CN102146135A - Recombinant human-like collagen and production method thereof - Google Patents
Recombinant human-like collagen and production method thereof Download PDFInfo
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Abstract
The invention discloses a recombinant human-like collagen. The recombinant human-like collagen has high hydrophilicity and stability. The structure of the recombinant human-like collagen is totally the same with the structure of the corresponding part of a natural collagen gene sequence. When the recombinant human-like collagen is applied to a human body, immunological rejection cannot be caused. The recombinant human-like collagen can be widely applied to the fields of biomedical materials, cosmetics and the like. The invention also discloses a production method of the recombinant human-like collagen, which comprises the following steps of: constructing pichia pastoris gene engineering bacteria; fermenting and cultivating the pichia pastoris gene engineering bacteria; inducing and expressing the recombinant human-like collagen; and purifying the recombinant human-like collagen. The human-like collagen produced by the method has high expression and is convenient to purify. The expression in a fermentation tank can achieve 5.0g/L. In a single batch, about 150g of crude collagens can be produced by a tank with the volume of 50L. Due to high expression, the purity of the crude collagens is over 80 percent and few impurity proteins exist.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of recombination human collagen and production method thereof.
Background technology
Collagen protein (collagen) is a histone matter family that is distributed widely in human connective tissue, also be the maximum a kind of protein of in-vivo content, account for 25%～33% of total protein, it plays an important role to normal physiological function and the injury repairing of safeguarding cell, tissue, organ.Collagen protein is as natural Biological resources, have incomparable biocompatibility of synthesized polymer material and biodegradability, can be widely used in the industry such as medicine, makeup, but natural collagen protein can not be water-soluble, and the collagen protein that extracts in the animal body is the mixture of the collagen peptide that do not wait from big to small of various molecular weight, character is very complicated, is difficult to further process, and has limited it as the application of biomaterial in fields such as organizational projects.And zoogenous collagen protein is difficult to avoid the suspicion of virus infection, is applied to the mankind simultaneously and also can brings side effects such as immunological rejection.In order to obtain the collagen protein in people source, have the investigator to adopt people's placenta to extract, but this mode not only is subjected to the restriction of raw material supply more will face the censure of ethics as raw material.
The collagen protein series products of current market sale or take from large animal tissues such as pigskin, ox-hide or take from the skin histology of fish.Because animal tissues's extract often has the hidden danger of virus infection, this series products only can use as the raw material that is used for makeup and healthcare products, has limited its application as high-end product aspects such as medical supplies.
What the production collagen protein was traditional also is that present topmost method is to utilize acid, alkaline process to handle in the tissue of animal-origin to extract collagen protein, also have enzymolysis process to extract collagen protein in addition, these methods have the higher rate of recovery, but collagen protein poorly water-soluble, difficulty that these methods are produced are separated to pure product, and show rejection clinically, make its application be subjected to considerable restraint as aspects such as biomedical material or pharmaceutical carriers.
Development along with modern biotechnology, utilize transgenic technology and gene recombination technology, can obtain recombinant human collagen albumen in animal, plant and microbial expression system, solve the shortcomings such as viral hidden danger that traditional extraction process exists, also improved the wetting ability of collagen protein, immune rejection property etc. simultaneously.Some research institutions and biotech firm drop into research and development research recombined collagen in succession both at home and abroad, as utilize transgenic method, cultivation contain Human-like Collagen small white mouse, obtain containing the Mammals of recombination human collagen milk by transgenosis micro-injection, but by mammals or the insect cell line production cost is high, the production cycle is long.The employing pcr amplifications such as Fan Daidi of NORTHWEST CHINA university obtain several sections human collagen gene fragments, then splice repetition, transformed into escherichia coli, and, obtain the recombination human collagen of high expression level by high density fermentation cultivation production Human-like Collagen.2004, the Yao J of Tokyo agricultural science and technology university etc. did research to effect of collagen protein cell attachment and self-crosslinking spiral correlated series, and having designed length is 240 amino acid whose Human-like Collagens, and has obtained expression in intestinal bacteria.But bacterial expression system causes pyrogen remnants easily, cause expression product impure, be difficult to be applied to clinical, and target protein is usually with the inclusion body formal representation, product purification process complexity, the quality of disadvantages affect products such as the translation post-treatment of prokaryotic expression system is modified the system imperfection in addition, and the biological activity of expression product is lower.
