations.Except that Oligo 13, other 23 dna fragmentations are 40bp entirely.Underscore is depicted as the beginning of IFN-γ gene coded sequence among the Oligo 1, and the TAG that adds frame among the Oligo 12 is a terminator sequence.Reference dna shuffling method, according to the full-length gene order (Fig. 1) of following steps pcr amplification IFN-γ:

24 oligonucleotide fragments of equivalent are mixed, form the primer pond.Then become the PCR reaction solution: primer pond 1.56 μ l (the about 20ng/ μ of DNA final concentration l) according to following mixed; 10 * pfu damping fluid, 5 μ l; Pfu polysaccharase 1 μ l; DNTP (10mmol/L each) 1.5 μ l; H ₂O 40.9 μ l.This reaction solution on Hybaid PCR Sprint amplification instrument prior to 94 ℃ of sex change 5min; 94 ℃ of 30sec then, 55 ℃ of 45sec, 72 ℃ of 30sec+3sec/ circulations; Carry out 45 pcr amplification circulations altogether.Extend the IFN-γ gene (Fig. 1) that 10min obtains total length at 72 ℃ at last.

The structure of 3 pPIC3.5K-ifn

Cloned plasmids pUC18-ifn the EcoR I and the Avr II double digestion that will contain synthetic IFN-γ gene, 1% agarose gel electrophoresis also reclaims the fragment that contains IFN-γ gene, then be connected with the pPIC3.5K that separates and reclaim with identical enzymes double zyme, ammonia benzyl resistance screening obtains pPIC3.5K-ifn (Fig. 5).

4 expression plasmids transform the P.pastoris cell

Electric shock conversion and colony screening method are with reference to Invitrogen pichia spp laboratory manual, and wherein host cell adopts the pichia spp SMD1163 of proteolytic enzyme defective.The pPIC3.5K-ifn plasmid DNA contains IFN-γ expression casette fragment with Bgl II elder generation's linearization for enzyme restriction and recovery, and the expression cassette fragment of this recovery is used for electric shock and transforms.PPIC3.5K empty plasmid with Bgl II enzymolysis transforms contrast as electric shock simultaneously.

5 IFN-γ expression methods

Transform the recombinant clone that the back obtains with pPIC3.5K-ifn plasmid electric shock, carry out IFN-γ and express; Its expression method is with reference to Invitrogen pichia spp laboratory manual.

6 immunoblottings and SDS-PAGE detection method

Immunoblotting is substantially with the Western-blot method, and only sample is directly put on power socket of the eye teacher Xiao acid tunica fibrosa, subsequent disposal again after the oven dry.Wherein gamma-interferon antibody adopts the anti-people of rabbit how anti-; Two anti-couplings are the horseradish peroxidase oxydase; Chromogenic substrate is an O-Phenylene Diamine.

7 P.pastoris genome DNA extraction methods

P.pastoris reconstitution cell inoculation 3ml YPD, 30 ℃ of shaking tables are cultivated OD ₆₀₀Between about 5-8: the centrifugal collection thalline of room temperature is in 1.5ml Eppendorf pipe; With the SCED solution suspension thalline of using 0.5ml after the aqua sterilisa washing once again, and add Zymolase and the 10 μ l 2 mercapto ethanols that 40 μ l concentration are 1mg/ml: the centrifugal supernatant that goes behind the 1h is handled in 37 ℃ of 200r/min concussions, sedimentary thalline suspends again with 0.5ml Tris/EDTA (containing 50mmol/L Tris-HCl, 20mmol/L EDTA) and adds 50 μ l 10%SDS; Solution becomes gets thickness behind 65 ℃ of processing 20min, add the KAc of 200 μ l5M this moment, be inverted and put 30-60min on ice behind the mixing: then room temperature is centrifugal and supernatant transferred in the new Eppendorf pipe, adds the dehydrated alcohol (visible thread DNA precipitates) of about 1ml; Centrifugal 10 seconds of room temperature goes to be inverted the dried DNA precipitation of filter behind the supernatant.The DNA of gained dissolves again with 300 μ l TE and adds 50 μ l RAase A.37 ℃ of insulation 1h are with degraded RNA wherein.Add the 0.5ml Virahol at last, DNA precipitation is separated out the back and is chosen with the toothpick of sterilization and insert in the new Eppendorf pipe, adds after 100 μ l TE dissolve, and is used for pcr amplification IFN-γ expression cassette.

Two experimental results

(1) IFN-γ gene design

When design gamma-interferon gene, the 3rd base of some amino acid whose triplet codon changed, be (being that frequency of utilization is high in the methanol yeast bacterium) codon that yeast is had a preference for.Proteinic amino-acid sequence can be do not changed like this, but the expression efficiency of gamma-interferon in methanol yeast can be improved.The main purpose that changes is in order to improve expression efficiency.Another purpose that changes the 3rd base of triplet codon is to wish to increase some restriction enzyme sites, so that be easy to cut out some fragment in the future, the protein engineering transformation is carried out in correct, with the structure-function relationship of research gamma-interferon.The restriction enzyme site such as the table 1 of synthetic gamma-interferon gene (Fig. 1).

