CN103555749A - Method for in vitro efficient construction of multi-copy Pichia expression vector - Google Patents

Abstract

The present invention provides a method for in vitro efficient construction of a multi-copy Pichia expression vector. The method comprises: 1) based on a pPIC9K expression vector, constructing a vector pHBM905A; 2) constructing a recombinant vector pHBM905BDM; 3) selecting exogenous gene protein requiring being expressed; 4) constructing exogenous gene protein-containing single-copy recombinant vector pHBM905BDM-gene-1; and 5) based on a Biobrick method, constructing a new multi-copy vector pHBM905BDM-gene-n having an artificial in vitro tandem duplication expression cassette type structure. According to the present invention, based on a series of transformations on the pPIC9K expression vector, the multi-copy recombinant vector pHBM905BDM suitable for in vitro construction is constructed; construction of the exogenous gene-containing single-copy recombinant vector pHBM905BDM-gene-1 and the multi-copy recombinant vector pHBM905BDM-gene-n is achieved; and the high-copy recombinant plasmids are obtained through digestion and enzyme linkage on the basis of the copy 1 with right sequencing, such that re-sequencing is not required so as to save cost, save time, save labor, and substantially increase expression ability of exogenous gene protein.

Description

A kind of in vitro efficient method that builds multi-copy Pichia Expression Vector

Technical field

The present invention relates to the in vitro efficient method that builds multi-copy Pichia Expression Vector, belong to gene engineering technology field.

Background technology

Pichia pastoris phaff expression system originates from the eighties in 19th century, relatively late, but later stage development fast.Pichia pastoris phaff has become a kind of exogenous protein expression system of maturation.

Conventional pichia pastoris phaff expression vector contains some common features.A lot of carriers all select pichia pastoris phaff HIS4 gene as selected marker.AOX1 3 ' the flanking sequence that derives from 3 ' end of pichia pastoris phaff genome AOX1 gene, is also present in a lot of carriers, and can be used as the site of exogenous origin gene integrator and insertion.Using maximum is from yeast saccharomyces cerevisiae MF α-SS leading peptide encoding sequence.

In pichia pastoris phaff expression system, alcohol oxidase (AOX) promotor is most popular.MF α-SS is most popular signal sequence, and after translation, MF α-SS signal peptide is identified by peptase, and is excised by kex2 proteolytic enzyme, thereby forms ripe protein.The content that affects pichia yeast expression system secretory protein mainly contains several factors: (1) promotor, and (2) signal peptide, (3) express the copy number that box structure is integrated in Host Strains.So the optimization of current pichia yeast expression system is mainly set about from these aspects.

It is reported, improve copy number that target gene box structure integrates in Host Strains and be the effective means that increases the expression amount of recombinant protein in individual cells.The high copy of screening bacterial strain has 2 kinds of methods conventionally: 1) depend on restriction enzyme, the high copy of artificial external structure recombinant plasmid; 2) select the expression vector that contains resistance screening mark.Use " biological brick " technology (" Biobrick "), utilize simple means, from the simplest assembly, start to set up, finally can build large-scale complicated multiple copied system.

One of current most popular yeast expression vector is pPIC9K carrier (sale of Invitrogen company), though this carrier at present successful expression a lot of foreign proteins, it also has the limitation of himself.

1) clone's mode is complicated.It is all the clone's mode that depends on digestion with restriction enzyme and ligase enzyme enzyme company of use that goal gene is cloned on this carrier, the multiple clone site place of this carrier only has four restriction enzyme sites, two restriction enzyme sites being of little use wherein, when this makes DCRP external source fragment, alternative insertion point greatly reduces; And if with PCR product cloning goal gene, must consider and in object fragment, whether have this restriction enzyme digestion sites, because the restriction enzyme site on this carrier is limited, so will be restricted to carrier for different gene clones, may will cause so a lot of genes inside also to include the required restriction enzyme site of clone, cause goal gene to be cloned into will suddenling change on this carrier some restriction enzyme sites of its gene inside, thereby it is complicated to make to clone step;