Summary of the invention
Technical problem to be solved by this invention is at above-mentioned the deficiencies in the prior art, and a kind of expression amount height is provided, good water solubility, and its structure and performance all are better than the recombination human collagen of animal source collagen protein.
For solving the problems of the technologies described above, the technical solution used in the present invention is: a kind of recombination human collagen is characterized in that its sequence is as follows:
AGNTGAPGSP GVSGPKGDAG QPGEKGSPGA QGPPGAPGPL GIAGITGARG LAGPPGMPGP 60
RGSPGPQGVK GESGKPGANG LSGERGPPGP QGLPGLAGTA GEPGRDGNPG SDGLPGRDGS 120
PGGKGDRGEN GSPGAPGAPG HPGPPGPVGP AGKSGDRGES GPAGPAGAPG PAGSRGAPGP 180
QGPRGDKGET GERGAAGIKG HRGFPGNPGA PGSPGPAGQQ GAIGSPGPAE FGADGVAGPK 240
GPAGERGSPG PAGPKGSPGE AGRPGEAGLP GAKGLTGSPG SPGPDGKTGP PGPAGQDGRP 300
GPPGPPGARG QAGVMGFPGP KGAAGEPGKA GERGVPGPPG AVGPAGKDGE AGAQGPPGPA 360
GPAGERGEQG PAGSPGFQGL PGPAGPPGEA GKPGEQGVPG DLGAPGPSGA RGERGFPGER 420
GVQGPPGPAG PRGANGAPGN DGAKGDAGAP GAPGSQGAPG LQGMPGERGA AGLPGPKGDR 480
GDAGPKGADG SPGKDGVRGL TGPIGPPGPA GAPGDKGESG PSGPAGPTGA RGAPGDRGEP 540
GPPGPAGFAG PPGADGQPGA KGEPGDAGAK GDAGPPGPAG PAGPPGPIGN VGAPGAKGAR 600
GSAGPPGATG FPGAAGRVGP PGPSGNAGPP GPPGPAGKEG GKGPRGETGP AGRPGEVGPP 660
GPPGPAGEKG SPGADGPAGA PGTPGPQGIA GQRGVVGLPG QRGERGFPGL PGPSGEPGKQ 720
GPSGASGERG PPGPMGPPGL AGPPGESGRE GAPGAEGSPG RDGSPGAKGD RGETGPAGPP 780
GAPGAPGAPG PVGPAGKSGD RGETGPAGPA GPVGPVGARG PAGPQGPRGD KGETGEQGD 839
The structure of described recombination human collagen is strand, single coil configuration, and its amino acid is 839, and wherein 1-241 is an III type peptide section, and 242-839 is an I type peptide section.
The present invention also provides a kind of method of producing this recombination human collagen, it is characterized in that, this method may further comprise the steps:
(1) structure of pichia yeast genetic engineering bacteria;
(2) fermentation culture of pichia yeast genetic engineering bacteria;
(3) recombination human collagen inducing and expressing;
(4) purifying of recombination human collagen.
The structure of pichia yeast genetic engineering bacteria is as follows described in the above-mentioned steps (1):
A. the fragment gene with known I type of human body and III collagen type gene helical region extracts respectively, sews up recombinant DNA then;
B. select for use pichia spp SMD1168 strain as host cell, recombinant DNA is changed over to and is incorporated in the genome of host cell, screening obtains pichia yeast genetic engineering bacteria (pichia pastoris phaff Pichia pastoris C13); This pichia yeast genetic engineering bacteria has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (preservation centre address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 8th, 2010, Institute of Microorganism, Academia Sinica, postcode: 100101), deposit number is CGMCC No.4437.
