CN101955957B - Gene engineered Pichia pastoris highly secreting and expressing phytase - Google Patents
Gene engineered Pichia pastoris highly secreting and expressing phytase Download PDFInfo
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- CN101955957B CN101955957B CN 200910017741 CN200910017741A CN101955957B CN 101955957 B CN101955957 B CN 101955957B CN 200910017741 CN200910017741 CN 200910017741 CN 200910017741 A CN200910017741 A CN 200910017741A CN 101955957 B CN101955957 B CN 101955957B
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Abstract
The invention discloses a new phytase gene. The phytase gene has a deoxyribonucleic acid (DNA) sequence with the size of 1,236bp shown as ID NO:1, is accordant with the codon bias of pichia pastoris and encodes phytase of an amino acid sequence shown as ID NO:2. A pichia pastoris engineering bacterium which contains an expression vector pKDN-1 of the phytase gene and is transformed by the expression vector pKDN-1 is provided and is collected in the China Center for Type Culture Collection with the collection number of CCTCC M 209130. The invention also provides a method for constructing the pichia pastoris bacterium CCTCC M 209130. The phytase gene provided by the invention is accordant with the codon bias of the pichia pastoris and can be efficiently expressed in the pichia pastoris. When the constructed high-expression engineering bacterium CCTCC M 209130 is subjected to high density fermentation, enzyme activity is up to 2,000 U/mL to the maximum extent.
Description
Technical field
The present invention relates to a kind of synthetic new phytase gene, is a kind of dna sequence dna that meets the escherichia coli phytase codon of pichia spp codon bias; Especially also made up a kind of Pichia yeast engineering of this synthetic gene of energy efficient secretory expression.Belong to technical field of microbial genetic engineering.
Background technology
Phytic acid (phytinic acid) is the main storage form of phosphorus in the crops such as cereal, beans and oil plant, and its content is up to total phosphorus content 18%-88% (Reddy NR etc., 1982).Although the content of crop phytate phosphorus is very high, owing to lack phytase in the monogastric animal digestive tube, can not in digestive tube, be hydrolyzed into inorganic phosphate to phytic acid, so the utilization ratio of phytic acid is low.Phytate phosphorus in phytase (EC3.1.3.8) the energy hydrolyzing plant forage, thus the monogastric animals such as raising pig fowl are to the utilization ratio of phosphorus.The interpolation of phytase can make the output of phosphorus in the feces of livestock and poultry reduce 40%～75%, can greatly alleviate the environmental pollutions such as rivers that aquaculture causes or waters, and is all significant to development aquaculture and environment protection.
Although phytase is used widely in fodder industry, obviously reduce the fodder production cost, improve environmental protection, produce considerable economic benefit and social benefit; But the deficiency of the production of feeding phytase is one of factor of restriction feed processing industry development.Therefore, become the developing direction of phytase industrialization with modern biotechnology transformation and improving yield, it also is that product competitiveness improves and one of the key factor of winning victory in each biological industry company.
The source of phytase is comparatively extensive, such as plant seed, rice bran, prokaryotic organism such as intestinal bacteria, fungi such as aspergillus niger, or even animal intestinal.Because the degeneracy of genetic code exists codon bias between different plant species, this also is the important factor that affects the gene heterogenous expression.The codon bias transformation is the important means that improves the exogenous gene expression amount.
Summary of the invention
For the deficiency that prior art exists, one of technical problem to be solved by this invention is to provide a kind of new phytase gene, makes its codon bias that meets pichia spp, can be in pichia spp efficient secretory expression.Two of technical problem to be solved by this invention is to provide a kind of expression vector of this phytase gene, and make up a kind of Pichia yeast engineering that produces this phytase, make it this phytase gene of efficient secretory expression, and can produce phytase for animal-feed by high density fermentation.
For solving the problems of the technologies described above, the present invention utilizes the method for synthetic gene, be the codon bias that meets pichia spp with the codon modify of escherichia coli phytase appA gene, in pichia spp, express over one's competence to lay the foundation for it, and make up the Pichia yeast engineering of high level expression phytase.One of the technical solution used in the present invention is: a kind of new phytase gene, and the codon bias that it meets pichia spp has size and is the following dna sequence dna of 1236bp:
1 ATGCAATCTG AACCAGAATT GAAGTTGGAA TCTGTTGTCA TCGTCTCTAG
51 ACATGGTGTT AGAGCACCAA CCAAGGCCAC CCAACTTATG CAAGATGTCA
101 CCCCAGACGC TTGGCCAACC TGGCCAGTCA AGCTGGGTTG GTTGACACCT
151 AGAGGTGGTG AGCTCATTGC TTACTTGGGT CACTACCAAA GACAGCGTCT
201 TGTTGCCGAC GGATTGTTGG CCAAGAAGGG TTGTCCACAA TCTGGTCAAG
251 TAGCTATTAT TGCTGACGTC GACGAAAGAA CCCGTAAGAC AGGTGAAGCC
301 TTCGCCGCCG GTCTTGCTCC TGACTGTGCC ATTACCGTTC ACACCCAAGC
351 TGACACTTCT TCTCCAGATC CATTGTTCAA CCCTTTGAAG ACTGGTGTTT
401 GCCAATTGGA CAACGCTAAC GTTACTGACG CTATCTTGTC CAGAGCTGGA
451 GGATCCATTG CTGACTTCAC CGGTCACAGA CAGACTGCCT TCAGAGAGTT
501 GGAAAGAGTT CTTAACTTCC CACAATCCAA CTTGTGCCTT AAGCGTGAGA
551 AGCAAGACGA ATCCTGTTCC TTGACTCAAG CATTACCATC TGAGTTGAAG
601 GTCTCCGCCG ACAACGTCTC TTTGACCGGT GCTGTCAGCT TGGCTTCCAT
651 GTTGACTAAA ATCTTTCTTC TGCAACAAGC TCAAGGTATG CCTGAGCCAG
701 GTTGGGGTAG AATCACCGAC TCTCACCAAT GGAACACCTT GTTGTCCTTG
751 CACAACGCTC AATTCTACTT GCTGCAGAGA ACTCCAGAGG TTGCTAGATC
801 CAGAGCCACC CCATTGTTGG ACTTGATCAA GACTGCTTTG ACTCCTCACC
851 CACCTCAAAA GCAAGCCTAC GGTGTTACCT TGCCCACTTC TGTCTTGTTC
901 ATTGCCGGTC ACGATACTAA CTTGGCAAAT CTCGGCGGTG CTTTGGAGTT
951 GAACTGGACT CTTCCTGGTC AACCTGATAA CACTCCACCA GGTGGTGAGC
1001 TCGTTTTCGA AAGATGGCGT AGACTATCTG ATAACTCTCA ATGGATTCAG
1051 GTTTCGTTGG TCTTCCAAAC TTTGCAGCAG ATGAGAGACA AGACTCCACT
1101 GTCTTTGAAC ACGCCTCCAG GAGAAGTCAA ATTGACCTTG GCTGGATGTG
1151 AAGAGAGAAA TGCTCAGGGT ATGTGTTCCT TGGCTGGTTT CACTCAAATT
1201 GTTAACGAAG CTAGAATTCC AGCTTGTTCT TTGTAG。
The present invention according to pichia spp preference codon table, converts the codon of coding phytase to the form of pichia spp preference according to the escherichia coli phytase gene order of having reported.Above-mentioned phytase gene is synthetic by following approach:
16 oligonucleotide primers of synthetic at first, namely forward primer is 8: F1-F8,8 of reverse primers: R1-R8, length between 95～106nt, contains the tumor-necrosis factor glycoproteins (seeing primer table 1) of 20 ~ 23bp respectively between primer.Get each 25pmol of primers F 1 and R1 and carry out PCR reaction, reaction volume 50 μ L, reaction conditions: 95 ℃ of 4min; 94 ℃ of 40s, 52 ℃ of 40s, 72 ℃ of 20s, totally 25 circulations; 72 ℃ are extended 7min.Get 0.1 μ L reaction product after the reaction as template, take F2 and R2 as primer, carry out the 2nd PCR reaction, reaction conditions: 94 ℃ of 4min; 94 ℃ of 1min, 52 ℃ of 30s, 72 ℃ of 30s, totally 25 circulations; 72 ℃ are extended 10min.The rest may be inferred, carries out all the other 6 PCR reactions, but the extension time of each reaction increases 10s.Carry out continuously altogether 8 PCR reactions, the synthetic phytase gene that meets the pichia spp codon bias with said gene sequence.