2) biological safety is inadequate.After goal gene is cloned on pPIC9K carrier, linearizing recombinant plasmid in the way to insert homologous recombination in pichia pastoris phaff host genome, due to the expression boxlike element " Amp " of the anti-penbritin on pPIC9K with in intestinal bacteria, for the element " Ori " of replication initiation, can get on along with destination gene expression box is incorporated into Yeast genome together, make like this genetic engineering bacterium building there is certain resistance, and copy number is higher, resistance is just stronger, if resistant gene is leaked in environment accidentally, just may increase the risk of environmental organism security, and copy number is higher, resistance is just stronger, 3) efficiency that linearizing fragment transforms is low.The recombinant plasmid of traditional pPIC9K vector construction is with after SalI linearizing, and because " Amp+Ori " element gets on along with destination gene expression box is incorporated into Yeast genome together, linear fragment is too large, and transformation efficiency just reduces greatly; 4) what pPIC9K carrier adopted is AOX1 promotor, although control methods are very rigorous, the transcriptional level of this promoters driven mRNA is not also very high, need further to strengthen the activity of promotor; 5) signal sequence that pPIC9K carrier is used carrys out the DNA sequence dna of own coding yeast saccharomyces cerevisiae MF α-SS sequence.Yeast saccharomyces cerevisiae and pichia spp belong to yeast, but aspect codon-bias, still have a certain distance, make it the expression and secretion of foreign protein may not reach best effect; 6) owing to there being the dosage effect of gene, most of genes, along with the increase of copy number, transcribing also of goal gene greatly improves, thereby likely improves the expression amount of goal gene.The frequency occurring due at present traditional yeast expression vector multiple copied is very low; under state of nature; if proceed to the expression vector of pichia pastoris phaff, it is single copy; the probability that occurs multi-copy integration after conversion is about 1-10%; and copy number uncontrollable (Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris.[J] FEMS Microbiol Rev. 2000; 24 (1): 45-66), have randomness, in body, method can be screened the His+ transformant that inserts multiple copied foreign gene, but efficiency is very low, the transformant that screens q.s just can obtain the transformant of high copy, has so greatly improved cost and the workload of the high copy of screening bacterial strain.Because that antibiotic expression cassette unit (" kan ") of the anti-card that pPIC9K carrier contains bacterial origin, give pichia spp G418 resistance, so now general use is all to screen high copy transformant with different G418 resistance levels, but this screening method defectiveness: the one, can cause false negative, because the new cell transforming needs the time to express the meta-bolites of the anti-G418 of q.s, because yeast growth is much slower than bacterium, most of recombination yeast has just been killed before microbiotic to resist on flat board at the meta-bolites of the abundant anti-G418 of accumulation; The 2nd, due to the increase along with copy number, the copy number of " kan " integrating on Yeast genome also increases, if " kan " is leaked in environment, brings certain risk may to the biological safety of environment.

Some defects based on more than existing pPIC9K expression vector, the present invention is based on a series of transformation of pPIC9K expression vector, a kind of in vitro efficient method that builds the yeast expression vector of multiple copied has been proposed, the multiple copied that can realize destination gene expression box in vitro, improves a lot for pichia yeast expression system marking protein.

Summary of the invention

The object of the invention is to propose a kind of in vitro efficient method that builds multi-copy Pichia Expression Vector, pichia pastoris phaff Expression vector pPIC9K is carried out to a series of transformation, to optimize the expression amount of clone's mode and raising target protein.

The following (see figure 1) of specific practice:

1. carrier construction pHBM905A.

First on the basis of original yeast expression vector pPIC9K, design the following primer different fragment that increases respectively, according to fragment corresponding to the primer amplification of table 1 (the corresponding NO 1,2,3,4,5,6 of sequence table), then utilize overlapping PCR method, by 1,2,3 fragment covers build up a large fragment called after A, by 4,5,6 one of fragment intussusception large fragment called after B.A, B fragment is all after Dpn I processes digestion template, with glue, reclaiming test kit reclaims after purifying, Segment A and fragment B are mixed according to 1:1, transform intestinal bacteria DH10 β competent cell, incubated overnight, while growing obvious bacterium colony on flat board, select conversion bacterium colony, extract plasmid, the evaluation of checking order respectively in a small amount.Check order correct recon with regard to called after carrier pHBM905A.

Table 1

The key distinction of the pHBM905A that transformation obtains like this and original pPIC9K carrier is: (1) " Amp+Ori " element is inserted in HIS4 gene order; (2) " Kan " resistant gene box structure is inserted between element " a-MF " and 3 ' AOX1 (TT) as stuffer, and the multiple clone site element " MCS " in original pPIC9K carrier is eliminated; (3) at " Kan " resistant gene box structure 3 ' and 5 ', hold the recognition site of introducing respectively restriction enzyme enzyme NotI and CpoI.

2. build recombinant vectors pHBM905BDM

1) adopt synthetic d1+2 * 201 of conventional gene engineering method AOX1 promoter mutation body fragment, MF4I-SS signal peptide sequence 4I-MF fragment, kana+3 ' AOX1(TT) " fragment (the corresponding NO 7,8,9 of sequence table);

Concrete process is as follows:

Synthetic d1+2 * 201 AOX1 promoter mutation body fragment

D1+2 * 201 AOX1 promoter mutation body is by-777 to-712 sequence deletion of the wild-type AOX1 promoter sequence on pPIC9k; and the sequence of tumor-necrosis factor glycoproteins-203 to-190 obtains (Hartner FS; Ruth C; Langenegger D; Johnson SN; Hyka P; Lin-Cereghino GP, et al. Promoter library designed for fine-tuned gene expression in Pichia pastoris.[J] Nucleic Acids Res. 2008; 36 (12): e76.).

First the above-mentioned carrier pHBM905A of take is template, and with primer pair 2 * 201-F, 2 * 201-R carries out PCR(primer pair sequence in Table 2), at 5 ' of d1+2 * 201 AOX1, hold the recognition site of introducing EcoRI+XbaI.With gel, reclaim test kit again this PCR product is carried out to gel recovery, obtain about 1kb fragment, called after d1+2 * 201 AOX1.

Synthetic MF4I-SS signal peptide sequence 4I-MF fragment

According to the aminoacid sequence of yeast saccharomyces cerevisiae MF α-SS signal peptide, utilize the online software of DNAWorks, it is carried out to sequence optimisation according to pichia spp codon-bias, then the encoding sequence of this signal peptide of synthetic (MF4I-SS).With software DNAman, the nucleotide sequence before and after optimizing is compared, result shows 54 Nucleotide and suddenlys change, and homology is 84.87%.The detailed process of transformation is, utilizes overlapping PCR method, with 14 primers (MF4I-1-MF4I-14) mutually each other template carry out pcr amplification, in reaction system, there will be the goal gene 4I-MF fragment of a small amount of total length.Take this PCR product is template again, carries out second and takes turns pcr amplification, obtains more object fragment.PCR product (408bp) is standby after V-gene gel reclaims test kit recovery purifying, and called after 4I-MF.