The fermentation culture process of pichia yeast genetic engineering bacteria is as follows described in the above-mentioned steps (2): picking pichia yeast genetic engineering bacteria list bacterium colony, place the 150mL triangular flask that 30mL BMGY substratum is housed, in 28 ℃～30 ℃, it is 2.0～6.0 that 250rpm～300rpm is cultured to OD600, the centrifugal 5min of 1500g～3000g under the room temperature, collect thalline, with the resuspended thalline of BMMY substratum, making the thalline OD600 after resuspended is 1.0; The bacterium liquid of resuspended thalline gained is placed the triangular flask of 500mL, seal, on 28 ℃～30 ℃, the shaking table of 250rpm～300rpm, continue to cultivate with four layers of gauze; Every 24h adds anhydrous methanol in substratum, the addition of anhydrous methanol is 1% of a substratum cumulative volume.
The purge process of the described recombination human collagen of above-mentioned steps (4) is: the pichia yeast genetic engineering bacteria of cultivation is collected supernatant liquor through after the centrifugation, the supernatant liquor of collecting is removed colour substance and fishy smell through chromatography column S100, remove unnecessary salinity through desalting column G75 again, ultrafiltration and concentration after the desalination is collected the protein frozen drying.
The production method of above-mentioned a kind of recombination human collagen, the sequence of described recombinant DNA is as follows:
GCGGGTAACA CTGGTGCTCC TGGCAGCCCT GGAGTGTCTG GACCAAAAGG TGATGCTGGC 60
CAACCAGGAG AGAAGGGATC GCCTGGTGCC CAGGGCCCAC CAGGAGCTCC AGGCCCACTT 120
GGGATTGCTG GGATCACTGG AGCACGGGGT CTTGCAGGAC CACCAGGCAT GCCAGGTCCT 180
AGGGGAAGCC CTGGCCCTCA GGGTGTCAAG GGTGAAAGTG GGAAACCAGG AGCTAACGGT 240
CTCAGTGGAG AACGTGGTCC CCCTGGACCC CAGGGTCTTC CTGGTCTGGC TGGTACAGCT 300
GGTGAACCTG GAAGAGATGG AAACCCTGGA TCAGATGGTC TTCCAGGCCG AGATGGATCT 360
CCTGGTGGCA AGGGTGATCG TGGTGAAAAT GGCTCTCCTG GTGCCCCTGG CGCTCCTGGT 420
CATCCAGGCC CACCTGGTCC TGTCGGTCCA GCTGGAAAGA GTGGTGACAG AGGAGAAAGT 480
GGCCCTGCTG GCCCTGCTGG TGCTCCCGGT CCTGCTGGTT CCCGAGGTGC TCCTGGTCCT 540
CAAGGCCCAC GTGGTGACAA AGGTGAAACA GGTGAACGTG GAGCTGCTGG CATCAAAGGA 600
CATCGAGGAT TCCCTGGTAA TCCAGGTGCC CCAGGTTCTC CAGGCCCTGC TGGTCAGCAG 660
GGTGCAATCG GCAGTCCAGG ACCTGCAGAA TTCGGCGCAG ATGGTGTTGC TGGTCCCAAG 720
GGTCCCGCTG GTGAACGTGG TTCTCCTGGC CCTGCTGGCC CCAAAGGATC TCCTGGTGAA 780
GCTGGTCGTC CCGGTGAAGC TGGTCTGCCT GGTGCCAAGG GTCTGACTGG AAGCCCTGGC 840
AGCCCTGGTC CTGATGGCAA AACTGGCCCC CCTGGTCCCG CCGGTCAAGA TGGTCGCCCC 900
GGACCCCCAG GCCCACCTGG TGCCCGTGGT CAGGCTGGTG TGATGGGATT CCCTGGACCT 960
AAAGGTGCTG CTGGAGAGCC CGGCAAGGCT GGAGAGCGAG GTGTTCCCGG ACCCCCTGGC 1020
GCTGTCGGTC CTGCTGGCAA AGATGGAGAG GCTGGAGCTC AGGGACCCCC TGGCCCTGCT 1080
