CN1459507A - Multi copy yeast expression carrier for proceeding correct secretory expression against exogenous gene - Google Patents

Multi copy yeast expression carrier for proceeding correct secretory expression against exogenous gene Download PDF

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CN1459507A
CN1459507A CN02117906A CN02117906A CN1459507A CN 1459507 A CN1459507 A CN 1459507A CN 02117906 A CN02117906 A CN 02117906A CN 02117906 A CN02117906 A CN 02117906A CN 1459507 A CN1459507 A CN 1459507A
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sequence
carrier
yeast expression
expression carrier
proceeding
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CN1212402C (en
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陈薇
齐连权
于长明
付玲
来大志
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Institute of Bioengineering Chinese Academy of Military Medical Sciences
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Institute of Microbiology and Epidemiology of AMMS
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Abstract

A multi-copy yeast expression carrier able to make exogeneous gene correctly secrete and express in yeast, its DNA sequence, and two pairs of special primers for configuring said expression carrier and their sequences are disclosed.

Description

Foreign gene is carried out the multiple copied Yeast expression carrier of correct secreting, expressing
Technical field
The present invention relates to a kind of Yeast expression carrier and make up primer, particularly relate to a kind of multiple copied Yeast expression carrier and structure primer thereof that foreign gene is carried out correct secreting, expressing.
Background technology
Pichia yeast expression system (Pichia pastoris) is used to one of the most successful expression system of expressing foreign protein, development in recent years is very rapid, at present existing 200 multiple protein are expressed (CreggJM in this expression system, Cereghino JL, Shi JY, et al.Recombinant protein expression in Pichiapastoris[J] .Mol Biotechnol, 2000,16 (1): 23-52.).Pichia spp is a kind of methyl alcohol nutritional type yeast, its growth rapidly, culture condition is simple, can the high-density cultured continuously, genetic manipulation is similar to yeast saccharomyces cerevisiae, technology is maturation quite.
Pichia yeast expression system comprises in the born of the same parents expresses and the secreting, expressing dual mode, and wherein secreting, expressing is owing to its expression amount height, and glycosylation is complete, and disulfide linkage and higher structure form correctly, and especially the later separation purifying is simple and convenient and enjoy favor.Proteic secreting, expressing needs the participation of signal peptide.Had some kinds of signal peptide sequences of the signal peptide sequence that comprises some foreign protein self to be successfully used to the secreting, expressing of foreign protein, but wherein the most successful be the signal peptide sequence that from yeast saccharomyces cerevisiae, separates the alpha factor that obtains.
Below be the partial sequence of signal peptide alpha factor and multiple clone site:
Xho?I?????????Kex2????????????????SnaB?I???EcoR?IATG?AGA?TTT?CCT?…?TCT CTC?GAG?AAA?AGA↓GAG?GCT??GAA?GCT TAC?GTA? GAA?TTC?CCT?…Met?Arg?Phe?Pro?…?Ser?Leu?Glu?Lys?Arg??Glu?Ala↑Glu?Ala↑Tyr?Val?Glu?Phe?Pro?…
Ste13
ATG is a translation initiation codon, and Kex2 is responsible for the shearing of signal peptide, and it is essential that the Glu-Ala-Glu-Ala tumor-necrosis factor glycoproteins is not Kex2 shearing institute, but its existence can improve shear efficiency; Another kind of proteolytic enzyme Ste13 can continue the Glu-Ala-Glu-Ala tumor-necrosis factor glycoproteins is sheared, but shears often also not exclusively.In addition, also have two amino-acid residues can stay the N end (Tyr and Val) of expressing protein at least.
For above-mentioned reasons, existing Yeast expression carrier, when foreign gene is carried out secreting, expressing, expressing protein often exists incorrect N terminal sequence, these amino-acid residues that have more can influence and change expressed proteic 26S Proteasome Structure and Function (Goda S, Takano K, Yamagata Y, et al.Effect of extra N-terminal residueson the stability and folding of human lysozyme expressed in Pichia pastoris[J] .Protein Eng, 2000,13 (4): 299-307.).
Summary of the invention
The purpose of this invention is to provide a kind of multiple copied Yeast expression carrier that can make foreign gene correct secreting, expressing in yeast.
A kind of multiple copied Yeast expression carrier provided by the present invention, its dna sequence dna is the nucleotide sequence of sequence 1 in the sequence table.