Amplification " kana+3 ' AOX1(TT) " fragment

Take carrier pHBM905A as template, with Kan-F and BS-3AOX1(TT) amplification of-R primer pair, at 3 ' AOX1(TT) 3 ' end introduce the recognition site of SpeI+BamHI.Obtain the fragment of 1.6kb left and right, then with gel, reclaim test kit this PCR product is carried out to gel recovery, called after " kana+3 ' AOX1(TT) ".

2) by the method for overlapping by d1+2 * 201 AOX1,4I-MF, " kana+3 ' AOX1(TT) " cover builds up a large fragment, approximately 2.9kb left and right, reclaims test kit with gel this PCR product is carried out to gel recovery, and called after 1 fragment.3) with the anti-method amplification vector skeleton expanding

Take pHBM905A as template, with primer pair AMP-F, AMP-R amplification, obtain the fragment (the corresponding NO 10 of sequence table) of 6kb left and right, with gel, reclaim test kit this PCR product is carried out to gel recovery, and called after 2 fragments.4) by mode (the Zhu D without enzyme clone; Zhong X; Tan R; Chen L; Huang G; Li J, et al. High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli.[J] Anal Biochem. 2010; 397 (2): 162-7) 1 fragment and 2 fragment assemblies are obtained to carrier pHBM905BDM.

This 1 fragment and 2 fragments are used to Dpn I digestion process 2 hours, again these 2 fragments being carried out to solution reclaims after purifying, volume mixture conversion intestinal bacteria DH10 β competent cell by these two fragments with 1:1, incubated overnight, while growing obvious bacterium colony on flat board, select conversion bacterium colony, extract plasmid, the evaluation of checking order respectively in a small amount.The correct person that checks order, can obtain destination carrier pHBM905BDM.

The difference of pHBM905BDM (the corresponding NO 11 of sequence table) carrier and pHBM905A (the corresponding NO 12 of sequence table) carrier: (1) replaces to d1+2 * 201 AOX1 promotor by 5 ' AOX1 promotor; (2) a-MF signal peptide is replaced to 4I-MF signal peptide; (3) at 5 ' end of d1+2 * 201 AOX1 promotor, introduce the recognition site of EcoRI+XbaI, at 3 ' AOX1(TT) 3 ' end introduce the recognition site of SpeI+BamHI.

Primer pair sequence table 2

The synthetic primer of MF4I-SS encoding gene:

MF4I-1?5'?ATAATTGCGACTGGTTCCAATTGACAAGTTGTTGATCTTGACTACTT?3'

MF4I-2?5'?CTTAGATCTTCTCAAGTTATCGTTAAAAGTAGTCAAGATCAACAACTT?3'

MF4I-3?5'?ACGATAACTTGAGAAGATCTAAGAACAACtaaCTGTTTGAAACTATGGCTA?3'

MF4I-4?5'?CTATAAAGATAGATGGAAATCTTGGAATAGCCATAGTTTCAAACAGTTAG?3'

MF4I-5?5'?CAAGATTTCCATCTATCTTTATAGCTGTCTTGTTTGCTGCATCTTCTG?3'

MF4I-6?5'?AGTAGTAGTGTTAACTGGAGCAGCCAAAGCAGAAGATGCAGCAAACAA?3'

MF4I-7?5'?GCTCCAGTTAACACTACTACTGAAGATGAAACTGCTCAAATTCCAGCT?3'

MF4I-8?5'?TTCCAAATCAGAGTAACCAATAACAGCCTCAGCTGGAATTTGAGCAGT?3'

MF4I-9?5'?ATTGGTTACTCTGATTTGGAAGGTGATTTTGATGTTGCTGTTTTGCCA?3'

MF4I-10?5'?TAACAAACCGTTGTTAGTAGAGTTAGAAAATGGCAAAACAGCAACATC?3'

MF4I-115'?CTCTACTAACAACGGTTTGTTAGAGGAAGCCGAGGCTGAAGCTGAACC?3'

MF4I-12?5'?AATAGAGGCAATAGTAGTATTAATGAACTTTGGTTCAGCTTCAGCCTC?3'

MF4I-13?5'?CATTAATACTACTATTGCCTCTATTGCTGCTAAGGAAGAAGGTGTTTC?3'

MF4I-14?5'GCGCCGGACCGGGATCTTAATTCTACTTCCAAAGAAACACCTTCTTCCTTAGC?3'

Plasmid pHBM905BDM transforms primer

AMP-F:?5'ATCGATAAGCTTTAATGCGGTAGTTTA?3'

AMP-R:?5'?AGATCTTGAGATAAATTTCACGTTTAA?3'

2×201-F:?5'ttatctcaagatcGAATTCTCTAGAAGATCTAACATCCAAAGACGAAAGGTT3'

2×201-R:?5'?tataaagatagatggAAATCTTGGAATAGCCATAGTTTCAA?3'

Kan-F:?5'gaaggtgtttctttggaaGTAGAATTAAGATCCCGGTCCG?3'

BS-3AOX1（TT）-R:?5'attaaagcttatcgatGGATCCACTAGTAAGCTTGCACAAAC?3'

3, select to need the exogenous gene albumen of expression, the carrier pHBM905BDM that the present invention builds is suitable for can be at the gene protein of Pichia anomala expression.For example chitosanase gene (chi, GenBank:BBZ88800.1), mannase gene (man, GenBank:AF324506.1), inscribe β-N-acetyl-glucosamine Glycosylase gene (Endo H, GenBank:P04067.1) etc.

