CN102120967A - Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain - Google Patents

Abstract

The invention discloses preparation and application of an alpha-1,6 mannose transferase gene (och1) flaw type P. pastoris X-33 bacterial strain, belonging to the technical field of biological engineering. The engineering bacterial strain P. pastoris X-33 (delta och1) is characterized in that a target gene knockout method by double-exchange homologous recombination is adopted, and a URA3 gene serves as a selective marker to knock out the alpha-1,6 mannose transferase gene (och1) of the P. pastoris X-33 (delta och1) to obtain the P. pastoris X-33 (delta och1) bacterial strain with OCH1 gene knockout. The bacterial strain provides a P. pastoris expression system which modifies protein by virtue of low N-glycosylation modification and provides a favourable basis for further glycosylation modification. The bacterial strain has the advantage of great potential application prospect.

Description

The structure and the application of OCH1 gene defection type pichia spp X-33 bacterial strain

Technical field

The invention discloses the structure and the utilization of strain pichia spp X-33 (Δ och1) bacterial strain, belong to technical field of bioengineering.

Background technology

Glycoprotein medicine (as antibody, cytokine, vaccine etc.) has become one of the fastest medicine of development in recent years, and demand increases rapidly.At present the production of these glycoprotein mainly is use mammalian cell, yet there are many shortcomings in mammalian cell production pharmaceutical protein, as the cell cultures complexity, expressing quantity is low, production cost is high.

Pichia yeast expression system have as the prokaryotic organism fast growth, be convenient to genetic manipulation, with low cost, be convenient to characteristics such as large scale culturing and high density fermentation, has simultaneously eukaryotic most of translation post-treatment rhetorical function (as glycosylation process etc.) again, be widely used in various proteic expression, thereby caused people's favor day by day with pichia spp cells produce pharmaceutical protein.But pichia spp is the high mannose type sugar chain to proteic glycosylation modified generation, the complexity sugar chain that is different from mammalian cell, cause the immunogenicity of glycoprotein to strengthen thus, a series of detrimentally affects such as the transformation period shortens, active reduction, this is the major cause that pichia spp can not be used for most of glycoprotein medication preparation.

The N-glycosylation of yeast and mammalian cell all originates in endoplasmic reticulum.In endoplasmic reticulum, polysaccharide chain on the glycosylated common lipopolysaccharides precursor Glc3Man9GlcNAc2-dol-p-p of N-is transferred on the Asn in new polypeptide chain Asn-X-Thr/Ser (X for except that Pro the arbitrary amino acid) conserved sequence, then form the Man8GlcNAc2 structure under the effect of Glycosylases such as glucuroide I and II, the forming process of this oligonucleotide chain all is high conservative from the yeast to the mammalian cell.This intermediate is transported in the golgi body subsequently, and the sugar chain processing of yeast and mammalian cell is obviously different with the modification approach.In the mammalian cell, under the effect of a series of Glycosylases and glycosyltransferase, form various complexity N-glycan structures; And in the pichia spp, at first at a-1,6-mannose transferase (Och1p) effect down, on the Man8GlcNAc2 sugar chain, add an a-1,6 seminoses form the Man9GlcNAc2 sugar chain structure, a series of then mannose transferases and phosphomannose transferring enzyme continue to add seminose as substrate, finally form the high mannose type sugar chain structure.This shows that a-1,6-mannose transferase (Och1p) are that the first step of formation high mannose baseization also is the most key enzyme, block the high mannose modification approach of pichia spp, must knock out its OCH1 gene earlier.

P.pastoris X-33 is that the pichia spp original strain that wild-type that Invitrogen company releases did not transform any gene is that methyl alcohol utilizes type fast, tolerance is relatively good, the higher comparatively speaking expression that is widely used in foreign protein of transformation efficiency and expression amount.The present invention knocks out the P.pastoris X-33 engineering bacteria (X-33 (Δ och1)) that has obtained the OCH1 defective type to the OCH1 gene of pichia spp X-33 first.This strain growth proterties is fine, stabilization characteristics of genetics, and the glycoprotein N that gives expression to end mannose groupization obviously alleviates, and can be used for expressing the various glycoprotein of the low seminose of N end, and it is glycosylation engineered to can be used for further people source.