A kind of multiple copied Yeast expression carrier provided by the present invention, contain two resistant genes of intestinal bacteria replication orgin Col E1 and penbritin and kantlex, wherein kalamycin resistance gene can make pichia spp that G418 is produced resistance, is used for the screening that multiple copied inserts.
A kind of multiple copied Yeast expression carrier provided by the present invention is an inducible promoter with the promotor (pAOX1) of alcohol oxidase gene, suppressed by glucose, and just transcriptional start, translation is very suitable for expression of exogenous gene behind the methanol induction.
Multiple copied Yeast expression carrier of the present invention is called pMEX9K.
Second purpose of the present invention provides a kind of primer special that makes up above-mentioned multiple copied Yeast expression carrier.
Two pairs of primers provided by the present invention, they are nucleotide sequences of sequence 2 in the sequence table, sequence 3 and sequence 4, sequence 5.
Utilize carrier of the present invention, make foreign protein N hold first amino acid to be close to after Lys-Arg, foreign protein energy secreting, expressing is correct, and N end sequencing result shows that the albumen of secreting, expressing is consistent with the native protein structure.
Description of drawings
Fig. 1 is the structure synoptic diagram of expression vector of the present invention.
Fig. 2 cuts evaluation for the enzyme of expression vector of the present invention.
Fig. 3-Fig. 9 is that foreign gene (people's ω-Interferon, rabbit) is measured figure at the N terminal sequence of the recombinant protein of expression vector pMEX9K secreting, expressing of the present invention
Embodiment
The structure of embodiment 1, carrier pMEX9K of the present invention
As shown in Figure 1, the structure of carrier pMEX9K of the present invention may further comprise the steps:
(1) the plasmid pPIC9K DNA that sells with Invitrogen company is a template, is primer pyrobest enzymatic amplification purpose fragment with sequence in the sequence table 2 and sequence 3.The condition of PCR is: 94 ℃ of pre-sex change 180s; 94 ℃ of sex change 40s; 55 ℃ of annealing 40s; 72 ℃ are extended 60s; 30 circulations; Add general T aq enzyme again and extend 10min;
(2) 1% agarose gel electrophoresis are cut glue and are reclaimed the PCR product and be connected into the pGEMT of TaKaRa company carrier.Behind the transformed into escherichia coli DH5 α, screening positive clone pGEMTXho.
(3) be that primer utilizes point mutation test kit MutanBEST Kit that pGEMTXho is suddenlyd change to remove Xho I restriction enzyme site wherein with sequence in the sequence table 4 and sequence 5.The operation by specification carries out.
(4) the recombinant plasmid pGEMTMuXho after screening and the evaluation sudden change.
(5) downcut the external source fragment MuXho (0.6kb) of sudden change with restriction endonuclease sma I and Sph I from recombinant plasmid pGEMTMuXho, and cut the dna fragmentation (8.7kb) that glue reclaims after being connected into the above-mentioned two kinds of endonuclease digestion pPIC9K of same usefulness.
(6) behind the transformed into escherichia coli DH5 α, screening positive clone pMEX9K and order-checking.Sequencing result shows that sequence is correct.
The enzyme of embodiment 2, expression vector of the present invention is cut evaluation
As shown in Figure 2, confirm that through restriction endonuclease analysis and sequencing vector construction is correct, among the figure: M1.DL15000 Marker; 1.pPIC9K; 2.pMEX9K; 3.pPIC9K/Nae I; 4.pPIC9K/Xho I; 5.pMEX9K/Xho I; M2.DL2000 Marker.Behind the transformed into escherichia coli DH5 α, this carrier makes recipient bacterium obtain penbritin and kalamycin resistance, confirms that the kalamycin resistance gene function is normal.
Embodiment 3, utilize carrier of the present invention to make foreign gene correct secreting, expressing in pichia spp
Adopt the ordinary method in the biotechnology, utilize carrier of the present invention that foreign gene people ω-Interferon, rabbit (Interferon ω) is imported in the pichia spp and carry out secreting, expressing, expressed proteins is carried out N terminal sequence mensuration (utilize 491 sequential analysis of protein instrument of PE company, adopt the automatic edman degradation method), the result shows (Fig. 3): preceding 5 amino-acid residues of target protein N end, its sequence is: LGCDL, and consistent with native protein N terminal amino acid sequence.