The gamma-interferon gene that designs also will be got rid of unnecessary secondary structure with calculating machine check, especially will get rid of the secondary structure of 5 ' end, in order to avoid influence the interpretative function of mRNA.The codon-bias statistics of P.pastoris has had relevant report, according to this codon bias table a small amount of rare password that exists in the known array is carried out point mutation to realize the high expression level of gene, and this is a kind of feasible method.But when there being more rare password during the complete synthesis gene of needs, only adopt the preference password may make synthetic gene phenomenons such as the secondary structure of G+C% imbalance, non-expectation and aminoacyl-tRNA be crowded occur, net result may can not get higher expression amount on the contrary.We design synthetic IFN-γ gene in accordance with the following methods to reject the rare codon in the gene in this experiment:

1. by online data retrieval, from Http: //www.kazusa.or.jp/codonThe place obtains the codon usage frequency table of P.pastoris.This table has been added up 39 sub-service conditions of proteinic amino acid code (table slightly) among the P.pastoris altogether.Show that by analysis there is very big dependency in this codon usage frequency table with the codon-bias table of having reported.

2. the P.pastoris codon usage frequency table of above acquisition is suitably revised the back as parameter input CODOP biosoftware, and, former gamma-interferon gene order is redesigned based on human gama-interferon aminoacid sequence (Fig. 1).Wherein (Effective number of codons, threshold value setting Nc) is 0.7 to judge rare password to the codon significant figure.(annotate: the major function of CODOP biosoftware is automatically rare password to be replaced in proportion at random the same amino acid whose non-rare password of coding)

The restriction endonuclease sites of table 1 synthetic gamma-interferon gene

Xho I EcoR I Pae R7I(6) Ava I(6) Taq I(7) Alu I(27) Drd I(39) Eco 57I(43) Ssp I(69) Tru 9I(73) Mse I(73) Ksp 632I(136) Ear I(136) Bsr I(138) Hinf I(144) Ple I(152) Sau 3AI(172) Nde II(172) Mbo I(172) Dpn II(172) Dpn I(174) Bsm AI(182) Esp 3I(182) Hind III(192)

Alu I(194) Fok I(210) Sau 3AI(215) Nde I(215) Mbo I(215) Dpn II(215) Bcl I(215) Dpn I(217) Bsm AI(234) Tru 9I(274) Mse I(274) Hind II(313) Hinc II(313) Tsp 509I(317) Dde I(323) Mae III(326) Drd I(339) Bsl I(350) Bsi YI(350) Alu I(365) Hinf I(368) Tfi I(368) Bpm I(376)

Bbv I(383) Alu I(396) Pvu II(396) Msp A1I(396) Ita I(397) Fnu 4HI(397) Bsa WI(405) Msp I(406) Hpa II(406) Bsm AI(427) Bsa I(427) Bpu AI(449) Bbs I(449) Sty I(462) Bsa JI(462) Bln I(462) Avr II(462) Mae I(463) Bfa I(463) Xba I(475) Mae I(476) Bfa I(476) Sfa NI(476)

3. adopt Biosuite VectorNTI biosoftware to make the restriction mapping of primary design gene, and inner Sac I, PshA I, Sal I, BglII, BamH I, Avr II, Xho I, Xba I, the EcoR I restriction enzyme site that may exist of manual removal, with convenient follow-up genetic manipulation.

4. according to the tumor-necrosis factor glycoproteins of (G+C) %, gene inside with whether there is mRNA splicing site (Fig. 3) etc., combining cipher uses table, the further manual gene order of revising design.

5. draw 24 primer sequences of required synthetic (Fig. 2) according to the complete synthesis scheme of determining of PCR method, the modification by gene order makes the fusing point of all primers all more than 50 ℃.

6. add suitable restriction enzyme site as required to make things convenient for genetic manipulation at the gene order two ends of design.

IFN-γ gene (G+C) % that obtains according to above principle redesign is 40.37; Inner potential mRNA split-join model of gene and tumor-necrosis factor glycoproteins are removed; The codon service condition of composition sequence and former sequence is as shown in table 2, and wherein codon significant figure Nc is (ratio %/100 that a certain amino acid whose a certain codon occupies in these amino acid whose all codons) * this amino acid whose password subnumber; What highlight is the rare password of judging according to Nc among the design; Fusing point usefulness [4 * C+G+2 * A+T] calculating of 4 primers of required Synthetic 2 (Fig. 2) all between 52-60 ℃, adopts [81.5+0.41 * (G+C/ (A+C+GT+T) * 100)-675/ (A+C+G+T)] algorithm all between 60-70 ℃.