A kind of expression vector pKDN-1 that contains above-mentioned phytase gene obtains by following approach:
Design primer Phy-F and Phy-R (seeing primer table 1), Eco RI and Not I restriction enzyme site are introduced respectively in the primer two ends; Carry out pcr amplification with above-mentioned synthetic phytase gene as template.Sepharose reclaims pcr amplification product, then carries out the reaction of Eco RI and Not I double digestion, and then gel recovery enzyme is cut product; This enzyme is cut the product orientation be connected to Eco RI and Not I double digestion carrier pPIC9K, then obtain expression vector, called after pKDN-1.
A kind of Pichia Pastoris KDN-1 (Pichiapastoris KDN-1) that contains above-mentioned expression vector pKDN-1, be deposited in the Chinese Typical Representative culture collection center that is positioned at Wuhan University on June 20th, 2009, its deposit number is CCTCC M 209130.It obtains by following approach:
Electric shock behind the carrier pKDN-1 linearization for enzyme restriction is imported pichia yeast bacterium GS115, and screening transforms the recombinant pichia yeast strain of phytase gene high copy number, then carries out the high density fermentation checking, obtains to efficiently express at last the Pichia yeast engineering of phytase.
The synthetic phytase gene of table 1 and construction of expression vector the primer table
| The primer name | Sequence forms |
| F1 | 5’-CACAATCCAACTTGTGCCTTAAGCGTGAGAAGCAAGACGAATCCTGTTCCTT GACTCAAGCATTACCATCTGAGTTGAAGGTCTCCGCCGACAACGTCTC-3’ |
| F2 | 5’-GAGGATCCATTGCTGACTTCACCGGTCACAGACAGACTGCCTTCAGAGAG TTGGAAAGAGTTCTTAACTTCCCACAATCCAACTTGTGCCTTAAGC-3’ |
| F3 | 5’-CATTGTTCAACCCTTTGAAGACTGGTGTTTGCCAATTGGACAACGCTAAC GTTACTGACGCTATCTTGTCCAGAGCTGGAGGATCCATTGCTGACTTC-3’ |
| F4 | 5’-AAGCCTTCGCCGCCGGTCTTGCTCCTGACTGTGCCATTACCGTTCACAC CCAAGCTGACACTTCTTCTCCAGATCCATTGTTCAACCCTTTGAAGAC-3’ |
| F5 | 5’-TTGTCCACAATCTGGTCAAGTAGCTATTATTGCTGACGTCGACG AAAGAACCCGTAAGACAGGTGAAGCCTTCGCCGCCGGTCTTG-3’ |
| F6 | 5’-GTGGTGAGCTCATTGCTTACTTGGGTCACTACCAAAGACAGCGTCTTG TTGCCGACGGATTGTTGGCCAAGAAGGGTTGTCCACAATCTGGTCAAG-3’ |
| F7 | 5’-CACCCAACTTATGCAAGATGTCACCCCAGACGCTTGGCCAACCTGGCCAG TCAAGCTGGGTTGGTTGACACCTAGAGGTGGTGAGCTCATTGCTTAC-3’ |
| F8 | 5’-ATGCAATCTGAACCAGAATTGAAGTTGGAATCTGTTGTCATCGTCTCTAG ACATGGTGTTAGAGCACCAACCAAGGCCACCCAACTTATGCAAGATG-3’ |
| R1 | 5’-CTCAGGCATACCTTGAGCTTGTTGCAGAAGAAAGATTTTAGTCAACAT GGAAGCCAAGCTGACAGCACCGGTCAAAGAGACGTTGTCGGCGGAGAC-3’ |
| R2 | 5’-CAGCAAGTAGAATTGAGCGTTGTGCAAGGACAACAAGGTGTTCCATTGGT GAGAGTCGGTGATTCTACCCCAACCTGGCTCAGGCATACCTTGAGCTT-3’ |
| R3 | 5’-GGTGAGGAGTCAAAGCAGTCTTGATCAAGTCCAACAATGGGGTGGCTC TGGATCTAGCAACCTCTGGAGTTCTCTGCAGCAAGTAGAATTGAGCGT-3’ |
| R4 | 5’-CCAAGTTAGTATCGTGACCGGCAATGAACAAGACAGAAGTGGGCAAGGTA ACACCGTAGGCTTGCTTTTGAGGTGGGTGAGGAGTCAAAGCAGTCTT-3’ |
| R5 | 5’-GCTCACCACCTGGTGGAGTGTTATCAGGTTGACCAGGAAGAGTCCAGT TCAACTCCAAAGCACCGCCGAGATTTGCCAAGTTAGTATCGTGACCG-3’ |
| R6 | 5’-GCAAAGTTTGGAAGACCAACGAAACCTGAATCCATTGAGAGTTATCAG ATAGTCTACGCCATCTTTCGAAAACGAGCTCACCACCTGGTGGAGTG-3’ |
| R7 | 5’-CACATCCAGCCAAGGTCAATTTGACTTCTCCTGGAGGCGTGTTCAAAG ACAGTGGAGTCTTGTCTCTCATCTGCTGCAAAGTTTGGAAGACCAAC-3’ |
| R8 | 5’-CTACAAAGAACAAGCTGGAATTCTAGCTTCGTTAACAATTTGAGTGAAACCAG CCAAGGAACACATACCCTGAGCATTTCTCTCTTCACATCCAGCCAAGGTCAAT-3’ |
| Phy -F | 5’-AAGAATTCCAATCTGAACCAGAATTGAAG-3’ |
| Phy -R | 5’-AAGCGGCCGCCTACAAAGAACAAGCTGGAAG-3’ |
A kind ofly make up the method that above-mentioned deposit number is the pichia yeast bacterium of CCTCC M 209130, comprise following content:
1,16 oligonucleotide primers of synthetic at first, namely forward primer is 8, and 8 of reverse primers carry out 8 PCR reactions, the synthetic phytase gene that meets the pichia spp codon bias with aforementioned gene order continuously;
2, design primer Phy-F and Phy-R (seeing primer table 1), Eco RI and Not I restriction enzyme site are introduced respectively in the primer two ends; Carry out pcr amplification with above-mentioned synthetic phytase gene as template.Sepharose reclaims pcr amplification product, then carries out the reaction of Eco RI and Not I double digestion, and then gel recovery enzyme is cut product; This enzyme is cut the product orientation be connected to Eco RI and Not I double digestion carrier pPIC9K, then obtain expression vector pKDN-1;
3, electric shock behind the carrier pKDN-1 linearization for enzyme restriction is imported pichia yeast bacterium GS115, screening transforms the recombinant pichia yeast strain of phytase gene high copy number, obtains to efficiently express the Pichia yeast engineering of phytase.