Sequence Table <160> 5 <210> 1 <211> 9276 <212> DNA <213> Artificial Sequence <220> <223> <400> Serial Number agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcgcca taccgtttgt 540 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900 caacttgaga agatcaaaaa acaactaatt attcgaagga tccaaacgat gagatttcct 960 tcaattttta ctgcagtttt attcgcagca tcctccgcat tagctgctcc agtcaacact 1020 acaacagaag atgaaacggc acaaattccg gctgaagctg tcatcggtta ctcagattta 1080 gaaggggatt tcgatgttgc tgttttgcca ttttccaaca gcacaaataa cgggttattg 1140 tttataaata ctactattgc cagcattgct gctaaagaag aaggggtatc tctcgagaaa 1200 agagaggctg aagcttacgt agaattccct agggcggccg cgaattaatt cgccttagac 1260 atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa 1320 tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt 1380 ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc 1440 ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa 1500 tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta 1560 agtgagaagt tcgtttgtgc aagcttatcg ataagcttta atgcggtagt ttatcacagt 1620 taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc 1680 tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc 1740 tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg ctgctagcgc 1800 tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg 1860 gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac gcgatcatgg 1920 cgaccacacc cgtcctgtgg atctatcgaa tctaaatgta agttaaaatc tctaaataat 1980 taaataagtc ccagtttctc catacgaacc ttaacagcat tgcggtgagc atctagacct 2040 tcaacagcag ccagatccat cactgcttgg ccaatatgtt tcagtccctc aggagttacg 2100 tcttgtgaag tgatgaactt ctggaaggtt gcagtgttaa ctccgctgta ttgacgggca 2160 tatccgtacg ttggcaaagt gtggttggta ccggaggagt aatctccaca actctctgga 2220 gagtaggcac caacaaacac agatccagcg tgttgtactt gatcaacata agaagaagca 2280 ttctcgattt gcaggatcaa gtgttcagga gcgtactgat tggacatttc caaagcctgc 2340 tcgtaggttg caaccgatag ggttgtagag tgtgcaatac acttgcgtac aatttcaacc 2400 cttggcaact gcacagcttg gttgtgaaca gcatcttcaa ttctggcaag ctccttgtct 2460 gtcatatcga cagccaacag aatcacctgg gaatcaatac catgttcagc ttgagacaga 2520 aggtctgagg caacgaaatc tggatcagcg tatttatcag caataactag aacttcagaa 2580 ggcccagcag gcatgtcaat actacacagg gctgatgtgt cattttgaac catcatcttg 2640 gcagcagtaa cgaactggtt tcctggacca aatattttgt cacacttagg aacagtttct 2700 gttccgtaag ccatagcagc tactgcctgg gcgcctcctg ctagcacgat acacttagca 2760 ccaaccttgt gggcaacgta gatgacttct ggggtaaggg taccatcctt cttaggtgga 2820 gatgcaaaaa caatttcttt gcaaccagca actttggcag gaacacccag catcagggaa 2880 gtggaaggca gaattgcggt tccaccagga atatagaggc caactttctc aataggtctt 2940 gcaaaacgag agcagactac accagggcaa gtctcaactt gcaacgtctc cgttagttga 3000 gcttcatgga atttcctgac gttatctata gagagatcaa tggctctctt aacgttatct 3060 ggcaattgca taagttcctc tgggaaagga gcttctaaca caggtgtctt caaagcgact 3120 ccatcaaact tggcagttag ttctaaaagg gctttgtcac cattttgacg aacattgtcg 3180 acaattggtt tgactaattc cataatctgt tccgttttct ggataggacg acgaagggca 3240 tcttcaattt cttgtgagga ggccttagaa acgtcaattt tgcacaattc aatacgacct 3300 tcagaaggga cttctttagg tttggattct tctttaggtt gttccttggt gtatcctggc 3360 ttggcatctc ctttccttct agtgaccttt agggacttca tatccaggtt tctctccacc 3420 tcgtccaacg tcacaccgta cttggcacat ctaactaatg caaaataaaa taagtcagca 3480 cattcccagg ctatatcttc cttggattta gcttctgcaa gttcatcagc ttcctcccta 3540 attttagcgt tcaacaaaac ttcgtcgtca aataaccgtt tggtataaga accttctgga 3600 gcattgctct tacgatccca caaggtggct tccatggctc taagaccctt tgattggcca 3660 aaacaggaag tgcgttccaa gtgacagaaa ccaacacctg tttgttcaac cacaaatttc 3720 aagcagtctc catcacaatc caattcgata cccagcaact tttgagttgc tccagatgta 3780 gcacctttat accacaaacc gtgacgacga gattggtaga ctccagtttg tgtccttata 3840 gcctccggaa tagacttttt ggacgagtac accaggccca acgagtaatt agaagagtca 3900 gccaccaaag tagtgaatag accatcgggg cggtcagtag tcaaagacgc caacaaaatt 3960 tcactgacag ggaacttttt gacatcttca gaaagttcgt attcagtagt caattgccga 4020 gcatcaataa tggggattat accagaagca acagtggaag tcacatctac caactttgcg 4080 gtctcagaaa aagcataaac agttctacta ccgccattag tgaaactttt caaatcgccc 4140 agtggagaag aaaaaggcac agcgatacta gcattagcgg gcaaggatgc aactttatca 4200 accagggtcc tatagataac cctagcgcct gggatcatcc tttggacaac tctttctgcc 4260 aaatctaggt ccaaaatcac ttcattgata ccattattgt acaacttgag caagttgtcg 4320 atcagctcct caaattggtc ctctgtaacg gatgactcaa cttgcacatt aacttgaagc 4380 tcagtcgatt gagtgaactt gatcaggttg tgcagctggt cagcagcata gggaaacacg 4440 gcttttccta ccaaactcaa ggaattatca aactctgcaa cacttgcgta tgcaggtagc 4500 aagggaaatg tcatacttga agtcggacag tgagtgtagt cttgagaaat tctgaagccg 4560 tatttttatt atcagtgagt cagtcatcag gagatcctct acgccggacg catcgtggcc 4620 gacctgcagg gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca 4680 taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga 4740 gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct 4800 gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa 4860 caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac caattaacca 4920 attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat 4980 