Summary of the invention

The objective of the invention is to make up P.pastoris X-33 (Δ och1) bacterial strain that obtains the OCH1 transgenation.

The method that the present invention makes up acquisition OCH1 mutant strain is following, and (wherein used P.pastoris X-33 (Δ ura3) bacterial strain is that this laboratory makes up preservation.)

1) clone of OCH1 dna homolog arm: according to P.pastoris OCH1 gene order design two couples of Oligonucleolide primers OCH5F, OCH5R and OCH3F, the OCH3R of report among the GenBank (E12456), introduce SalI, MluI, NheI restriction enzyme site respectively at two ends, pcr amplification OCH1 gene two ends homology arm OCH5 ' and OCH3 ' and fusion fragment OCH3 '-5 ' thereof are respectively about 1000bp, 700bp and 1700bp.Primer sequence (underscore mark part is restriction enzyme site) is respectively:

OCH5F：5’-CTTCCCATTGACACTTTCCGA ACGCGTCGAGAGAACTCTGGATTAGCAGTC-3’

OCH5R：5’-TAGA GTCGACCTGATGATATTTGCTACGAACACTG-3’

OCH3F：5’-TAGA GCTAGCGTTGGAAGGACTAAAGAAAGCTAGAG-3’

OCH3R：5’-GACTGCTAATCCAGAGTTCTCTCG ACGCGTTCGGAAAGTGTCAATGGGAAG-3’

2) knock out the structure of plasmid pYXZ-OCH1: with plasmid pYES2 is template, design primer pYES2F and pYES2R carry out the PCR reaction product are reclaimed purifying, carry out ligation after cutting with the SalI enzyme, form plasmid pYXZ from connecting, transformed into escherichia coli JM109 selects positive colony with the LB flat board that contains penbritin.Primer sequence (underscore mark part is restriction enzyme site) is respectively:

pYES2F：5’-TAGA GTCGACATCGATAAGCTAGCTTTTCAATTCAATTC-3’

pYES2R：5’-TAGA GTCGACGAGCAAAAGGCCAGCAAAAGC-3’

OCH1 two ends homology arm is merged fragment OCH3 '-5 ' cut, be cloned on the plasmid pYXZ that cuts with SalI and NheI enzyme, form and knock out plasmid pYXZ-OCH1 with SalI and NheI enzyme.

3) the knocking out of OCH1 gene among the P.pastoris X-33: will knock out the MluI restriction enzyme site linearizing between last two homology arms of plasmid pYXZ-OCH1, electric shock changes P.pastoris X-33 (Δ ura3) competent cell over to, on the coating MD substratum, cultivate an about week for 25 ℃.With the single bacterium colony that grows on the MD substratum, with the corresponding dibbling of toothpick in two YPD substratum (peptones 2%, yeast extract 1%, glucose 2%, agar powder 1.5%) on, cultivated several days 25 ℃ and 37 ℃ respectively, select then at 37 ℃ and can not grow and 25 ℃ of well-grown single bacterium colonies are carried genome, use OCH1 gene two ends primer OCH5F, OCH3R and inner primer (in) 5F, (in) 3R to carry out PCR respectively and identify.Primer sequence is respectively:

(in)5F：5’-CAGCCTTAAAGAGCCCGCTAAA-3’

(in)3R：5’-GCCTCAATACCAATGACCAACCC-3’

The P.pastoris X-33 bacterial classification that the present invention obtains is through repeatedly going down to posterity, stabilization characteristics of genetics, and X-33 compares with wild-type, and this bacterium suits to grow under as 26 ℃ condition at low temperature.At high temperature, by adding 0.3M NaCl or 1.0M sorbitol also can obtain good growth characteristics.Utilize the quantity of the glycoprotein seminose of this bacterial strain expression obviously to reduce, molecular weight obviously reduces.Simultaneously P.pastoris X-33 (Δ och1) bacterium can also to be used for further people source N-glycosylation engineered, by introducing the needed enzyme of a series of synthetic complexity sugar chain albumen in yeast, thereby obtain to be more suitable for producing the Yeast engineering bacteria of medicinal glycoprotein.