The method of above-mentioned structure pichia yeast bacterium CCTCC M 209130, particular content is as follows:
1, it is 44.8kD at the molecular weight that the 23rd amino acid Gln place forms after enzyme is cut, has a sequence of the mature protein of 410 amino-acid residues according to coding in the CDS coded amino acid complete sequence of escherichia coli phytase, design meets the primer of pichia spp codon bias from the 23rd Gln residue, synthetic 16 oligonucleotide primers, i.e. 8 forward primer F1 to F8,8 reverse primer R1 to R8; Get each 25pmol of primers F 1 and R1 and carry out PCR reaction, reaction volume 50 μ L, reaction conditions: 95 ℃ of 4min; 94 ℃ of 40s, 52 ℃ of 40s, 72 ℃ of 20s, totally 25 circulations; 72 ℃ are extended 7min; Get 0.1 μ L reaction product after the reaction as template, take F2 and R2 as primer, carry out the 2nd PCR reaction, reaction conditions: 95 ℃ of 4min; 94 ℃ of 1min, 52 ℃ of 30s, 72 ℃ of 30s, totally 25 circulations; 72 ℃ of 7min; The rest may be inferred, carries out all the other 6 PCR reactions, but the extension time of each reaction increases 10s; Carry out continuously 8 PCR reactions, the synthetic phytase mature protein coding region dna fragmentation that meets the pichia spp codon bias;
2, design primer Phy-F as shown in table 1 and Phy-R, sequence is as follows:
Phy-F：5’-AA
CAATCTGAACCAGAATTGAAG-3’.................Eco RI；
Phy-R：5’-AA
CTACAAAGAACAAGCTGGAAG-3’..........Not I；
Take synthetic phytase gene as template, carry out pcr amplification take Phy-F and Phy-R as primer; The PCR condition is: 95 ℃ of 4min; 94 ℃ of 40s, 52 ℃ of 40s, 72 ℃ of 1min, totally 30 circulations; 72 ℃ are extended 7min; Gel reclaims the PCR product and with Eco RI and Not I double digestion; Equally, with Eco RI and Not I double digestion pPIC9K; Then enzyme is cut latter two double digestion product and connect acquisition recombinant expression plasmid pKDN-1;
3, the screening of efficient secretory expression Pichia yeast engineering:
(1) linearizing of recombinant plasmid pKDN-1: concentrated through the ethanol precipitation after recombinant plasmid pKDN-1 identifies with Bgl II restriction enzyme digestion and electrophoresis, measure DNA concentration, save backup with 3 μ g/ μ L concentration dilution plasmid fragments;
(2) preparation of Pichia pastoris GS115 competent cell: the single bacterium colony of picking GS115,30 ℃ of shaking culture are spent the night in the YPD substratum, transfer in the 100mL YPD substratum again, be cultured to OD600=1.5, ice bath 10min, 4 ℃ of centrifugal 3min of 5000rpm collect thalline.Aseptic double-distilled water with precooling washs thalline 2 times, then is resuspended in the electrophoretic buffer of 1mL precooling, and this damping fluid contains 1mM MgCl2,10mM HEPES, and 250mM sucrose, pH 7.8;
(3) electric shock transforms: add 5 μ L linearizing recombinant plasmid pKDN-1 in 80 μ L competent cells.Transform at the EMC830 electroporation with 1mm electric shock cup, its electric shock condition is 300V, 16ms; Coat at last the dull and stereotyped cultivation of MM, select recombinant bacterial strain; MM nutrient media components: 1.34%YNB, 4 * 10
-5The % vitamin H, 0.5% methyl alcohol;
(4) the high copy of Southern Blot screening recombinant conversion is sub: nylon membrane is relayed at the filter paper that soaked denaturation liquid hatch 15min, this denaturation liquid is 50mM EDTA, 2.5% beta-mercaptoethanol pH 9.0; Then nylon membrane is relayed and soaking on the filter paper of enzymolysis solution, hatched 4 hours for 37 ℃, this enzymolysis solution is 1mg/ml Zymolyase 100T; Nylon membrane relays 5min on the filter paper that soaked sex change liquid, and this sex change liquid is 0.5M NaOH, 1.5M NaCl mixed solution; Nylon membrane relayed soaking 5min on the filter paper of neutralizer, this neutralizer is 1.5M NaCl, 0.5M Tris-Cl mixed solution; Nylon membrane is relayed 5min on the filter paper that soaked 2 * SSC (trisodium citrate); Then drying at room temperature nylon membrane 30min; Nylon membrane placed under the ultraviolet lamp shine 4min; Carry out Southern hybridization, screening obtains the bacterial classification that hybridization signal is strong, with its called after Pichia pastoris KDN-1, its deposit number CCTCC M 209130.
Phytase gene provided by the present invention, the codon of its Expressing Recombinant Phytase goal gene meets the codon bias of pichia spp, can efficiently express in pichia spp; Although its nucleotide sequence of the phytase gene that is synthesized has changed, the zymologic property of the phytase of its coding does not change, and its optimum pH is 4.5.When the constructed high expression engineering CCTCC M 209130 of the present invention carried out high density fermentation, enzyme work reached as high as 2000U/mL.
Pichia pastoris GS115 involved in the present invention and plasmid pPIC9K are the products of Invitrogen company, can buy to it at any time when needing.Other raw materials and reagent involved in the present invention are industry ordinary articles.Except having particularly pointed out, equipment involved in the present invention, detection method are industry conventional equipment and method.
Description of drawings
Fig. 1 is the synthetic synoptic diagram of phytase gene;
Fig. 2 is that expression plasmid pKDN-1 structure iron is general;
Fig. 3 escherichia coli phytase Protein S ignalP-NN signal peptide prediction figure;
The impact that Fig. 4 pH lives on enzyme;
The SDS-PAGE that Fig. 5 expresses supernatant detects.
Embodiment
The bioinformatic analysis of escherichia coli phytase albumen: as shown in Figure 3, by on-line analysis software SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/) the CDS coded amino acid complete sequence of escherichia coli phytase is carried out signal peptide prediction, find that there is the signal peptide restriction enzyme site in it at the 23rd amino acid Gln place, form the mature protein with 410 amino-acid residues after enzyme is cut, its molecular weight is 44.8kD.