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc 5040 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa 5100 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag 5160 tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa 5220 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc 5280 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag 5340 gaatcgaatg caaccggogc aggaacactg ccagcgcatc aacaatattt tcacctgaat 5400 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc 5460 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca 5520 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt 5580 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt 5640 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta 5700 atcgcggcct ctagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac 5760 tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt 5820 aacatcagag attttgagac acaacgtggc tttccccccc ccccctgcag gtcggcatca 5880 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc 5940 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg 6000 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc 6060 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc 6120 gtcgagtatc tatgattgga agtatgggaa tggtgatacc cgcattcttc agtgtcttga 6180 ggtctcctat cagattatgc ccaactaaag caaccggagg aggagatttc atggtaaatt 6240 tctctgactt ttggtcatca gtagactcga actgtgagac tatctcggtt atgacagcag 6300 aaatgtcctt cttggagaca gtaaatgaag tcccaccaat aaagaaatcc ttgttatcag 6360 gaacaaactt cttgtttcga actttttcgg tgccttgaac tataaaatgt agagtggata 6420 tgtcgggtag gaatggagcg ggcaaatgct taccttctgg accttcaaga ggtatgtagg 6480 gtttgtagat actgatgcca acttcagtga caacgttgct atttcgttca aaccattccg 6540 aatccagaga aatcaaagtt gtttgtctac tattgatcca agccagtgcg gtcttgaaac 6600 tgacaatagt gtgctcgtgt tttgaggtca tctttgtatg aataaatcta gtctttgatc 6660 taaataatct tgacgagcca aggcgataaa tacccaaatc taaaactctt ttaaaacgtt 6720 aaaaggacaa gtatgtctgc ctgtattaaa ccccaaatca gctcgtagtc tgatcctcat 6780 caacttgagg ggcactatct tgttttagag aaatttgcgg agatgcgata tcgagaaaaa 6840 ggtacgctga ttttaaacgt gaaatttatc tcaagatctc tgcctcgcgc gtttcggtga 6900 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 6960 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 7020 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca 7080 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 7140 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 7200 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 7260 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 7320 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 7380 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 7440 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 7500 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 7560 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 7620 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 7680 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 7740 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 7800 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 7860 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 7920 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 7980 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 8040 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 8100 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 8160 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 8220 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 8280 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 8340 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 8400 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct 8460 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 8520 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 8580 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 8640 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 8700 agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa 8760 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 8820 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 8880 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 8940 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 9000 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 9060 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 9120 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt 9180 ctcatgtttg acagcttatc atcgataagc tgactcatgt tggtattgtg aaatagacgc 9240 agatcgggaa cactgaaaaa taacagttat tattcg 9276 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> <400> 2 tacgcgatcg ctgttaaa 18 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> <400> 3 gaggccgttg agcac 15 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> <400> 4 tgctctaggc cgcgatta 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> <400> 5 agacgtttcc cgttgaat 18 ...