Description of drawings

Fig. 1, pYXZ-OCH1 knock out plasmid

The PCR of the outside primer OCH5F of Fig. 2, P.pastoris X-33 (Δ och1) bacterium OCH1, OCH3R is the M:DNA molecular weight standard as a result; 1:P.pastoris X-33 (Δ och1) bacterium; 2:P.pastoris X-33 wild-type bacteria.

The PCR of inner primer (in) 5F of Fig. 3, P.pastorisX-33 (Δ och1) bacterium OCH1, (in) 3R is the M:DNA molecular weight standard as a result; 1:P.pastoris X-33 (Δ och1) bacterium; 2:P.pastoris X-33 wild-type bacteria.

Fig. 4, expression vector pPICZaA/GM-CSF

Fig. 5, the GM-CSF expression in P.pastoris X-33 (och1-) bacterium

M: albumen lower molecular weight standard; The 1:pPICZaA/GM-CSF electricity changes the protein expression of P.pastoris X-33 wild-type bacteria; The 2:pPICZaA/GM-CSF electricity changes the protein expression of P.pastorisX-33 (Δ och1) bacterium; 3:pPICZaA empty carrier electricity changes the protein expression of P.pastorisX-33 wild-type bacteria expresses.

GM-CSF after Fig. 6, Glycosylase F (PNGaseF) enzyme are cut

M: albumen lower molecular weight standard; 1:P.pastoris the GM-CSF that the X-33 wild-type bacteria is expressed; 2:P.pastoris the GM-CSF that the X-33 wild-type bacteria is expressed handles through Glycosylase F; 3:P.pastoris the GM-CSF that X-33 (Δ och1) bacterium is expressed; 4:P.pastoris the GM-CSF that X-33 (Δ och1) bacterium is expressed handles through Glycosylase F.

Embodiment:

Embodiment one:

The knocking out and identifying of OCH1 gene: will knock out plasmid pYXZ-OCH1 (Fig. 1) changes X-33 (Δ ura3) competent cell with electricity after the MluIization, with purpose generation double exchange homologous recombination.Knock out plasmid and contain the URA3 marker gene, what therefore can grow on the MD substratum is the bacterial strain that homologous recombination takes place; Simultaneously och1 knock out bacterium at 25 ℃ of well-growns and 37 ℃ can not grow, select the bacterial strain that meets above-mentioned two kinds of proterties, carry out genome PCR and identify.Because the sequence of about 1000bp is replaced by the selective marker sequence of 2.8kb in the OCH1 gene reading frame, identify that with outside primer OCH5F, OCH3R X-33 bacterium pcr amplification product is that 2.8kb, X-33 (Δ och1) bacterium is 4.4kb (Fig. 2); (in) 3R among inner primer (in) 5F, (in) 3R is arranged in by metathetical reading frame sequence, therefore the X-33 bacterial strain can amplify the fragment of 1.6kb, and the amplification of X-33 (Δ och1) bacterium is less than fragment (Fig. 3), electrophoresis result shows that the OCH1 gene is knocked out, screening be P.pastoris X-33 (Δ och1) bacterium.

Embodiment two:

The structure of GM-CSF expression vector and expression

1) structure of expression vector: the plasmid pUC19/GM-CSF that this chamber is preserved is with EcoRI, NotI double digestion, reclaim GM-CSF gene fragment (its terminal 6xHis of introducing sequence label), be cloned on the yeast expression vector pPICZaA that cuts with same enzyme, form expression vector pPICZaA/GM-CSF (Fig. 4), screening positive clone, enzyme are cut and are identified and order-checking.