4, build the single copy recombinant vectors pHBM905BDM-gene-1 that contains exogenous gene albumen.

Detailed process following (seeing accompanying drawing 2):

Pass through design of primers, 5 ' the end at goal gene gene forward primer adds GTCA, 5 ' end of reverse primer adds GGCCA, uses after high-fidelity enzymatic amplification goal gene fragment, reclaims, then under the condition existing at dTTP, with T4 archaeal dna polymerase, process this fragment 20min in 12 ℃, generate sticky end, pHBM905BDM plasmid is cut through restriction enzyme Cop I and Not I successively enzyme simultaneously, produce two with the fixing strand sticky end of base, agarose gel reclaims object fragment.Finally, after carrier after double digestion and T4 archaeal dna polymerase are processed, fragment is connected after 2h through 16 ℃ of connection test kit (Solution I) ligase enzymes of Takara company, transform intestinal bacteria DH10 β bacterial strain, amicillin resistance LB plate screening transformant, some transformants of picking extract plasmid in a small amount, through the comparison of plasmid size, PCR evaluation and Restriction Enzyme enzyme, cut the recombinant plasmid of identifying that acquisition is complete, to identify correct recon order-checking, correct person's called after pHBM905BDM-gene-1 checks order.

5, based on Biobrick method, built the novel vector pHBM905BDM-gene-n(that the outer series connection of prosthesis repeats to express box structure multiple copied and seen accompanying drawing 3).

By part recombinant plasmid pHBM905BDM-gene-1 EcoRI+SpeI double digestion, agarose gel reclaims and obtains fragment 1, by part pHBM905BDM-gene-1 XbaI+BamHI double digestion, agarose gel reclaims and obtains fragment 2, by part pHBM905BDM-gene EcoRI+BamHI double digestion, agarose gel reclaims and obtains pHBM905BDM carrier framework.Carrier framework after double digestion, fragment 1 and fragment 2 are mixed with 1:2:2, through 16 ℃ of Solution I ligase enzymes, connect after 2h, transform intestinal bacteria DH10 β bacterial strain, amicillin resistance LB plate screening transformant, cuts the 2 copy recombinant plasmid pHBM905BDM-gene-2 that identify that acquisition is correct through the comparison of plasmid size, PCR evaluation and enzyme.3 copies just, on the basis of 2 copies, just can obtain by similar above-mentioned steps, by that analogy, just can obtain recombinant plasmids more than 3 copies.And recombinant plasmids of these high copies just on the basis of 1 correct copy of order-checking enzyme cut that enzyme gets continuously, without checking order again, so just can be cost-saving, time saving and energy saving.

The advantage of the improved carrier pHBM905BDM of the present invention

(1) improve the security of genetic engineering bacterium, linearizing clip size reduces, and has improved transformation efficiency.First on the basis of original yeast expression vector pPIC9K, element on carrier pPIC9K " Amp+Ori " is inserted in HIS4 gene order, with " Amp " resistant gene screening recon, recombinant plasmid is with after SalI linearizing, " Amp+Ori " element is cut off, only having HIS4 unit and destination gene expression box to insert is incorporated in pichia spp host genome, resistant gene Amp expression cassette does not insert and is incorporated in pichia spp host genome, with regard to having reduced resistant gene, be accidentally leaked to risk and the disadvantageous effect that environment brings to the biological safety of environment like this, recombinant plasmid is with after SalI linearizing, and because element " Amp+Ori " is all cut off, effectively fragment reduces, and has greatly improved transformation efficiency.

(2) exonuclease activity based on T4 archaeal dna polymerase, optimizes clone's mode, makes to clone step and simplifies.Improved carrier pHBM905BDM introduces respectively restriction enzyme NotI and CpoI recognition site in the upstream and downstream of " Kan " tolerant gene expression box.The PCR product of external source fragment, under the protection of dTTP, with T4 archaeal dna polymerase, process, utilize its 3 '-5 ' exonuclease activity, produce the sticky end identical with linear carrier skeleton after NotI and CpoI double digestion, then after the carrier framework after double digestion and T4 archaeal dna polymerase being processed, external source fragment is connected after 2h through 16 ℃ of connection test kit (Solution I) ligase enzymes of Takara company, transform intestinal bacteria DH10 β bacterial strain, amicillin resistance LB plate screening transformant, through the comparison of plasmid size, PCR evaluation and Restriction Enzyme enzyme are cut evaluation just can obtain complete recombinant plasmid.This method has been avoided the complex operations of PCR fragment double digestion; but also can consider the restriction enzyme site of gene inside; the complex operations such as sudden change of the inner restriction enzyme site of gene have been saved; and need to not add restriction enzyme recognition sequence and extra protection base at PCR fragment 5 ' end; reduced primer length; reduce cost, and because outstanding sticky end has 4 different bases, just greatly reduced carrier from the probability connecting.