Embodiment 2
The synthetic of phytase gene
According to signal estimation result in the example 1, design meets the primer of pichia spp codon bias from its 23 Gln residues, design altogether the oligonucleotide primer of 16 95-106nt, by 8 consecutive PCR reactions, obtain the phytase mature protein coding region dna fragmentation (being called for short the phy gene) of synthetic.Concrete steps are as follows:
Get each 25pmol of primers F 1 and R1 and carry out PCR reaction, reaction volume 50 μ L, reaction conditions: 95 ℃ of 4min; 94 ℃ of 40s, 52 ℃ of 40s, 72 ℃ of 20s, totally 25 circulations; 72 ℃ of 7min.Get 0.1 μ L reaction product after the reaction as template, take F2 and R2 as primer, carry out the 2nd PCR reaction, reaction conditions: 95 ℃ of 4min; 94 ℃ of 1min, 52 ℃ of 30s, 72 ℃ of 30s, totally 25 circulations; 72 ℃ of 7min.The rest may be inferred, carries out all the other 6 PCR reactions, but the extension time of each reaction increases 10s.Carry out continuously 8 PCR reactions.
(1) for the first time PCR reaction composition sequence
Utilize the primers F 1/R1 shown in the table 1 to carry out the synthetic following sequence of PCR reaction:
Size: 176bp
CACAATCCAA CTTGTGCCTT AAGCGTGAGA AGCAAGACGA ATCCTGTTCC TTGACTCAAG
CATTACCATC TGAGTTGAAG GTCTCCGCCG ACAACGTCTC TTTGACCGGT GCTGTCAGCT
TGGCTTCCAT GTTGACTGAA ATCTTTCTTC TGCAACAAGC TCAAGGTATG CCTGAG；
(2) for the second time PCR reaction composition sequence
Take the first time PCR reactant as template, carry out PCR reaction take the F2/R2 shown in the table 1 as primer, synthetic following sequence:
Size: 326bp
GAGGATCCAT TGCTGACTTC ACCGGTCACA GACAGACTGC CTTCAGAGAG TTGGAAAGAG
TTCTTAACTT CCCACAATCC AACTTGTGCC TTAAGCGTGA GAAGCAAGAC GAATCCTGTT
CCTTGACTCA AGCATTACCA TCTGAGTTGA AGGTCTCCGC CGACAACGTC TCTTTGACCG
GTGCTGTCAG CTTGGCTTCC ATGTTGACTG AAATCTTTCT TCTGCAACAA GCTCAAGGTA
TGCCTGAGCC AGGTTGGGGT AGAATCACCG ACTCTCACCA ATGGAACACC TTGTTGTCCT
TGCACAACGC TCAATTCTAC TTGCTG
(3) for the third time PCR reaction composition sequence
Take the second time PCR reactant as template, carry out PCR reaction take the F3/R3 shown in the table 1 as primer, synthetic following sequence:
Size: 480bp
CATTGTTCAA CCCTTTGAAG ACTGGTGTTT GCCAATTGGA CAACGCTAAC GTTACTGACG
CTATCTTGTC CAGAGCTGGA GGATCCATTG CTGACTTCAC CGGTCACAGA CAGACTGCCT
TCAGAGAGTT GGAAAGAGTT CTTAACTTCC CACAATCCAA CTTGTGCCTT AAGCGTGAGA
AGCAAGACGA ATCCTGTTCC TTGACTCAAG CATTACCATC TGAGTTGAAG GTCTCCGCCG
ACAACGTCTC TTTGACCGGT GCTGTCAGCT TGGCTTCCAT GTTGACTGAA ATCTTTCTTC
TGCAACAAGC TCAAGGTATG CCTGAGCCAG GTTGGGGTAG AATCACCGAC TCTCACCAAT
GGAACACCTT GTTGTCCTTG CACAACGCTC AATTCTACTT GCTGCAGAGA ACTCCAGAGG
TTGCTAGATC CAGAGCCACC CCATTGTTGG ACTTGATCAA GACTGCTTTG ACTCCTCACC
(4) the 4th PCR reaction composition sequences
Take PCR reactant for the third time as template, carry out the PCR reaction take the F4/R4 shown in the table 1 as primer, synthetic following sequence:
Size: 630bp
AAGCCTTCGC CGCCGGTCTT GCTCCTGACT GTGCCATTAC CGTTCACACC CAAGTTGACA
CTTCTTCTCC AGATCCATTG TTCAACCCTT TGAAGACTGG TGTTTGCCAA TTGGACAACG
CTAACGTTAC TGACGCTATC TTGTCCAGAG CTGGAGGATC CATTGCTGAC TTCACCGGTC
ACAGACAGAC TGCCTTCAGA GAGTTGGAAA GAGTTCTTAA CTTCCCACAA TCCAACTTGT
GCCTTAAGCG TGAGAAGCAA GACGAATCCT GTTCCTTGAC TCAAGCATTA CCATCTGAGT
TGAAGGTCTC CGCCGACAAC GTCTCTTTGA CCGGTGCTGT CAGCTTGGCT TCCATGTTGA
CTGAAATCTT TCTTCTGCAA CAAGCTCAAG GTATGCCTGA GCCAGGTTGG GGTAGAATCA
CCGACTCTCA CCAATGGAAC ACCTTGTTGT CCTTGCACAA CGCTCAATTC TACTTGCTGC
AGAGAACTCC AGAGGTTGCT AGATCCAGAG CCACCCCATT GTTGGACTTG ATCAAGACTG
CTTTGACTCC TCACCCACCT CAAAAGCAAG CCTACGGTGT TACCTTGCCC ACTTCTGTCT
TGTTCATTGC CGGTCACGAT ACTAACTTGG
(5) the 5th PCR reaction composition sequences
Take the 4th PCR reactant as template, carry out the PCR reaction take the F5/R5 shown in the table 1 as primer, synthetic following sequence:
Size: 770bp
TTGTCCACAA TCTGGTCAAG TAGCTATTAT TGCTGACGTC GACGAAAGAA CCCGTAAGAC
AGGTGAAGCC TTCGCCGCCG GTCTTGCTCC TGACTGTGCC ATTACCGTTC ACACCCAAGT
TGACACTTCT TCTCCAGATC CATTGTTCAA CCCTTTGAAG ACTGGTGTTT GCCAATTGGA
CAACGCTAAC GTTACTGACG CTATCTTGTC CAGAGCTGGA GGATCCATTG CTGACTTCAC
CGGTCACAGA CAGACTGCCT TCAGAGAGTT GGAAAGAGTT CTTAACTTCC CACAATCCAA
CTTGTGCCTT AAGCGTGAGA AGCAAGACGA ATCCTGTTCC TTGACTCAAG CATTACCATC
TGAGTTGAAG GTCTCCGCCG ACAACGTCTC TTTGACCGGT GCTGTCAGCT TGGCTTCCAT
GTTGACTGAA ATCTTTCTTC TGCAACAAGC TCAAGGTATG CCTGAGCCAG GTTGGGGTAG
AATCACCGAC TCTCACCAAT GGAACACCTT GTTGTCCTTG CACAACGCTC AATTCTACTT
GCTGCAGAGA ACTCCAGAGG TTGCTAGATC CAGAGCCACC CCATTGTTGG ACTTGATCAA