Table 2.The codon usage of synthetic IFN-γ gene and original IFN-γ gene

		Nc	A a	E b			Nc	A	B			Nc	A	B			Nc	A	B
																				Phe Leu Leu Ile Met Val	TTT TTC TTA TTG CTT CTC CTA CTG ATT ATC ATA ATG GTT GTC GTA GTG	1.092 0.908 0.924 1.948 1.018 0.443 0.681 0.986 1.457 0.965 0.578 1.000 1.672 0.917 0.630 0.780	6 4 1 2 3 1 0 3 2 3 2 4 0 3 3 2	5 5 1 6 2 0 0 1 3 4 0 4 2 4 0 2	Ser Pro Thr Ala	TCT TCC TCA TCG CCT CCC CCA CCG ACT ACC ACA ACG GCT GCC GCA GCG	1.743 1.227 1.152 0.434 1.379 0.570 1.664 0.386 1.523 1.039 1.046 0.393 1.824 1.004 0.963 0.209	0 2 1 2 0 0 2 0 3 1 1 0 2 0 5 1	5 4 2 0 0 0 2 0 0 2 3 0 4 0 4 0	Tyr Ter His Gln Asn Lys Asp Glu	TAT TAC TAA TAG CAT CAC CAA CAG AAT AAC AAA AAG GAT GAC GAA GAG	0.905 1.095 1.313 1.313 1.061 0.939 1.222 0.778 0.968 1.032 0.913 1.087 1.108 0.892 1.133 0.867	3 1 1 0 2 0 4 5 7 3 12 8 4 6 6 3	1 3 0 1 1 1 8 1 5 5 6 14 5 5 3 6	Cys Ter Trp Arg Ser Arg Gly	TGT TGC TGA TGG CGT CGC CGA CGG AGT AGC AGA AGG GGT GGC GGA GGG	1.245 0.755 0.375 1.000 0.981 0.303 0.519 0.260 0.905 0.539 2.986 0.952 1.786 0.550 1.309 0.355	0 0 0 1 0 1 4 0 3 3 2 1 2 1 1 1	0 0 0 1 3 0 0 0 0 0 4 1 2 0 3 0

a：Codon usage of the original IFN-γgene；b：Codon usage of the synthetic IFN-γgene.

(2) IFN-γ expression vector establishment

The method that reference dna shuffles, we have promptly finally obtained dna fragmentation (Fig. 4) about 500bp by a step PCR assembling and amplification.Because adopted pfu polysaccharase skin to answer, the dna fragmentation that obtains is the flush end product, can directly the PCR product that obtains be packed in the pUC of Sma I enzymolysis 18 cloning vectors.After the blue hickie screening of X-Gal/I PTG, clone of picking delivers the order-checking of match hundred victory companies, and the IFN-gamma gene sequences of result and Design Theory is in full accord.

Behind the pUC18-ifn of sequence verification plasmid amplification, make up the P.pastoris expression plasmid pPIC3.5K-ifn (Fig. 5) that obtains IFN-γ according to preceding method.

(3) expression of reconstitution cell

In this optimum experimental sequence was expressed, we used Pichia pastoris SMD 1163 as host cell.The pPIC3.5K-ifn electric shock is screened the recombinant clone that contains the multiple copied expression vector with G418 concentration incremental method after transforming Pichia pastoris SMD1163, and the recombinant clone that obtains is carried out a small amount of fermentation expression.

4 RDB flat boards of cell coating after electric shock transforms, host cell Pichia pastoris SMD1163 is a histidine defect, expression vector contains the gene of encoding histidine, utilizes its complementary effect to filter out the Histidine positive colony.Respectively add in 2ml sterile distilled water to 4 flat board, wash out the Histidine positive colony and merge it, drawing 200 μ l thalline liquid coats and contains 1,2,4, on the G418 YPD flat board of 6mg/ml, picks out G418 positive colony totally 511 strains on G418 concentration 4 and 6mg/ml two flat boards.