(3) based on AOX1 promotor, optimize promoter sequence, improve the transcriptional level of mRNA, thereby may improve the expression amount of foreign protein.In pichia pastoris phaff expression system, the most frequently used promotor is wild-type AOX1 promotor at present.Research report, some are derived from the mutant of wild-type AOX1 promotor, can affect the expression level of foreign protein.There is bibliographical information; d1+2 * 201 AOX1 promoter mutation body (Hartner FS; Ruth C; Langenegger D; Johnson SN; Hyka P, Lin-Cereghino GP, et al. Promoter library designed for fine-tuned gene expression in Pichia pastoris.[J] Nucleic Acids Res. 2008; 36 (12): e76.) than original AOX1 promotor, more can effectively improve transcribing of mRNA, thereby likely improve the expression amount of foreign protein, so the present invention has selected one to make the promoter mutation body d1+2 * 201 AOX1 promotor of exogenous protein expression amount increase rate maximum replace the AOX1 promotor on carrier pHBM905A, transformation obtains novel vector pHBM905BDM, and select chitosanase gene (chi, GenBank:BBZ88800.1) etc. gene is verified, to determine the impact of d1+2 * 201 AOX1 saltant type promotor on foreign protein expression amount.

(4) the MF α-SS signal peptide based on deriving from yeast saccharomyces cerevisiae, optimizes signal peptide sequence, improves to a certain extent protein expression amount.Because different systems exists the preferences of codon, the method is according to the aminoacid sequence that derives from MF α-SS signal peptide of yeast saccharomyces cerevisiae, after codon optimized with pichia spp, this signal coding sequence of synthetic, to replace the MF α-SS signal coding sequence in initial carrier pPIC9K, build a novel vector pHBM905BDM, and select chitosanase gene (chi, GenBank:BBZ88800.1) etc. gene pairs its clone and functional verification, to determine signal peptide MF4I-SS and the impact of wild-type MF α-SS on foreign protein expression amount after optimization.

(5) novel vector pHBM905BDM is applicable to the outer series connection of Biobrick method structure prosthesis and repeats to express box structure multiple copied.By this carrier, can realize in vitro structure multiple copied, can greatly increase like this probability of multiple copied, and the copy number of gene is determined in vitro, builds as required the recombinant plasmid of different copy numbers.

Accompanying drawing explanation:

Fig. 1 is that the present invention builds the in vitro building process of realizing high copy yeast expression vector.First on the basis of original Expression vector pPIC9K, " Kan " tolerant gene expression box in carrier pPIC9K is inserted into multiple clone site, in the upstream and downstream of " Kan " tolerant gene expression box, adds restriction enzyme noti and cpoi recognition site, and element " Ampr+Ori " is inserted in HIS4 gene order, carrier pHBM905A obtained.On the basis of pHBM905A carrier, 5 ' AOX1 promotor is replaced to d1+2 * 201 AOX1 promotor again, signal peptide a-MF replaces to 4I-MF, and introduces at the 3 ' end of d1+2 * 201 AOX1 ecoRi+ xbathe restriction enzyme site of I, introduces at the 3 ' end of 3 ' AOX1 (TT) spei+ bamHthe restriction enzyme site of I.

Fig. 2 in the present invention links goal gene (gene) whole process of the clone's mode on carrier pHBM905A.By design of primers, at 5 ' end of forward primer, add GTCA, 5 ' end of reverse primer adds GGCCA, uses after high-fidelity enzymatic amplification goal gene fragment, reclaims, and under the condition then existing at dTTP, uses t4archaeal dna polymerase is processed this fragment 20min in 12 ℃, generates sticky end, simultaneously by pHBM905A plasmid through restriction enzyme copi and noti successively enzyme is cut, and produces two with the fixing strand sticky end of base, and agarose gel reclaims object fragment.Finally, the carrier after double digestion and T4 archaeal dna polymerase are processed after fragment through the connection test kit of Takara company ( solutioni) after 16 ℃ of connection 2h of ligase enzyme, transform intestinal bacteria DH10 β bacterial strain, amicillin resistance LB plate screening transformant, cuts evaluation through the comparison of plasmid size, PCR evaluation and Restriction Enzyme enzyme and obtains complete recombinant plasmid.Clone's mode of goal gene (gene) being linked to carrier pHBM905BDM is the same with carrier pHBM905A.

Fig. 3 is from the process of the plasmid pHBM905BDM-gene 1 copy external structure multiple copy expression cassette of goal gene is housed.Plasmid pHBM905BDM-gene uses ecoRi+ spei double digestion, agarose gel reclaims and obtains Fragment1, and pHBM905BDM-gene uses xbai+ bamHi double digestion, agarose gel reclaims and obtains Fragment2, and pHBM905BDM-gene uses ecoRi+ bamHi double digestion, agarose gel reclaims and obtains pHBM905BDM carrier framework.By the carrier after double digestion, fragment Fragment1 and fragment Fragment2, through 16 ℃ of Solution I ligase enzymes, connect after 2h, transform intestinal bacteria DH10 β bacterial strain, amicillin resistance LB plate screening transformant, cuts 2 copies of identifying that acquisition is correct through the comparison of plasmid size, PCR evaluation and enzyme.3 copies are just on the basis of 2 copies, and similar above-mentioned steps just can obtain, and by that analogy, just can obtain recombinant plasmids more than 3 copies.And plasmids of these high copies just on the basis of 1 correct copy of order-checking enzyme cut that enzyme gets continuously, without checking order again, so just can be cost-saving, time saving and energy saving.