GACTGCTTTG ACTCCTCACC CACCTCAAAA GCAAGCCTAC GGTGTTACCT TGCCCACTTC
TGTCTTGTTC ATTGCCGGTC ACGATACTAA CTTGGCAAAT CTCGGCGGTG CTTTGGAGTT
GAACTGGACT CTTCCTGGTC AACCTGATAA CACTCCACCA GGTGGTGAGC
(6) the 6th PCR reaction composition sequences
Take the 5th PCR reaction product as template, carry out the PCR reaction take the F6/R6 shown in the table 1 as primer, synthetic following sequence:
Size: 921bp
GTGGTGAGCT CATTGCTTAC TTGGGTCACT ACCAAAGACA GCGTCTTGTT GCCGACGGAT
TGTTGGCCAA GAAGGGTTGT CCACAATCTG GTCAAGTAGC TATTATTGCT GACGTCGACG
AAAGAACCCG TAAGACAGGT GAAGCCTTCG CCGCCGGTCT TGCTCCTGAC TGTGCCATTA
CCGTTCACAC CCAAGTTGAC ACTTCTTCTC CAGATCCATT GTTCAACCCT TTGAAGACTG
GTGTTTGCCA ATTGGACAAC GCTAACGTTA CTGACGCTAT CTTGTCCAGA GCTGGAGGAT
CCATTGCTGA CTTCACCGGT CACAGACAGA CTGCCTTCAG AGAGTTGGAA AGAGTTCTTA
ACTTCCCACA ATCCAACTTG TGCCTTAAGC GTGAGAAGCA AGACGAATCC TGTTCCTTGA
CTCAAGCATT ACCATCTGAG TTGAAGGTCT CCGCCGACAA CGTCTCTTTG ACCGGTGCTG
TCAGCTTGGC TTCCATGTTG ACTGAAATCT TTCTTCTGCA ACAAGCTCAA GGTATGCCTG
AGCCAGGTTG GGGTAGAATC ACCGACTCTC ACCAATGGAA CACCTTGTTG TCCTTGCACA
ACGCTCAATT CTACTTGCTG CAGAGAACTC CAGAGGTTGC TAGATCCAGA GCCACCCCAT
TGTTGGACTT GATCAAGACT GCTTTGACTC CTCACCCACC TCAAAAGCAA GCCTACGGTG
TTACCTTGCC CACTTCTGTC TTGTTCATTG CCGGTCACGA TACTAACTTG GCAAATCTCG
GCGGTGCTTT GGAGTTGAAC TGGACTCTTC CTGGTCAACC TGATAACACT CCACCAGGTG
GTGAGCTCGT TTTCGAAAGA TGGCGTAGAC TATCTGATAA CTCTCAATGG ATTCAGGTTT
CGTTGGTCTT CCAAACTTTG C
(7) the 7th PCR reaction composition sequences
Take the 6th PCR reaction product as template, carry out the PCR reaction take the F7/R7 shown in the table 1 as primer, synthetic following sequence:
Size: 1073bp
CACCCAACTT ATGCAAGATG TCACCCCAGA CGCTTGGCCA ACCTGGCCAG TCAAGCTGGG
TTGGTTGACA CCTAGAGGTG GTGAGCTCAT TGCTTACTTG GGTCACTACC AAAGACAGCG
TCTTGTTGCC GACGGATTGT TGGCCAAGAA GGGTTGTCCA CAATCTGGTC AAGTAGCTAT
TATTGCTGAC GTCGACGAAA GAACCCGTAA GACAGGTGAA GCCTTCGCCG CCGGTCTTGC
TCCTGACTGT GCCATTACCG TTCACACCCA AGTTGACACT TCTTCTCCAG ATCCATTGTT
CAACCCTTTG AAGACTGGTG TTTGCCAATT GGACAACGCT AACGTTACTG ACGCTATCTT
GTCCAGAGCT GGAGGATCCA TTGCTGACTT CACCGGTCAC AGACAGACTG CCTTCAGAGA
GTTGGAAAGA GTTCTTAACT TCCCACAATC CAACTTGTGC CTTAAGCGTG AGAAGCAAGA
CGAATCCTGT TCCTTGACTC AAGCATTACC ATCTGAGTTG AAGGTCTCCG CCGACAACGT
CTCTTTGACC GGTGCTGTCA GCTTGGCTTC CATGTTGACT GAAATCTTTC TTCTGCAACA
AGCTCAAGGT ATGCCTGAGC CAGGTTGGGG TAGAATCACC GACTCTCACC AATGGAACAC
CTTGTTGTCC TTGCACAACG CTCAATTCTA CTTGCTGCAG AGAACTCCAG AGGTTGCTAG
ATCCAGAGCC ACCCCATTGT TGGACTTGAT CAAGACTGCT TTGACTCCTC ACCCACCTCA
AAAGCAAGCC TACGGTGTTA CCTTGCCCAC TTCTGTCTTG TTCATTGCCG GTCACGATAC
TAACTTGGCA AATCTCGGCG GTGCTTTGGA GTTGAACTGG ACTCTTCCTG GTCAACCTGA
TAACACTCCA CCAGGTGGTG AGCTCGTTTT CGAAAGATGG CGTAGACTAT CTGATAACTC
TCAATGGATT CAGGTTTCGT TGGTCTTCCA AACTTTGCAG CAGATGAGAG ACAAGACTCC
ACTGTCTTTG AACACGCCTC CAGGAGAAGT CAAATTGACC TTGGCTGGAT GTG
(8) the 8th PCR reaction composition sequences
Take the 7th PCR reaction product as template, carry out the PCR reaction take the F8/R8 shown in the table 1 as primer, synthetic following sequence:
Size: 1236bp
ATGCAATCTG AACCAGAATT GAAGTTGGAA TCTGTTGTCA TCGTCTCTAG ACATGGTGTT
AGAGCACCAA CCAAGGCCAC CCAACTTATG CAAGATGTCA CCCCAGACGC TTGGCCAACC
TGGCCAGTCA AGCTGGGTTG GTTGACACCT AGAGGTGGTG AGCTCATTGC TTACTTGGGT
CACTACCAAA GACAGCGTCT TGTTGCCGAC GGATTGTTGG CCAAGAAGGG TTGTCCACAA
TCTGGTCAAG TAGCTATTAT TGCTGACGTC GACGAAAGAA CCCGTAAGAC AGGTGAAGCC
TTCGCCGCCG GTCTTGCTCC TGACTGTGCC ATTACCGTTC ACACCCAAGT TGACACTTCT
TCTCCAGATC CATTGTTCAA CCCTTTGAAG ACTGGTGTTT GCCAATTGGA CAACGCTAAC
GTTACTGACG CTATCTTGTC CAGAGCTGGA GGATCCATTG CTGACTTCAC CGGTCACAGA
CAGACTGCCT TCAGAGAGTT GGAAAGAGTT CTTAACTTCC CACAATCCAA CTTGTGCCTT
AAGCGTGAGA AGCAAGACGA ATCCTGTTCC TTGACTCAAG CATTACCATC TGAGTTGAAG
GTCTCCGCCG ACAACGTCTC TTTGACCGGT GCTGTCAGCT TGGCTTCCAT GTTGACTGAA
ATCTTTCTTC TGCAACAAGC TCAAGGTATG CCTGAGCCAG GTTGGGGTAG AATCACCGAC
TCTCACCAAT GGAACACCTT GTTGTCCTTG CACAACGCTC AATTCTACTT GCTGCAGAGA
ACTCCAGAGG TTGCTAGATC CAGAGCCACC CCATTGTTGG ACTTGATCAA GACTGCTTTG
ACTCCTCACC CACCTCAAAA GCAAGCCTAC GGTGTTACCT TGCCCACTTC TGTCTTGTTC
ATTGCCGGTC ACGATACTAA CTTGGCAAAT CTCGGCGGTG CTTTGGAGTT GAACTGGACT
CTTCCTGGTC AACCTGATAA CACTCCACCA GGTGGTGAGC TCGTTTTCGA AAGATGGCGT
AGACTATCTG ATAACTCTCA ATGGATTCAG GTTTCGTTGG TCTTCCAAAC TTTGCAGCAG
ATGAGAGACA AGACTCCACT GTCTTTGAAC ACGCCTCCAG GAGAAGTCAA ATTGACCTTG
GCTGGATGTG AAGAGAGAAA TGCTCAGGGT ATGTGTTCCT TGGCTGGTTT CACTCAAATT
GTTAACGAAG CTAGAATTCC AGCTTGTTCT TTGTAG
(9) sequential analysis