Above-mentioned multiple copied clone is carried out a small amount of fermentation expression respectively.With the strain of sterilization toothpick picking mono-clonal, be inoculated in the test tube that contains 3ml YPD 30 ℃ of 300rpm overnight incubation.Get 150 μ l thalline and be seeded in the 50ml centrifuge tube that contains 15ml BMGY, 28-30 ℃ of 300rpm cultivated 16 hours; On whizzer with the centrifugal collection thalline of the speed of 4000rpm.In super clean bench, add 5ml BMMY, and change centrifugal lid into 4 layers of sterile gauze.Continue at 28-30 ℃ of 300rpm and cultivate the 24 little pure methyl alcohol of 50 μ l that add later; Cultivate again and add the pure methyl alcohol of 50 μ l after 24 hours; Continue to cultivate the centrifugal collection thalline of 5000rpm after 24 hours again, with carrying out ultrasonication after the Zymolase enzymic digestion again, the supernatant liquor after centrifugal is directly put nitrocellulose membrane and is carried out immunoblot experiment.We screen a lot of tangible positive reaction SMD1163/pPIC3.5K-ifn recombinant clones (Fig. 6) that present.Centrifugal collection thalline and supernatant carry out SDS-PAGE electrophoresis detection intracellular protein and fermented liquid secretory protein respectively, found that the specific proteins informal voucher band that exists in the born of the same parents about 17kD, and fermented liquid supernatant does not almost have the specific proteins band to occur.The cell strain that this explanation screening obtains is for expressing in the born of the same parents.Total DNA of the reconstitution cell of extracting simultaneously SMD 1163/pPIC3.5K-ifn, use synthetic DNA fragment Oligo 1 and Oligo 13 to carry out pcr amplification as primer, the result can both obtain the specific band (figure slightly) of about 500bp, shows to be integrated with synthetic IFN-γ expression casette in the reconstitution cell.Positive colony is cloned with the dull and stereotyped repeated screening multiple copied of the YPD that contains 5-15mg/ml G418, and carry out a small amount of fermentation expression; Thalline and broken bacterium are collected in centrifugal back; The centrifugal back of broken bacterium liquid supernatant is directly put nitrocellulose membrane and is carried out immunoblot experiment and SDS-PAGE electrophoresis, detects expressing protein in the born of the same parents; The interior homogeneous of bacterial strain energy born of the same parents that wherein is numbered pPIC3.5K-ifn-181 is expressed the IFN-γ target protein of 17KD, and expression amount accounts for about 25% of yeast body protein.With pPIC3.5K-ifn-181 bacterial strain called after Pichia pastoris SMD1163 (pPIC 3.5K-ifn, his4 pep4 Δ prb1)-181 and preserve standby.

Embodiment 2: the human gama-interferon production industrial fermentation of barms

One, experiment material and instrument

1. bacterial classification: methanol yeast bacterium Pichia pastoris SMD1163 (pPIC 3.5K-ifn, his4 pep4 Δ prb1)-181.

2. fermentor tank: 5 liters of fermentor tanks of Biostate E of German B.Bruan company.

3. electrophoresis apparatus: the Phast System electrophoresis system of Pharmacia company.

4 spectrophotometers: the DU-68 type spectrophotometer of Beckman company.

5. substratum and substratum are prepared and are used reagent: (1) YPD, YPB; (2) eutrophy substratum: press 100ug/ml among the YPD and add VITAMIN B4, leucine, uridylic; (3) poor nutritional medium; The poor nutritional medium of (4) 1% methyl alcohol (W/V); (5) the substratum preparation is all analytical pure or biochemical level reagent with other reagent.

Two, cultivation and fermentation method

Yeast is inoculated in the 25ml YPB substratum in 30 ℃ of incubation growth.To contain 57.8 gram (NH ₄) ₂SO ₄, 46.6 gram KCl, 30.8 gram MgSO ₄7H ₂XO, 8.6 gram CaSO ₄2H ₂2.5 liters of solution of O and 2.0 gram NaCl add in the fermentor tank, autoclaving.Be cooled to 29 ℃, add 350ml 50% glucose, the trace element solution of 210ml 30% hempa acid esters sodium salt solution and 10 times of concentration of 250ml.The microelement concentration of 10 times of concentration is every liter and contains 27.8 gram FeSO ₄7H ₂O, 0.5 gram CuSO ₄5H ₂O, 1.09 gram ZnCl ₂, 1.35 gram MnSO ₄H ₂O, 0.48 gram CoGl ₂6H ₂O, 0.24 gram Na ₂MoO ₄2H ₂O, 0.5 gram H ₃BO ₃, 0.08 gram KI, 5 milligrams of vitamin Hs, 0.5 gram VITMAIN B1,2.5 milliliters of sulfuric acid are used 10%NH ₄OH and 10%H ₃PO ₄PH transfers to 5 with fermented liquid, regulates aseptic compressed air, and flow velocity is 50 liters/minute, and rotor speed is 300rpm; Regulate dissolved oxygen to 30%, stirring velocity 300-800rpm when stirring speed greater than 800rpm, will supply pure oxygen automatically.Cultivate after 24-36 hour, add liquid with 60ml/h speed and (add liquid by 50% glucose, 300mM (NH ₄) ₂SO ₄Form with the trace element of 1 times of concentration), this stage cultivated 24 hours altogether; Add 50% grape glucose with 20ml/h speed, add pure methyl alcohol with 25ml/h speed, this stage cultivated 36-48 hour altogether, and this stage starts for inducing the AOX1 promotor, carries out gamma-interferon and synthesizes.Stop to cultivate, collect fermented liquid.

Three fermentation results

The gamma-interferon biosynthesizing of the used bacterial classification of the present invention, be by this yeast regulated and control with the AOX1 gene on the expression vector because this gene product is subjected to methanol concentration control, so can utilize methyl alcohol to control the synthetic of gamma-interferon.Discover that this bacterium was cultivated 48 hours at 30 ℃ earlier, cultivated 48 hours then, and interferon gene is had than high expression level in 1% methyl alcohol.Table 3 is three crowdes of results of continuously fermenting.Fig. 7-8 is three batches of SDS-PAGE electrophorograms that continuously ferment.