Fig. 4 is for exogenous gene albumen--multi-copy vector pHBM905BDM that chitoanase (chi) gene the is verified structure impact on protein expression amount.Get the yeast supernatant of induction after 5 days, SDS-PAGE electrophoresis detection.M represents protein Marker, 0 swimming lane represents unloaded contrast (GS905A), and 1 swimming lane represents GS905A-chi, and 2 swimming lanes represent GS905BDM-chi-1 (1 copy), 3 swimming lanes represent GS905BDM-chi-2(2 copy), 4 swimming lanes represent GS905BDM-chi-3(3 copy).From scheming, can see that promotor and signal peptide improve to protein expression amount, along with the gene copy number increase also can increase protein expression amount.

Fig. 5 is for exogenous gene albumen--multi-copy vector pHBM905BDM that inscribe β-N-acetyl-glucosamine Glycosylase (EndoH) gene the is verified structure impact on protein expression amount.Get the yeast supernatant of induction after 5 days, SDS-PAGE electrophoresis detection.M represents protein Marker, and 0 swimming lane represents unloaded contrast (GS905A), and 1 swimming lane represents GS905A-EndoH, and 2 swimming lanes represent GS905BDM-EndoH-1, and 3 swimming lanes represent GS905BDM-EndoH-2, and 4 swimming lanes represent GS905BDM-EndoH-3.From scheming, can see that promotor and signal peptide improve to protein expression amount, along with the gene copy number increase also can increase protein expression amount.

embodiment

With example, the present invention is further described below

Embodiment 1: carrier construction pHBM905A as stated above, pHBM905BDM.

Embodiment 2: build and contain exogenous gene albumen--the multiple copied recombinant vectors pHBM905BDM-chi-n of chitoanase (chi).

By pHBM905A, pHBM905BDM plasmid is cut through restriction enzyme Cop I and Not I successively enzyme, produces with two fixing carriers of base strand sticky end, and agarose gel reclaims this carrier.Secondly, according to known chitosanase gene sequence, adopt GeneTool software design chi pF:chi-F:5' gTCAAtGCTGGCAGTCCCAGCCTCT 3' and chi-R:5' gGCCAtwo primers of TTAAGCAAAACACTCAGCCCAAT 3', use primerstar archaeal dna polymerase to carry out pcr amplification, obtain chi(650bb) gene fragment, after this fragment agarose gel is reclaimed, under the condition existing at dTTP, with 12 ℃ of T4 archaeal dna polymerases, process 20min, generate and pHBM905A, the sticky end that pHBM905BDM carrier matches, reclaims fragment.Then carrier is connected after 2 hours through 16 ℃ of Solution I enzymes with fragment, is converted into intestinal bacteria DH10 β competent cell, penbritin LB plate screening transformant, cuts and identifies acquisition recombinant plasmid through the comparison of plasmid size, PCR evaluation and enzyme.Random choose recombinant plasmid is served the order-checking of extra large Sani Bioisystech Co., Ltd, by the correct recombinant plasmid of sequencing result called after pHBM905A chi respectively, pHBM905BDM-chi-1.

With the plasmid of the correct pHBM905BDM-chi-1 plasmid construction multiple copied of order-checking, concrete operation step is as follows again:

1) pHBM905BDM-chi-1 plasmid is divided into three parts.A recombinant plasmid pHBM905BDM chi-1 EcoRI+SpeI double digestion, agarose gel reclaims and obtains fragment 1; Another part of pHBM905BDM-chi-1 XbaI+BamHI double digestion, agarose gel reclaims and obtains fragment 2; With EcoRI and BamH are two, cut for the 3rd part, agarose reclaims and obtains pHBM905BDM carrier framework,

2), by above-mentioned carrier framework, fragment 1 is mixed and is connected 2 hours through 16 ℃ of Solution I enzymes with 1:2:2 with fragment 2.3) transform intestinal bacteria DH10 β competent cell, penbritin LB plate screening transformant, cuts evaluation through the comparison of plasmid size, PCR evaluation and enzyme and obtains restructuring 2 copy plasmids, without checking order again, and called after pHBM905BDM-chi-2.4) plasmid that builds 3 copies just just can obtain by the plasmids of 2 copies and the plasmid repetition above-mentioned steps of 1 copy, called after pHBM905BDM-chi-3.