Synthesized phytase gene with pichia spp preferences password.Although composition sequence and colibacillary phytase sequence (AF537219) only have 74% homology; But synthetic phytase N-end has lacked signal peptide, and the homology of its maturation protein sequence is 100%.It contrasts respectively as follows:
1, the contrast of synthetic phytase and escherichia coli phytase nucleotide sequence is as follows:
synthetic phytase------------------------------------------------------------
E.coli phytase ATGAAAGCGATCTTAATCCCATTTTTATCTCTTCTGATTCCGTTAACCCCGCAATCTGCA
Synthetic phytase---ATGCAATCTGAACCAGAATTGAAGTTGGAATCTGTTGTCATCGTCTCTAGACATGGT
E.coli phytase TTCGCTCAGAGTGAGCCGGAGCTGAAGCTGGAAAGTGTGGTGATTGTCAGTCGTCATGGT
** *** ** ** ***** ***** *** ** ** *** * * ******
Synthetic phytase GTTAGAGCACCAACCAAGGCCACCCAACTTATGCAAGATGTCACCCCAGACGCTTGGCCA
E.coli phytase GTGCGTGCTCCAACCAAGGCCACGCAACTGATGCAGGATGTCACCCCAGACGCATGGCCA
** * ** ************** ***** ***** ***************** ******
Synthetic phytase ACCTGGCCAGTCAAGCTGGGTTGGTTGACACCTAGAGGTGGTGAGCTCATTGCTTACTTG
E.coli phytase ACCTGGCCGGTAAAACTGGGTTGGCTGACACCGCGCGGTGGTGAGCTAATCGCCTATCTC
******** ** ** ********* ******* * *********** ** ** ** *
Synthetic phytase GGTCACTACCAAAGACAGCGTCTTGTTGCCGACGGATTGTTGGCCAAGAAGGGTTGTCCA
E.coli phytase GGACATTACCAACGCCAGCGTCTGGTAGCCGACGGATTGCTGGCGAAAAAGGGCTGCCCG
** ** ****** * ******** ** ************ **** ** ***** ** **
Synthetic phytase CAATCTGGTCAAGTAGCTATTATTGCTGACGTCGACGAAAGAACCCGTAAGACAGGTGAA
E.coli phytase CAGTCTGGTCAGGTCGCGATTATTGCTGATGTCGACGAGCGTACCCGTAAAACAGGCGAA
** ******** ** ** *********** ******** * ******** ***** ***
Synthetic phytase GCCTTCGCCGCCGGTCTTGCTCCTGACTGTGCCATTACCGTTCACACCCAAGTTGACACT
E.coli phytase GCCTTCGCCGCCGGGCTGGCACCTGACTGTGCAATAACCGTACATACCCAGGCAGATACG
************** ** ** *********** ** ***** ** ***** * ** **
Synthetic phytase TCTTCTCCAGATCCATTGTTCAACCCTTTGAAGACTGGTGTTTGCCAATTGGACAACGCT
E.coli phytase TCCAGTCCCGATCCGTTATTTAATCCTCTAAAAACTGGCGTTTGCCAACTGGATAACGCG
** *** ***** ** ** ** *** * ** ***** ********* **** *****
Synthetic phytase AACGTTACTGACGCTATCTTGTCCAGAGCTGGAGGATCCATTGCTGACTTCACCGGTCAC
E.coli phytase AACGTGACTGACGCGATCCTCAGCAGGGCAGGAGGGTCAATTGCTGACTTTACCGGGCAT
***** ******** *** * *** ** ***** ** *********** ***** **
Synthetic phytase AGACAGACTGCCTTCAGAGAGTTGGAAAGAGTTCTTAACTTCCCACAATCCAACTTGTGC
E.coli phytase CGGCAAACGGCGTTTCGCGAACTGGAACGGGTGCTTAATTTTCCGCAATCAAACTTGTGC
* ** ** ** ** * ** ***** * ** ***** ** ** ***** *********
Synthetic phytase CTTAAGCGTGAGAAGCAAGACGAATCCTGTTCCTTGACTCAAGCATTACCATCTGAGTTG
E.coli phytase CTTAAACGTGAGAAACAGGACGAAAGCTGTTCATTAACGCAGGCATTACCATCGGAACTC
***** ******** ** ****** ****** ** ** ** *********** ** *
Synthetic phytase AAGGTCTCCGCCGACAACGTCTCTTTGACCGGTGCTGTCAGCTTGGCTTCCATGTTGACT
E.coli phytase AAGGTGAGCGCCGACAATGTCTCATTAACCGGTGCGGTAAGCCTCGCATCAATGCTGACG
***** ********* ***** ** ******** ** *** * ** ** *** ****
Synthetic phytase GAAATCTTTCTTCTGCAACAAGCTCAAGGTATGCCTGAGCCAGGTTGGGGTAGAATCACC
E.coli phytase AAGATATTTCTCCTGCAACAAGCACAGGGAATGCCGGAGCCGGGGTGGGGAAGGATCACC
* ** ***** *********** ** ** ***** ***** ** ***** ** ******
Synthetic phytase GACTCTCACCAATGGAACACCTTGTTGTCCTTGCACAACGCTCAATTCTACTTGCTGCAG
E.coli phytase GATTCACACCAGTGGAACACCTTGCTAAGTTTGCATAACGCGCAATTTTATTTGTTACAA
** ** ***** ************ * ***** ***** ***** ** *** * **
Synthetic phytase AGAACTCCAGAGGTTGCTAGATCCAGAGCCACCCCATTGTTGGACTTGATCAAGACTGCT
E.coli phytase CGCACGCCAGAGGTTGCCCGCAGCCGCGCCACCCCGTTATTAGATTTGATCAAGACAGCG
* ** *********** * * * ******** ** ** ** *********** **
Synthetic phytase TTGACTCCTCACCCACCTCAAAAGCAAGCCTACGGTGTTACCTTGCCCACTTCTGTCTTG
E.coli phytase TTGACGCCCCATCCACCGCAAAAACAGGCGTATGGTGTGACATTACCCACTTCAGTGCTG
***** ** ** ***** ***** ** ** ** ***** ** ** ******** ** **
Synthetic phytase TTCATTGCCGGTCACGATACTAACTTGGCAAATCTCGGCGGTGCTTTGGAGTTGAACTGG
E.coli phytase TTTATCGCCGGACACGATACTAATCTGGCAAATCTCGGCGGCGCACTGGAGCTCAACTGG
** ** ***** *********** **************** ** ***** * ******
Synthetic phytase ACTCTTCCTGGTCAACCTGATAACACTCCACCAGGTGGTGAGCTCGTTTTCGAAAGATGG
E.coli phytase ACGCTTCCCGGTCAGCCGGATAACACGCCGCCAGGTGGTGAACTGGTGTTTGAACGCTGG
** ***** ***** ** ******** ** *********** ** ** ** *** * ***
Synthetic phytase CGTAGACTATCTGATAACTCTCAATGGATTCAGGTTTCGTTGGTCTTCCAAACTTTGCAG
E.coli phytase CGTCGGCTAAGCGATAACAGCCAGTGGATTCAGGTTTCGCTGGTCTTCCAGACTTTACAG
*** * *** ****** ** *************** ********** ***** ***
Synthetic phytase CAGATGAGAGACAAGACTCCACTGTCTTTGAACACGCCTCCAGGAGAAGTCAAATTGACC
E.coli phytase CAGATGCGTGATAAAACGCCGCTGTCATTAAATACGCCGCCCGGAGAGGTGAAACTGACC