Table 3 yeast fermentation and gamma-interferon output

Lot number	Volume of culture (liter)	Biomass (gram)	Gamma-interferon gross activity (IU)	Total protein (mg)	Interferon, rabbit crude product (mg)	Interferon, rabbit accounts for total protein ratio (%)
							2001-1 2001-2 2001-3	3.23 3.05 3.58	194 213 232	8.7×10 10 8.3×10 10 9.3×10 10	12554 12688 15066	3326.9 3172.0 3615.8	26.5 25.0 24.0

Embodiment 3: the purifying of human gama-interferon

One material and instrument

1. liquid chromatograph: the 650E high performance liquid chromatograph of Waters company.

2.Waters 510 high performance liquid chromatographs of company.

3.Pharmacia the Biopilot chromatographic system of company.

4. electrophoresis apparatus: the Phast System electrophoresis system of Pharmacia company.

3.Pharmacia the Biopilot chromatographic system of company.

4. electrophoresis apparatus: the Phast System electrophoresis system of Pharmacia company.

5. spectrophotometer: the DU-68 type spectrophotometer of Beckman company.

Two purification process

Divided for four steps carried out.

The absorption of the first step resin anion(R.A): fermented liquid is centrifugal, collect yeast.This yeast is washed 2 times by aquae destillata suspension-centrifugally operated step.Add the damping fluid [20mmol/L TrisCl, the pH7.9 that are equivalent to the thalline volume; 10mmol/L MgCl ₂1mmol/L EDTA; 5% (v/v) glycerine; 1mmol/L DTT; 0.3mol/L (NH ₄) ₂ 5O ₄1 * protease inhibitor cocktail] and be equivalent to the pickling glass pearl (450-500 μ m) of thalline volume, in 1-4 ℃ of carrying out ultrasonic bacteria breaking; 12000rpm (10min) centrifuging and taking supernatant liquor, supernatant liquor be by Sepharose DEAE (Fast Flow) resin, uses damping fluid (0.8M NaCl, 0.1M PB, pH8) the gamma-interferon crude product that is adsorbed of wash-out at last.

The second step gel permeation chromatography: with going up sample behind the above-mentioned interferon solution dialysis desalination, then with damping fluid (0.1M NH ₄Ac, 0.05%Tween, pH7.0) wash-out, flow velocity are the 8ml/ branch, two peaks occur, second peak is active peak, collects it.

The 3rd step anion exchange chromatography:, use 0.1M NH with DEAE Sepharose (FastFlow) the dress post of Pharmacia company ₄Ac, 2.5% sucrose, the solution equilibria post of pH7.0; With sample on second peak sample collecting in the above-mentioned chromatographic step, gamma-interferon is not retained with this understanding, and impurity is retained, and collects effluent liquid.

The 4th step cation exchange column chromatography: with CM Sepharose (FastFlow) the dress post of Pharmacia company, with sample on the effluent liquid of above-mentioned collection, gamma-interferon is adsorbed on the post, then with A liquid (20mM NH ₄Ac, 5% sucrose is pH8) with B liquid (20mM NH ₄Ac, 5% sucrose, 0.7M NaCl, pH8) ionic strength of Zu Chenging increases progressively eluant solution, collects the 3rd peak, gets the pure product of gamma-interferon.

Three purification result

Continuous three batches of purification result are listed in table 4.

Table 4 extracts from the P.pastoris thalline, the result of purifying gamma-interferon

Lot number	The mensuration project	Purification step
							Yeast expression	DEAE absorption	Sephacryl S-200	DEAE Sepharose	CM Sepharose
		2001-1 2001-2 2001-3	Total protein (mg) gross activity (IU) specific activity purification yield (%) total yield % total protein (mg) gross activity (IU) specific activity purification yield (%) total yield % total protein (mg) gross activity (IU) specific activity purification yield (%) total yield %	12554 8.7×10 10 0.69×10 7 1 100 12688 8.31×10 10 0.65×10 7 1 100 15066 9.3×10 10 0.62×10 7 1 100	4629 6.8×10 10 1.47×10 7 2.1 78 4433 6.65×10 10 1.50×10 7 2.3 80 4685 6.98×10 10 1.49×10 7 2.4 75	2375 4.42×10 10 2.1×10 7 1.4 65 2095 4.19×10 10 2.00×10 7 1.3 63 2457 4.67×10 10 1.90×10 7 1.3 67						996 2.79×10 10 2.8×10 7 1.3 63 929 2.76×10 10 2.93×10 7 1.5 66 1010 2.89×10 10 2.86×10 7 1.5 62	524 2×10 10 3.82×10 7 1.4 72 23.0 526 1.94×10 10 3.69×10 7 1.3 70 23.3 527 1.97×10 10 3.74×10 7 1.3 68 21.2

Embodiment 4: the calibrating of the human gama-interferon of purifying

1. titration

Measure with micro-cytopathic-effect inhibition assay.Different times of cell of people's amnion (WISH) is for measuring cell, and vesicular stomatitis virus (VSV) is a challenge virus, and calculating with Japanese reference material school is international unit.