Claims (2)

1, a kind of multiple copied Yeast expression carrier, its dna sequence dna is the nucleotide sequence of sequence 1 in the sequence table.
2, make up two pairs of primers of the described carrier of claim 1, they are nucleotide sequences of sequence 2 in the sequence table, sequence 3 and sequence 4, sequence 5.
CNB021179069A 2002-05-20 2002-05-20 Multi copy yeast expression carrier for proceeding correct secretory expression against exogenous gene Expired - Lifetime CN1212402C (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102643758A (en) * 2012-04-23 2012-08-22 陈战 Recombined yeast strain expressing cellulase and applications thereof
CN105237640A (en) * 2015-10-15 2016-01-13 中国人民解放军军事医学科学院生物工程研究所 Anthrax toxin receptor CMG2 and human serum albumin fused protein

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102643758A (en) * 2012-04-23 2012-08-22 陈战 Recombined yeast strain expressing cellulase and applications thereof
CN102643758B (en) * 2012-04-23 2015-04-22 陈战 Recombined yeast strain expressing cellulase and applications thereof
CN105237640A (en) * 2015-10-15 2016-01-13 中国人民解放军军事医学科学院生物工程研究所 Anthrax toxin receptor CMG2 and human serum albumin fused protein
CN105237640B (en) * 2015-10-15 2019-01-18 中国人民解放军军事医学科学院生物工程研究所 The fusion protein of anthrax toxin acceptor CMG2 and human serum albumins

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