By the unloaded pHBM905A building, pHBM905A chi, pHBM905BDM-chi-1, pHBM905BDM-chi-2, Sal I linearization for enzyme restriction for pHBM905BDM-chi-3 plasmid, cuts product electric shock by the enzyme after reclaiming and is converted into Pichia pastoris GS115 yeast competent cell, is then coated with histidine defect substratum MD dull and stereotyped, cultivate 3 days for 28 ℃, obtain recombinant conversion.The random transformant genome that extracts, take it as template, chi pF and chi pR are that primer carries out PCR evaluation, the transformant that amplification obtains 650bp product is positive colony, by the correct recombinant clone called after GS905A-chi of checking, GS905BDM-chi-1, GS905BDM-chi-2, GS905BDM-chi-3.Finally, will identify that correct recombinant conversion of checking is inoculated in 50 mL BMGY substratum, 28 ℃, 200 r/min cultivation 48h, to OD ₆₀₀for 6-9, in centrifugal 5000 r/min of room temperature, 5 min, collect thalline, subsequently, thalline is transferred in 25 mL BMMY substratum, 28 ℃, 200 r/min cultivations, every 24 h, add methyl alcohol (0.5% that dosage is culture volume), GS905A-chi, GS905BDM-chi-1, GS905BDM-chi-2, GS905BDM-chi-3 positive transformant, after abduction delivering, is induced after 5 days the protein expression level of sampling analysis different carriers.(see figure 4) M represents protein Marker, 0,1,2,3,4 swimming lanes represent respectively unloaded contrast (GS905A), GS905A-chi, GS905BDM-chi-1, GS905BDM-chi-2, GS905BDM-chi-3 can see that from scheming promotor and signal peptide improve to protein expression amount, along with the gene copy number increase also can increase protein expression amount.

Embodiment 3: build and contain exogenous gene albumen--the multiple copied recombinant vectors pHBM905BDM-EndoH-n of inscribe β-N-acetyl-glucosamine Glycosylase (EndoH)

By pHBM905A, pHBM905BDM plasmid is cut through restriction enzyme Cop I and Not I successively enzyme, produces with two fixing carriers of base strand sticky end, and agarose gel reclaims this carrier.Secondly, according to known sequence, adopt two primers of GeneTool software design:

EndoH-pF:5’ GTCAATGGCTCCAGTTAAACAAGGTCCAACTTCCGTT3’

EndoH-pR:?5’ GGCCATTATGGAGTTCTAACAGCTTCAGATCCGTACA3’

Use primerstar archaeal dna polymerase to carry out pcr amplification, obtain EndoH(1kb) gene fragment, after this fragment agarose gel is reclaimed, under the condition existing at dTTP, with 12 ℃ of T4 archaeal dna polymerases, process 20min, generate and pHBM905A, the sticky end that pHBM905BDM carrier matches, reclaims fragment.Then carrier is connected after 2 hours through 16 ℃ of Solution I enzymes with fragment, is converted into intestinal bacteria DH10 β competent cell, penbritin LB plate screening transformant, cuts and identifies acquisition recombinant plasmid through the comparison of plasmid size, PCR evaluation and enzyme.Random choose recombinant plasmid is served the order-checking of extra large Sani Bioisystech Co., Ltd, by the correct recombinant plasmid of sequencing result called after pHBM905A EndoH respectively, pHBM905BDM-EndoH-1.

With the plasmid of the correct pHBM905BDM-EndoH-1 plasmid construction multiple copied of order-checking, concrete operation step is as follows again:

1) pHBM905BDM-EndoH-1 plasmid is divided into two parts.A recombinant plasmid pHBM905BDM EndoH-1 EcoRI+SpeI double digestion, agarose gel reclaims and obtains fragment 1; Another part of pHBM905BDM-EndoH-1 XbaI+BamHI double digestion, agarose gel reclaims and obtains fragment 2.

2) will with EcoRI and BamHI are two, cut the pHBM905BDM carrier framework that agarose reclaims, fragment 1 is mixed and is connected 2 hours through 16 ℃ of Solution I enzymes with fragment 2.

3) transform intestinal bacteria DH10 β competent cell, penbritin LB plate screening transformant, cuts evaluation through the comparison of plasmid size, PCR evaluation and enzyme and obtains restructuring 2 copy plasmids, without checking order again, and called after pHBM905BDM-EndoH-2.4) plasmid that builds 3 copies just just can obtain by the plasmids of 2 copies and the plasmid repetition above-mentioned steps of 1 copy, called after pHBM905BDM-EndoH-3.

By the unloaded pHBM905A building, pHBM905A EndoH, pHBM905BDM-EndoH-1, pHBM905BDM-EndoH-2, Sal I linearization for enzyme restriction for pHBM905BDM-EndoH-3 plasmid, cuts product electric shock by the enzyme after reclaiming and is converted into Pichia pastoris GS115 yeast competent cell, is then coated with histidine defect substratum MD dull and stereotyped, cultivate 3 days for 28 ℃, obtain recombinant conversion.The random transformant genome that extracts, take it as template, EndoH-pF and EndoH-pR are that primer carries out PCR evaluation, the transformant that amplification obtains 1kb product is positive colony, by the correct recombinant clone called after GS905A-EndoH of checking, GS905BDM-EndoH-1, GS905BDM-EndoH-2, GS905BDM-EndoH-3.Finally, will identify that correct recombinant conversion of checking is inoculated in 50 mL BMGY substratum, 28 ℃, 200 r/min cultivation 48h, to OD ₆₀₀for 6-9, in centrifugal 5000 r/min of room temperature, 5 min, collect thalline, subsequently, thalline is transferred in 25 mL BMMY substratum, 28 ℃, 200 r/min cultivations, every 24 h, add methyl alcohol (0.5% that dosage is culture volume), GS905A-EndoH, GS905BDM-EndoH-1, GS905BDM-EndoH-2, GS905BDM-EndoH-3 positive transformant, after abduction delivering, is induced after 5 days the protein expression level of sampling analysis different carriers.(see figure 5) M represents protein Marker, 0,1,2,3,4 swimming lanes represent respectively unloaded contrast (GS905A), GS905A-EndoH, GS905BDM-EndoH-1, GS905BDM-EndoH-2, GS905BDM-EndoH-3 can see that from scheming promotor and signal peptide improve to protein expression amount, along with the gene copy number increase also can increase protein expression amount.