2. protein quantification

Use the Lowry method, do standard test with the standard protein of Nat'l Pharmaceutical ﹠ Biological Products Control Institute.

3. purity testing

Use the SDS-polyacrylamide gel electrophoresis, application of sample 5.0 μ g reduce and non-reduced electrophoresis simultaneously, with argentation dyeing (Fig. 9), carry out gel scanning on spectrophotometer, and it the results are shown in Table 5.The purity of finding out them thus is all more than 95%.

Way is: make the typical curve of the molecular weight-swimming distance of known protein, measure the swimming distance of purified product again, (Figure 10) finds their molecular weight from curve.Table 5 is the molecular weight of three batch samples, this and reported in literature 17126 approaching.

The purity and the molecular weight of table 5 SDS-PAGE cataphoretic determination gamma-interferon

Lot number	Purity scanning	Molecular weight
			2001-1 2001-2 2001-3	96.6％ 97.2％ 96.4％	17100 16900 16800

5. efficient liquid phase chromatographic analysis

With Waters 650E high performance liquid phase instrument, Protein-Pak 200SW post, analyze three batches of purification of samples, with 10mM PB, the pH7.0 damping fluid is a moving phase.Use normalization method calculation result, obtain the purity of three batch samples; Be respectively 97.9%, 98.5%, 97.6%, collection of illustrative plates is seen Figure 11.

6. residual DNA assay

Pichia pastoris SMD1163 (pPIC3.5K-ifn with [α 32p] dCTP mark, his4 pep4 Δ prb1) the full DNA of-181 yeast makes probe, carry out molecular hybridization with three batch samples of purifying, the results are shown in Figure 12, from scheming as seen, the contained residual DNA amount of the gamma-interferon of every dose of purifying all is lower than 5pg.

7. the iso-electric point scope is measured in isoelectrofocusing

With Phast System electrophoresis apparatus, measured the iso-electric point scope of three batch samples, measurement result is seen Figure 13, their iso-electric point scope meets reported in literature gamma-interferon iso-electric point and is 8.6 result between 8.65 to 8.45.

8. UV scanning

Three batch samples scan in 200nm～320nm wavelength region with ultraviolet spectrophotometer, and tangible albumen absorption peak is arranged at the 279.5nm place, and the strong absorption peak of peptide chain is also arranged near 228.5nm, the results are shown in Figure 14, and this is consistent with reported in literature.

9. peptide figure

9. peptide figure

When handling gamma-interferon albumen with cyanogen bromide, peptide chain can rupture at the methionine(Met) place.Gamma-interferon albumen has 4 methionine(Met), so can produce 5 peptide sections, but (Figure 15) can only see three bands in electrophorogram, this is because one 9 peptide disappears, another contains more basic aminoacids, the peptide section that mobility speed and molecular weight are bigger is overlapping, and (Figure 16) can know and see 5 peaks in the HPLC collection of illustrative plates, and is consistent with theoretical value.

10.N-terminal amino acid is measured

Use the Edman edman degradation Edman, at the 470A of ABI company amino acid sequence analysis instrument, analyzed 15 amino acid of N-terminal of the gamma-interferon of purifying, it the results are shown in Figure 17, and this and reported in literature are in full accord.

11. immunity seal stain test

In order to confirm that purified albumen is human gama-interferon really, we have carried out electrophoresis with the sample of three batches of purifying, gel behind the electrophoresis is transferred to albumen on the nitrocellulose membrane with Western blotting method, then with the human gama-interferon monoclonal antibody action of Boehringer Mannheim company, at last the sheep anti-mouse antibody with the alkali phosphatase enzyme mark of Promega company reacts with it, and colour developing, the results are shown in Figure 18, this shows that this three batch sample all shows strong positive reaction.

12. telling test

In the monoclonal anti physical efficiency of human gama-interferon and the activity of human gama-interferon, can differentiate thus whether purified product is human gama-interferon.The way of experiment is, we human gama-interferon and the monoclonal antibody (Boehringer Mannheim product) of preparation are done a series of dilutions, respectively gets 100 μ l then and mixes mutually, and 37 ℃ of insulations 1 hour and are measured it and tired.The results are shown in Figure 19-20, from scheming as seen along with the increase of monoclonal antibody concentration is tired also along with reduction, and the monoclonal antibody of alpha-interferon does not have this neutralization activity, and the activity that the Interferon, rabbit that we prepare is described is the human gama-interferon activity, none other than Interferon, rabbit or other protein.