GGTCCCGCTG GCGAGAGAGG TGAACAAGGC CCTGCTGGCT CCCCCGGATT CCAGGGTCTC 1140
CCTGGTCCTG CTGGTCCTCC AGGTGAAGCA GGCAAACCTG GTGAACAGGG TGTTCCTGGA 1200
GACCTTGGCG CCCCTGGCCC CTCTGGAGCA AGAGGCGAGA GAGGTTTCCC TGGCGAGCGT 1260
GGTGTGCAAG GTCCCCCTGG TCCTGCTGGT CCCCGAGGGG CCAACGGTGC TCCCGGCAAC 1320
GATGGTGCTA AGGGTGATGC TGGTGCCCCT GGAGCTCCCG GTAGCCAGGG CGCCCCTGGC 1380
CTTCAGGGAA TGCCTGGTGA ACGTGGTGCA GCTGGTCTTC CAGGGCCTAA GGGTGACAGA 1440
GGTGATGCTG GTCCCAAAGG TGCTGATGGC TCTCCTGGCA AAGATGGCGT CCGTGGTCTG 1500
ACTGGCCCCA TTGGTCCTCC TGGCCCTGCT GGTGCCCCTG GTGACAAGGG TGAAAGTGGT 1560
CCCAGCGGCC CTGCTGGTCC CACTGGAGCT CGTGGTGCCC CCGGAGACCG TGGTGAGCCT 1620
GGTCCCCCCG GCCCTGCTGG CTTTGCTGGC CCCCCTGGTG CTGACGGCCA ACCTGGTGCT 1680
AAAGGCGAAC CTGGTGATGC TGGTGCTAAA GGCGATGCTG GTCCCCCTGG CCCTGCCGGA 1740
CCCGCTGGAC CCCCTGGCCC CATTGGTAAT GTTGGTGCTC CTGGAGCCAA AGGTGCTCGC 1800
GGCAGCGCTG GTCCCCCTGG TGCTACTGGT TTCCCTGGTG CTGCTGGCCG AGTCGGTCCT 1860
CCTGGCCCCT CTGGAAATGC TGGACCCCCT GGCCCTCCTG GTCCTGCTGG CAAAGAAGGC 1920
GGCAAAGGTC CCCGTGGTGA GACTGGCCCT GCTGGACGTC CTGGTGAAGT TGGTCCCCCT 1980
GGTCCCCCTG GCCCTGCTGG CGAGAAAGGA TCCCCTGGTG CTGATGGTCC TGCTGGTGCT 2040
CCTGGTACTC CCGGGCCTCA AGGTATTGCT GGACAGCGTG GTGTGGTCGG CCTGCCTGGT 2100
CAGAGAGGAG AGAGAGGCTT CCCTGGTCTT CCTGGCCCCT CTGGTGAACC TGGCAAACAA 2160
GGTCCCTCTG GAGCAAGTGG TGAACGTGGT CCCCCTGGTC CCATGGGCCC CCCTGGATTG 2220
GCTGGACCCC CTGGTGAATC TGGACGTGAG GGGGCTCCTG GTGCCGAAGG TTCCCCTGGA 2280
CGAGACGGTT CTCCTGGCGC CAAGGGTGAC CGTGGTGAGA CCGGCCCCGC TGGACCCCCT 2340
GGTGCTCCTG GTGCTCCTGG TGCCCCTGGC CCCGTTGGCC CTGCTGGCAA GAGTGGTGAT 2400
CGTGGTGAGA CTGGTCCTGC TGGTCCCGCC GGTCCTGTCG GCCCTGTTGG CGCCCGTGGC 2460
CCCGCCGGAC CCCAAGGCCC CCGTGGTGAC AAGGGTGAGA CAGGCGAACA GGGCGAC 2517
The present invention compared with prior art has the following advantages: the present invention adopts pichia spp eukaryotic expression system scale operation recombination human collagen, and it is compared with traditional coli expression system and has the following advantages:
1, expression vector of the present invention utilizes the alcohol oxidase gene promoter, high cell growth speed, so the foreign protein output that this expression system is expressed is very high, its expression amount is higher 10 times even 100 times than other system, this is a quantum jump aspect expression amount, and the expression amount in fermentor tank can reach 5.0g/L, and single batch of 50 liters of jars can be produced about crude protein 150g, crude protein purity is more than 80%, and foreign protein seldom.
2, substratum of the present invention adopts inorganic salt, prepares simple, cheap.
3, expression vector of the present invention is not to exist with the free plasmid, but is incorporated on the karyomit(e), and the bacterial strain of structure is very stable.