Claims (3)

1. efficiently build a method for multi-copy Pichia Expression Vector in vitro, it is characterized in that step is:

1). carrier construction pHBM905A

First on the basis of original yeast expression vector pPIC9K, design the following primer different fragment that increases respectively, according to fragment corresponding to the primer amplification of table 1, then utilize overlapping PCR method, by 1,2,3 one of fragment intussusception large fragment called after A, by 4,5,6 one of fragment intussusception large fragment called after B; A, B fragment is all passed through dpni processes after digestion template, reclaims test kit reclaim after purifying with glue, and Segment A and fragment B are mixed according to 1:1, transforms intestinal bacteria

Table 1

DH10 β competent cell, incubated overnight, while growing obvious bacterium colony on flat board, selects conversion bacterium colony, extracts plasmid in a small amount, the evaluation of checking order respectively, the correct recon that checks order is with regard to called after carrier pHBM905A;

2). build recombinant vectors pHBM905BDM

A) adopt synthetic d1+2 * 201 of conventional gene engineering method AOX1 promoter mutation body fragment, mF4I-SSsignal peptide sequence 4I-MF fragment, kana+3 ' AOX1(TT) " fragment;

B) by the method for overlapping by d1+2 * 201 AOX1,4I-MF, " kana+3 ' AOX1(TT) " cover builds up a large fragment, approximately 2.9kb left and right, reclaims test kit with gel this PCR product is carried out to gel recovery, and called after 1 fragment;

C) with the anti-method amplification vector skeleton expanding

Take pHBM905A as template, with primer pair AMP-F, AMP-R amplification, obtain the fragment of 6kb left and right, with gel, reclaim test kit this PCR product is carried out to gel recovery, and called after 2 fragments;

D) this 1 fragment and 2 fragments are used dpni digestion process 2 hours, again these 2 fragments being carried out to solution reclaims after purifying, volume mixture conversion intestinal bacteria DH10 β competent cell by these two fragments with 1:1, incubated overnight, while growing obvious bacterium colony on flat board, selects conversion bacterium colony, extract in a small amount plasmid, the evaluation of checking order respectively, the correct person that checks order, can obtain destination carrier pHBM905BDM;

3). select to need the exogenous gene albumen gene of expression;

4). build the single copy recombinant vectors pHBM905BDM-gene-1 that contains exogenous gene albumen

5). based on Biobrick method, built outer series connection of prosthesis and repeated to express the novel vector pHBM905BDM-gene-n of box structure multiple copied.

2. a kind of in vitro efficient method that builds multi-copy Pichia Expression Vector according to claim 1, the step that it is characterized in that building the single copy recombinant vectors pHBM905BDM-gene-1 that contains exogenous gene albumen is:

By design of primers, at 5 ' of goal gene gene forward primer, to hold and add GTCA, 5 ' end of reverse primer adds GGCCA, uses after high-fidelity enzymatic amplification goal gene fragment, reclaim, under the condition then existing at dTTP, use t4archaeal dna polymerase is processed this fragment 20min in 12 ℃, generates sticky end, simultaneously by pHBM905BDM plasmid through restriction enzyme copi and noti successively enzyme is cut, and produces two with the fixing strand sticky end of base, and agarose gel reclaims object fragment; Finally, the carrier after double digestion and T4 archaeal dna polymerase are processed after fragment through the connection test kit of Takara company ( solutioni) after 16 ℃ of connection 2h of ligase enzyme, transform intestinal bacteria DH10 β bacterial strain, amicillin resistance LB plate screening transformant, some transformants of picking extract plasmid in a small amount, through the comparison of plasmid size, PCR evaluation and Restriction Enzyme enzyme, cut the recombinant plasmid of identifying that acquisition is complete, to identify correct recon order-checking, correct person's called after pHBM905BDM-gene-1 checks order.

3. a kind of in vitro efficient method that builds multi-copy Pichia Expression Vector according to claim 1 and 2, is characterized in that the step that builds the multiple copied recombinant vectors pHBM905BDM-gene-n that contains exogenous gene albumen is:

Part recombinant plasmid pHBM905BDM-gene-1 is used ecoRi+ spei double digestion, agarose gel reclaims and obtains fragment 1, and part pHBM905BDM-gene-1 is used xbai+ bamHi double digestion, agarose gel reclaims and obtains fragment 2, and part pHBM905BDM-gene is used ecoRi+ bamHi double digestion, agarose gel reclaims and obtains pHBM905BDM carrier framework; Carrier framework after double digestion, fragment 1 and fragment 2 are mixed according to 1:2:2, through 16 ℃ of Solution I ligase enzymes, connect after 2h, transform intestinal bacteria DH10 β bacterial strain, amicillin resistance LB plate screening transformant, cuts the 2 copy recombinant plasmid pHBM905BDM-gene-2 that identify that acquisition is correct through the comparison of plasmid size, PCR evaluation and enzyme; 3 copies just, on the basis of 2 copies, just can obtain by similar above-mentioned steps, by that analogy, just can obtain recombinant plasmids more than 3 copies.

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