Sequence table

(1) general information

(i) denomination of invention: human's efficiently expressing in Pichia yeast, fermentation and purifying

(ii) sequence number: 2

(2) information of sequence SEQ ID NO.1:

(i) sequence signature:

(A) length: 435bp

(B) type: nucleic acid

(C) chain: strand

(D) topological structure: linearity

(ii) molecule type: DNA

(iii) sequence description: SEQ NO.1

ATG CAA GAC CCA TAC GTC AAG GAG GCA GAA AAC TTG AAG AAA TAT TTT AAC GCT GGT CAT

TCT GAC GTG GCA GAC AAT GGA ACA TTA TTT TTG GGT ATT TTG AAG AAC TGG AAA GAA GAG

TCA GAT AGA AAG ATT ATG CAA TCC CAG ATC GTC TCC TTC TAC TTC AAG CTT TTC AAG AAC

TTC AAA GAT GAT CAA TCC ATC CAA AAA TCC GTT GAG ACA ATC AAA GAG GAT ATG AAT GTC

AAG TTC TTT AAC TCT AAT AAG AAG AAG CGT GAT GAC TTT GAG AAG TTG ACC AAT TAC TCA

GTT ACA GAC TTG AAT GTC CAA CGT AAG GCA ATC CAC GAG CTG ATT CAA GTG ATG GCA GAA

CTT TCT CCA GCT GCT AAA ACC GGA AAG AGA AAG AGG TCT CAA ATG TTG TTT AGA GGA AGA

CGT GCT TCT CAA TAG

(3) information of sequence SEQ NO.2:

(i) sequence signature:

(A) length: 144AA

(B) type: amino acid

(C) topological structure: linearity

(ii) molecule type: linearity

(iii) sequence description: SEQ NO.2

M Q D P Y V K E A E N L K K Y F N A G H

S D V A D N G T L F L G I L K N W K E E

S D R K I M Q S Q I V S F Y F K L F K N

F K D D Q S I Q K S V E T I K E D M N V

K F F N S N K K K R D D F E K L T N Y S

V T D L N V Q R K A I H E L I Q V M A E

L S P A A K T G K R K R A Q M L F R G R

R A S Q

Claims (6)

1, a kind of method by genetic engineering bacterium fermentative preparation human gama-interferon is characterized in that, human's sequence that gene structure is following,

ATG CAA GAC CCA TAC GTC AAG GAG GCA GAA AAC TTG AAG AAA TAT TTT AAC GCT GGT CAT

M Q D P Y V K E A E N L K K Y F N A G H

TCT GAC GTG GCA GAC AAT GGA ACA TTA TTT TTG GGT ATT TTG AAG AAG TGG AAA GAA GAG

S D V A D N G T L F L G I L K N W K E E

TCA GAT AGA AAG ATT ATG CAA TCC CAG ATC GTC TCC TTC TAC TTC AAG CTT TTC AAG AAC

S D R K I M Q S Q I V S F Y F K L F K N

TTC AAA GAT GAT CAA TCC ATC CAA AAA TCC GTT GAG ACA ATC AAA GAG GAT ATG AAT GTC

F K D D Q S I Q K S V E T I K E D M N V

AAG TTC TTT AAC TCT AAT AAG AAG AAG CGT GAT GAC TTT GAG AAG TTG ACC AAT TAC TCA

K F F N S N K K K R D D F E K L T N Y S

GTT ACA GAC TTG AAT GTC CAA CGT AAG GCA ATC CAC GAG CTG ATT CAA GTG ATG GCA GAA

V T D L N V Q R K A I H E L I Q V M A E

CTT TCT CCA GCT GCT AAA ACC GGA AAG AGA AAG AGG TCT CAA ATG TTG TTT AGA GGA AGA

L s P A A K T G K R K R A Q M L F R G R

CGT GCT TCT CAA TAG

R A S Q Term clone inserts and has among the carrier pPIC 3.5K of methyl alcohol adjustment type promotor AOX1, obtain expression vector pPIC3.5K-ifn, adopting the pichia spp SMD1163 of proteolytic enzyme defective is the host, with G418 resistance concentration incremental method screening multiple copied clone, get high expression level pichia spp strain (Pichia Pastoris) SMD1163 (pPIC3.5K-ifn, his4pep4 Δ prbl)-and 181CGMCC NO.0758, through fermentation, purifying gets human gama-interferon.

2, according to the described preparation human gama-interferon of claim 1 method, it is characterized in that may further comprise the steps:

A, human's is synthetic: at the pichia spp codon usage frequency, designer's gamma-interferon gene order draws 24 following primer sequences of structure, synthetic primer,