****** * ** ** ** ** ***** ** ** ***** ** ***** ** *** *****
Synthetic phytase TTGGCTGGATGTGAAGAGAGAAATGCTCAGGGTATGTGTTCCTTGGCTGGTTTCACTCAA
E.coli phytase CTGGCAGGATGTGAAGAGCGAAATGCGCAGGGCATGTGTTCGTTGGCAGGTTTTACGCAA
**** ************ ******* ***** ******** ***** ***** ** ***
Synthetic phytase ATTGTTAACGAAGCTAGAATTCCAGCTTGTTCTTTGTAG
E.coli phytase ATCGTGAATGAAGCACGCATACCGGCGTGCAGTTTGTAA
** ** ** ***** * ** ** ** ** ******。
2, the contrast of synthetic phytase and escherichia coli phytase protein sequence is as follows:
E.coli phytase MKAILIPFLSLLIPLTPQSAFAQSEPELKLESVVIVSRHGVRAPTKATQLMQDVTPDAWP
Synthetic phytase ---------------------MQSEPELKLESVVIVSRHGVRAPTKATQLMQDVTPDAWP
**************************************
E.coli phytase TWPVKLGWLTPRGGELIAYLGHYQRQRLVADGLLAKKGCPQSGQVAIIADVDERTRKTGE
Synthetic phytase TWPVKLGWLTPRGGELIAYLGHYQRQRLVADGLLAKKGCPQSGQVAIIADVDERTRKTGE
************************************************************
E.coli phytase AFAAGLAPDCAITVHTQADTSSPDPLFNPLKTGVCQLDNANVTDAILSRAGGSIADFTGH
Synthetic phytase AFAAGLAPDCAITVHTQADTSSPDPLFNPLKTGVCQLDNANVTDAILSRAGGSIADFTGH
************************************************************
E.coli phytase RQTAFRELERVLNFPQSNLCLKREKQDESCSLTQALPSELKVSADNVSLTGAVSLASMLT
Synthetic phytase RQTAFRELERVLNFPQSNLCLKREKQDESCSLTQALPSELKVSADNVSLTGAVSLASMLT
************************************************************
E.coli phytase KIFLLQQAQGMPEPGWGRITDSHQWNTLLSLHNAQFYLLQRTPEVARSRATPLLDLIKTA
Synthetic phytase KIFLLQQAQGMPEPGWGRITDSHQWNTLLSLHNAQFYLLQRTPEVARSRATPLLDLIKTA
************************************************************
E.coli phytase LTPHPPQKQAYGVTLPTSVLFIAGHDTNLANLGGALELNWTLPGQPDNTPPGGELVFERW
Synthetic phytase LTPHPPQKQAYGVTLPTSVLFIAGHDTNLANLGGALELNWTLPGQPDNTPPGGELVFERW
************************************************************
E.coli phytase RRLSDNSQWIQVSLVFQTLQQMRDKTPLSLNTPPGEVKLTLAGCEERNAQGMCSLAGFTQ
Synthetic phytase RRLSDNSQWIQVSLVFQTLQQMRDKTPLSLNTPPGEVKLTLAGCEERNAQGMCSLAGFTQ
************************************************************
E.coli phytase IVNEARIPACSL
Synthetic phytase IVNEARIPACSL
************
Embodiment 3: the zymologic property analysis
Measure phytase activity with the vanadium ammonium molybdate method.
Utilize phytase can Hydrolysis of Phytic Acid sodium to discharge the principle of inorganic phosphorus, by adding acid molybdenum one vanadium reagents hydrolysis reaction is stopped, while produces color reaction with the inorganic phosphorus that hydrolysis discharges, form yellow vanadium molybdenum complex, measure the content of phosphorus at the 415nm wavelength. take standard phosphorus as object of reference, calculating the content of phytase in the sample. the content of phytase represents with unit of enzyme activity, 1 phytase unit definition is: under 37 ℃, the condition of pH 5.0, decompose the needed enzyme amount of 1 μ mol inorganic phosphorus that discharges in the 1min from phytic acid.
Optimum pH standard buffer system: Gly-HCl (pH1.2-3.5), NaAc-HAc (pH4-6), Tris-HCl (pH6-7), Tris-HCl (pH6-8), CHES (pH 8.64-10.18) and CAPS (pH10.8).The mensuration of optimum pH: replace NaAc-HAc damping fluid in the conventional enzyme activity determination with above damping fluid, measure enzyme activity, take the gained maximum value as 100%.
Experimental result also has at least 90% enzyme work as shown in Figure 4 when the optimum pH of the phytase of vivoexpression is 4.5, pH5.0.
Embodiment 4
The structure of expression vector pKDN-1
Design primer Phy-F as shown in table 1 and Phy-R, sequence is as follows:
Phy-F：5’-AA
CAATCTGAACCAGAATTGAAG-3’ ……………..Eco RI；
Phy-R：5’-AA
CTACAAAGAACAAGCTGGAAG-3’……….Not I；
Take synthetic phytase gene as template, carry out pcr amplification take Phy-F and Phy-R as primer.The PCR condition is: 95 ℃ of 4min; 94 ℃ of 40s, 52 ℃ of 40s, 72 ℃ of 1min, totally 30 circulations; 72 ℃ of 7min.Gel reclaims the PCR product and with Eco RI and Not I double digestion; Equally, with EcoRI and Not I double digestion pPIC9K; Then enzyme is cut latter two double digestion product and connect acquisition recombinant expression plasmid pKDN-1, its collection of illustrative plates such as Fig. 2.
Embodiment 5
The screening of efficient secretory expression Pichia yeast engineering
(1) linearizing of recombinant plasmid pKDN-1
After recombinant plasmid pKDN-1 identifies with Bgl II restriction enzyme digestion and electrophoresis, concentrated through the ethanol precipitation, measure DNA concentration, save backup with 3 μ g/ μ L concentration dilution plasmid fragments.