2) expression of GM-CSF in P.pastoris X-33 and X-33 (Δ och1): with expression vector pPICZaA/GM-CSF with restriction enzyme SacI linearizing after respectively the electric shock change in X-33 and X-33 (Δ och1) competent cell, bacterium liquid coating YPDZ after power taking is hit (contains 100ug/mL respectively, 300ug/mL, the Zeocin of 500ug/mL) on the substratum, the X-33 transformant is cultivated 3-5d in 30 ℃, X-33 (Δ och1) transformant is cultivated an about week in 25 ℃, select well-grown single bacterium colony then and carry genome universal primer 5 ' AOX, 3 ' AOX carries out PCR to be identified, the screening positive transformant.The positive X-33/pPICZaA/GM-CSF bacterium of identifying is inoculated into the YPD (peptone 2% of 2ml, yeast extract 1%, glucose 2%) in the substratum, behind 30 ℃ of cultivation 24h, inoculum size with 5% is inoculated into the BMGY (peptone 2% of 10ml, yeast extract 1%, YNB1.34%, glycerine 2%, the phosphate buffered saline buffer PH 6.0 of 100mmol/L) in the substratum, cultivate 36h for 30 ℃, the centrifugal BMMY that removes supernatant and be resuspended in 3ml (yeast extract 1%, YNB1.34%, peptone 2%, the phosphate buffered saline buffer PH 6.0 of 100mmol/L) in the substratum, adds 2% methyl alcohol simultaneously and begin abduction delivering; X-33 (Δ och1)/pPICZaA/GM-CSF positive bacteria is 25 ℃ of cultivation 48h in the YPD substratum, cultivate 48h, 25 ℃ of abduction deliverings for 25 ℃ in the BMGY substratum, the same X-33 of other condition, every 24h adds methyl alcohol one time, induces 72h to finish back centrifuging and taking supernatant, is used for proteic expression analysis.The universal primer sequence is:

5’AOX1：5′-GACTGGTTCCAATTGACAAGC-3‘

3’AOX1：5′-GGCAAATGGCATTCTGACAT-3

3) proteic purifying of GM-CSF and N-Glycosylase F (PNGaseF) restriction analysis

The proteic purifying of a.GM-CSF: with the culture supernatant 500ml of P.pastoris X-33 bacterium and P.pastoris X-33 (Δ och1) bacterium with 10 times of Millipore LabscaleTM TFF System (retaining molecular weight is 10KD) ultrafiltration and concentration, through last sample level pad (20mmol/L Tris-HCl, 500mmol/L Nacl, pH 7.2) after the balance, utilize AKTA chromatographic instrument UPC-900 system, nickel affinity chromatography (Ni-affinity chromatography) column purification end has the GM-CSF of His6-tag label, moving phase (20mmol/L Tris-HCl, 500mmol/L NaCl, pH 7.2, and elutriant contains the 0.2mol/L imidazoles in addition).Then the GM-CSF that collects is lived in capable desalination with Sephadex G-50 gel permeation chromatography, moving phase (20mmol/L phosphate buffered saline buffer, pH 7.2).

The N-glucosides restriction analysis of b.GM-CSF: GM-CSF is 100 ℃ of sex change 10min in 0.5%SDS and 0.04mol/L DTT sex change damping fluid, then in 0.05mol/L sodium phosphate (pH 7.2), 1%NP-40,37 ℃ of enzymes are cut more than the 1h in the N-sugar enzyme F of the battalion system, enzyme is cut product carry out the SDS-PAGE analysis.

Claims (3)

1. the OCH1 deficient strain bacterial strain of a P.pastoris X-33.

2. according to the described genetic engineering bacterium of claim 1, its construction process be with URA3 as selective marker, adopt the double exchange homologous recombination to knock out the OCH1 gene.

3. claim 1 or 2 is being expressed the application aspect the low glycosylation modified glycoprotein of N-and the application of further glycosylation engineered aspect.

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