Oligo 1：CGACCTCGAGAAAAGAGAGGAATTCATGCAAGACCCATAC

Oligo 2：GTCAAGGAGGCAGAAAACTTGAAGAAATATTTTAACGCTG

Oligo 3：GTCATTCTGACGTGGCAGACAATGGAACATTATTTTTGGG

Oligo 4：TATTTTGAAGAACTGGAAAGAAGAGTCAGATAGAAAGATT

Oligo 5：ATGCAATCCCAGATCGTCTCCTTCTACTTCAAGCTTTTCA

Oligo 6：AGAACTTCAAAGATGATCAATCCATCCAAAAATCCGTTGA

Oligo 7：GACAATCAAAGAGGATATGAATGTCAAGTTCTTTAACTCT

Oligo 8：AATAAGAAGAAGCGTGATGACTTTGAGAAGTTGACCAATT

Oligo 9：ACTCAGTTACAGACTTGAATGTCCAACGTAAGGCAATCCA

Oligo 10：CGAGCTGATTCAAGTGATGGCAGAACTTTCTCCAGCTGCT

Oligo 11：AAAACCGGAAAGAGAAAGAGGTCTCAAATGTTGTTTAGAG

Oligo 12：GAAGACGTGCTTCTCAATAGCCTAGGCATCATTTCTAGAC

Oligo 13：GCAGTCTAGAAATGATGCCTAGG

Oligo 14：CTATTGAGAAGCACGTCTTCCTCTAAACAACATTTGAGAC

Oligo 15：CTCTTTCTCTTTCCGGTTTTAGCAGCTGGAGAAAGTTCTG

Oligo 16：CCATCACTTGAATCAGCTCGTGGATTGCCTTACGTTGGAC

Oligo 17：ATTCAAGTCTGTAACTGAGTAATTGGTCAACTTCTCAAAG

Oligo 18：TCATCACGCTTCTTCTTATTAGAGTTAAAGAACTTGACAT

Oligo 19：TCATATCCTCTTTGATTGTCTCAACGGATTTTTGGATGGA

Oligo 20：TTGATCATCTTTGAAGTTCTTGAAAAGCTTGAAGTAGAAG

Oligo 21：GAGACGATCTGGGATTGCATAATCTTTCTATCTGACTCTT

Oligo 22：CTTTCCAGTTCTTCAAAATACCCAAAAATAATGTTCCATT

Oligo 23：GTCTGCCACGTCAGAATGACCAGCGTTAAAATATTTCTTC

OLigo 24:AAGTTTTCTGCCTCCTTGACGTATGGGTCTTGCATGAATT adopts ordinary method that 24 oligonucleotide fragments mixing of equivalent are formed the primer pond, be mixed in proportion into the PCR reaction solution then, with the complete synthesis human's sequence of pcr amplification technology;

The structure of b, the strain of high expression level pichia spp: above-mentioned human's sequence clone importing is had among the expression vector pPIC3.5K of methyl alcohol adjustment type promotor AOX1, the pichia spp SMD1163 of electric shock transforming protein enzyme defect after the linearizing, with G418 resistance concentration incremental method screening multiple copied clone and the multiple copied clone is carried out a small amount of fermentation expression obtain high expression level Pichi strain (Pichia pastoris) SMD1163 (pPIC3.5K-ifn, his4 pep4 Δ prbl)-181 CGMCC NO.0758;

The fermentation of c, high expression level Pichi strain: the Pichi strain of above-mentioned gained high expression level in not containing the substratum YPB of methyl alcohol in 30 ℃ of incubation growth: seed liquor is seeded in the fermentor tank in 29-30 ℃ cultivated 48-60 hour, again through 1% methanol induction post-fermentation and culture 36-48 hour;

The purifying of d, human gama-interferon: medium centrifugal gets thalline, and the supernatant liquor behind the cellular lysate adsorbs through resin anion(R.A), gel permeation chromatography, and anion exchange chromatography, cation exchange column chromatography gets purified product.

3, according to the described method for preparing human gama-interferon of claim 2, the method that it is characterized in that multiple copied clone a small amount of fermentation expression is: multiple copied is cloned among the substratum YPD that does not contain methyl alcohol in 30 ℃ of overnight incubation, getting thalline is seeded among the substratum BMGY, in 28-30 ℃ of cultivation 40 hours, add 0.5% methanol induction at twice and cultivated 48 hours.

According to the described method for preparing human gama-interferon of claim 2, it is characterized in that 4, in the purifying of steps d, human gama-interferon, described cellular lysate is to add pickling glass pearl, carrying out ultrasonic bacteria breaking in thalline; DEAE Sepharose anionite-exchange resin is adopted in described resin anion(R.A) absorption; Described gel permeation chromatography adopts SephacrylS-200 to carry out the molecular sieve gel filtration chromatography; Described anion exchange chromatography adopts DEAE Sepharose resin; Described cation exchange column chromatography adopts the CM-Sepharose resin.

5, a kind of expression vector pPIC3.5K-ifn is characterized in that containing the described human's sequence of method of claim 1.

6, a kind of high expression level pichia spp strain (Pichia Pastoris) SMD1163 (pPIC3.5K-ifn, his4 pep4 Δ prbl)-181 CGMCC NO.0758, it is characterized in that the clone obtains through G418 resistance concentration incremental method screening multiple copied by the pichia spp SMD1163 of the described expression vector pPIC3.5K-ifn electric shock of claim 5 transforming protein enzyme defect.

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