(2) preparation of Pichia pastoris GS115 competent cell
The single bacterium colony of picking GS115,30 ℃ of shaking culture are spent the night in the YPD substratum, transfer in the 100mL YPD substratum again, are cultured to OD600=1.5, ice bath 10min, 4 ℃ of centrifugal 3min of 5000rpm collect thalline.Aseptic double-distilled water with precooling washs thalline 2 times, then is resuspended in the electrophoretic buffer of 1mL precooling, and this damping fluid contains 1mM MgCl2,10mM HEPES, and 250mM sucrose, pH 7.8.
(3) electric shock transforms
In 80 μ L competent cells, add 5 μ L linearizing recombinant plasmid pKDN-1.Transform at the EMC830 electroporation with 1mm electric shock cup, its electric shock condition is 300V, 16ms; Coat at last the dull and stereotyped cultivation of MM, select recombinant bacterial strain; MM nutrient media components: 1.34%YNB, 4 * 10
-5The % vitamin H, 0.5% methyl alcohol.
(4) the high copy of Southern Blot screening recombinant conversion
Nylon membrane relayed at the filter paper that soaked denaturation liquid hatch 15min, this denaturation liquid is 50mM EDTA, 2.5% beta-mercaptoethanol pH 9.0; Then nylon membrane is relayed and soaking on the filter paper of enzymolysis solution, hatched 4 hours for 37 ℃, this enzymolysis solution is 1mg/ml Zymolyase 100T; Nylon membrane relays 5min on the filter paper that soaked sex change liquid, and this sex change liquid is 0.5M NaOH, 1.5M NaCl mixed solution; Nylon membrane relayed soaking 5min on the filter paper of neutralizer, this neutralizer is 1.5MNaCl, 0.5M Tris-Cl mixed solution; Nylon membrane is relayed 5min on the filter paper that soaked 2 * SSC (trisodium citrate); Then drying at room temperature nylon membrane 30min; Nylon membrane placed under the ultraviolet lamp shine 4min; Carry out Southern hybridization, screening obtains the bacterial classification that hybridization signal is strong, with its called after Pichia pastoris KDN-1, its deposit number CCTCC M 209130.
Embodiment 6: the abduction delivering of phytase
Engineering bacteria P.pastoris KD-1 is inoculated in 5ml BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 4 * 10
-5The % vitamin H, 1% glycerine), 30 ℃ of overnight incubation, centrifugal collection thalline adds 50ml BMMY inducing culture (1% yeast extract, 2% peptone, 1.34%YNB, 4 * 10 to thalline
-5% vitamin H, 0.5% methyl alcohol), 30 ℃ of lower inducing culture 108hr that continue added 50 μ L methyl alcohol in per 12 hours.Sampling in per 12 hours detects the expression of inducing supernatant liquor by SDS-PAGE.The result shows, the target protein expression amount reached peak expression (Fig. 8) in 72 hours in continuous increase in the supernatant.This project bacterium is carried out high density fermentation in 10 tons of fermentor tanks, inducing culture 72 hours, its supernatant enzyme work reaches as high as 20000U/mL.
Sequence table
＜110〉Qingdao Continent Biotech Co., Ltd.
＜120〉a kind of new phytase gene and expression vector thereof
<160>2
<210>1
<211>1236
<212>DNA
＜213〉artificial sequence
<220>
＜223〉meet the phytase dna sequence dna of pichia spp codon bias
<400>1
atgcaatctg aaccagaatt gaagttggaa tctgttgtca tcgtctctag 50
acatggtgtt agagcaccaa ccaaggccac ccaacttatg caagatgtca 100
ccccagacgc ttggccaacc tggccagtca agctgggttg gttgacacct 150
agaggtggtg agctcattgc ttacttgggt cactaccaaa gacagcgtct 200
tgttgccgac ggattgttgg ccaagaaggg ttgtccacaa tctggtcaag 250
tagctattat tgctgacgtc gacgaaagaa cccgtaagac aggtgaagcc 300
ttcgccgccg gtcttgctcc tgactgtgcc attaccgttc acacccaagc 350
tgacacttct tctccagatc cattgttcaa ccctttgaag actggtgttt 400
gccaattgga caacgctaac gttactgacg ctatcttgtc cagagctgga 450
ggatccattg ctgacttcac cggtcacaga cagactgcct tcagagagtt 500
ggaaagagtt cttaacttcc cacaatccaa cttgtgcctt aagcgtgaga 550
agcaagacga atcctgttcc ttgactcaag cattaccatc tgagttgaag 600
gtctccgccg acaacgtctc tttgaccggt gctgtcagct tggcttccat 650
gttgactaaa atctttcttc tgcaacaagc tcaaggtatg cctgagccag 700
gttggggtag aatcaccgac tctcaccaat ggaacacctt gttgtccttg 750
cacaacgctc aattctactt gctgcagaga actccagagg ttgctagatc 800
cagagccacc ccattgttgg acttgatcaa gactgctttg actcctcacc 850
cacctcaaaa gcaagcctac ggtgttacct tgcccacttc tgtcttgttc 900
attgccggtc acgatactaa cttggcaaat ctcggcggtg ctttggagtt 950
gaactggact cttcctggtc aacctgataa cactccacca ggtggtgagc 1000
tcgttttcga aagatggcgt agactatctg ataactctca atggattcag 1050
gtttcgttgg tcttccaaac tttgcagcag atgagagaca agactccact 1100
gtctttgaac acgcctccag gagaagtcaa attgaccttg gctggatgtg 1150
aagagagaaa tgctcagggt atgtgttcct tggctggttt cactcaaatt 1200
gttaacgaag ctagaattcc agcttgttct ttgtag 1236
<210>2
<211>411
<212>PRT
＜213〉artificial sequence
<220>
＜223〉phytase of pichia spp secreting, expressing
<400>2
Met Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser 16
Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp 32
Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu 48
Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gln Arg 64
Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gln 80
Ser Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys 96
Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr 112
Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro 128
Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala Asn Val Thr Asp Ala 144
Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg 160
Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser 176
Asn Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr 192
Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu 208
Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr Lys Ile Phe Leu Leu 224
Gln Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp 240
Ser His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr 256
Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu 272
Leu Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln 288
Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His 304
Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr 320
Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe 336
Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser 352
Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser 368
Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu 384
Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile 390
Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu 411
Claims (1)
1. a plant height is imitated the Pichia yeast engineering of secreting, expressing phytase, it is characterized in that: described Pichia yeast engineering is pichia pastoris phaff (Pichia pastoris) KDN-1, deposit number is CCTCC NO:M 209130, and it contains the expression vector that can express the described phytase gene of SEQ ID NO:1.
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| CN1597961A (en) * | 2003-09-17 | 2005-03-23 | 广东肇庆星湖生物科技股份有限公司 | Production for phytase with high living rate high temp. resisting by pichia |
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| CN1597961A (en) * | 2003-09-17 | 2005-03-23 | 广东肇庆星湖生物科技股份有限公司 | Production for phytase with high living rate high temp. resisting by pichia |
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| Title |
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| J Dassa et.al..The complete nucleotide sequence of the Escherichia coli gene appA reveals significant homology between pH 2.5 acid phosphatase and glucose-1-phosphatase.《J. Bacteriol.》.1990,第172卷(第9期),5497-5500. * |
| 罗会颖 等.来源于Escherichia coli 的高比活植酸酶基因的高效表达.《生物工程学报》.2004,第20卷(第1期),78-84. * |
| 赵翔 等.毕赤酵母的密码子用法分析.《生物工程学报》.2000,第16卷(第3期),308-311. * |
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