4, recombination human collagen of the present invention has good hydrophilicity and stability, its amino acid is formed identical with natural collagen protein aminoacid sequence corresponding section 100%, compare with natural collagen protein, have close structure and biological activity, need not sex change and renaturation, be applied to can not cause immunological rejection in the middle of the human body, can be widely used in fields such as bio-medical material, makeup.
Below in conjunction with drawings and Examples, technical solution of the present invention is described in further detail.
Description of drawings
The route collection of illustrative plates that Fig. 1 makes up for carrier pPIC9K-co3a1-co1a1 of the present invention.
Fig. 2 identifies the electrophorogram of recombinant yeast pichia pastoris for bacterium colony PCR of the present invention.
Fig. 3 is the electrophorogram behind the pPIC9K-co3a1-co1a1 recombinant yeast pichia pastoris abduction delivering of the present invention.
Embodiment
A kind of recombination human collagen, (SEQ ID NO.1) is as follows for its sequence:
AGNTGAPGSP GVSGPKGDAG QPGEKGSPGA QGPPGAPGPL GIAGITGARG LAGPPGMPGP 60
RGSPGPQGVK GESGKPGANG LSGERGPPGP QGLPGLAGTA GEPGRDGNPG SDGLPGRDGS 120
PGGKGDRGEN GSPGAPGAPG HPGPPGPVGP AGKSGDRGES GPAGPAGAPG PAGSRGAPGP 180
QGPRGDKGET GERGAAGIKG HRGFPGNPGA PGSPGPAGQQ GAIGSPGPAE FGADGVAGPK 240
GPAGERGSPG PAGPKGSPGE AGRPGEAGLP GAKGLTGSPG SPGPDGKTGP PGPAGQDGRP 300
GPPGPPGARG QAGVMGFPGP KGAAGEPGKA GERGVPGPPG AVGPAGKDGE AGAQGPPGPA 360
GPAGERGEQG PAGSPGFQGL PGPAGPPGEA GKPGEQGVPG DLGAPGPSGA RGERGFPGER 420
GVQGPPGPAG PRGANGAPGN DGAKGDAGAP GAPGSQGAPG LQGMPGERGA AGLPGPKGDR 480
GDAGPKGADG SPGKDGVRGL TGPIGPPGPA GAPGDKGESG PSGPAGPTGA RGAPGDRGEP 540
GPPGPAGFAG PPGADGQPGA KGEPGDAGAK GDAGPPGPAG PAGPPGPIGN VGAPGAKGAR 600
GSAGPPGATG FPGAAGRVGP PGPSGNAGPP GPPGPAGKEG GKGPRGETGP AGRPGEVGPP 660
GPPGPAGEKG SPGADGPAGA PGTPGPQGIA GQRGVVGLPG QRGERGFPGL PGPSGEPGKQ 720
GPSGASGERG PPGPMGPPGL AGPPGESGRE GAPGAEGSPG RDGSPGAKGD RGETGPAGPP 780
GAPGAPGAPG PVGPAGKSGD RGETGPAGPA GPVGPVGARG PAGPQGPRGD KGETGEQGD 839
The structure of this recombination human collagen is strand, single coil configuration, and its amino acid is 839, and wherein 1-241 is an III type peptide section, and 242-839 is an I type peptide section.
The production method of this recombination human collagen comprises:
With pichia spp SMD1168 is starting strain, carries out the structure of pichia yeast genetic engineering bacteria; The fermentation culture of pichia yeast genetic engineering bacteria and inducing; The purification of recombination human collagen obtains target protein.
Related substratum composition is respectively:
The YPD substratum:
Yeast extract powder 10g/L Tryptones 20g/L
Glucose 20g/L.
The BMGY substratum:
Yeast extract powder 10g/L Tryptones 20g/L
PH6.0 buffer solution of potassium phosphate 100mmol/L YNB 13.4g/L
Biotin 0.2mg/L glycerine 10mL/L.
The BMMY substratum:
Yeast extract powder 10g/L Tryptones 20g/L
PH6.0 buffer solution of potassium phosphate 100mmol/L YNB 13.4g/L
Biotin 0.2mg/L methyl alcohol 10mL/L.
One, the structure of pichia yeast genetic engineering bacteria
(1), the structure of recombinant expression vector
Extract RNA from newborn infant's placenta tissue, reverse transcription obtains cDNA; Human collagen I type and the III type gene order delivered according to GenBank, according to the design of primers principle, utilize PrimerPremier 5.0 and DNAMAN software, design col1a1 and col3a1 gene PCR amplimer F1 (SEQ ID NO.3), R1 (SEQ ID NO.4), F2 (SEQ ID NO.5), R2 (SEQ IDNO.6), primer sequence is respectively:
F1：5’-CTGGCGCAGA TGGTGTTG-3’；
R1：5’-CCACGGTGAC CCTTTATGC-3’。
F2：5’-GAATCTGTGA ATCATGCCCT AC-3’；
R2：5’-GAAGTCATAA TCTCATCGGT GTT-3’。
I type and III collagen type gene helical region partly increased out and make joint at two of gene with proper restriction site obtains recombinant DNA, and is sewn onto and obtains recombinant expression vector pPIC9K-co3a1-co1a1 among the yeast expression vector pPIC9K; (SEQID NO.2) is as follows for the sequence of this recombinant DNA:
GCGGGTAACA CTGGTGCTCC TGGCAGCCCT GGAGTGTCTG GACCAAAAGG TGATGCTGGC 60
CAACCAGGAG AGAAGGGATC GCCTGGTGCC CAGGGCCCAC CAGGAGCTCC AGGCCCACTT 120
GGGATTGCTG GGATCACTGG AGCACGGGGT CTTGCAGGAC CACCAGGCAT GCCAGGTCCT 180
AGGGGAAGCC CTGGCCCTCA GGGTGTCAAG GGTGAAAGTG GGAAACCAGG AGCTAACGGT 240
CTCAGTGGAG AACGTGGTCC CCCTGGACCC CAGGGTCTTC CTGGTCTGGC TGGTACAGCT 300
GGTGAACCTG GAAGAGATGG AAACCCTGGA TCAGATGGTC TTCCAGGCCG AGATGGATCT 360
CCTGGTGGCA AGGGTGATCG TGGTGAAAAT GGCTCTCCTG GTGCCCCTGG CGCTCCTGGT 420
CATCCAGGCC CACCTGGTCC TGTCGGTCCA GCTGGAAAGA GTGGTGACAG AGGAGAAAGT 480
GGCCCTGCTG GCCCTGCTGG TGCTCCCGGT CCTGCTGGTT CCCGAGGTGC TCCTGGTCCT 540
CAAGGCCCAC GTGGTGACAA AGGTGAAACA GGTGAACGTG GAGCTGCTGG CATCAAAGGA 600
CATCGAGGAT TCCCTGGTAA TCCAGGTGCC CCAGGTTCTC CAGGCCCTGC TGGTCAGCAG 660
GGTGCAATCG GCAGTCCAGG ACCTGCAGAA TTCGGCGCAG ATGGTGTTGC TGGTCCCAAG 720
GGTCCCGCTG GTGAACGTGG TTCTCCTGGC CCTGCTGGCC CCAAAGGATC TCCTGGTGAA 780
GCTGGTCGTC CCGGTGAAGC TGGTCTGCCT GGTGCCAAGG GTCTGACTGG AAGCCCTGGC 840
AGCCCTGGTC CTGATGGCAA AACTGGCCCC CCTGGTCCCG CCGGTCAAGA TGGTCGCCCC 900
GGACCCCCAG GCCCACCTGG TGCCCGTGGT CAGGCTGGTG TGATGGGATT CCCTGGACCT 960
AAAGGTGCTG CTGGAGAGCC CGGCAAGGCT GGAGAGCGAG GTGTTCCCGG ACCCCCTGGC 1020
GCTGTCGGTC CTGCTGGCAA AGATGGAGAG GCTGGAGCTC AGGGACCCCC TGGCCCTGCT 1080
GGTCCCGCTG GCGAGAGAGG TGAACAAGGC CCTGCTGGCT CCCCCGGATT CCAGGGTCTC 1140
CCTGGTCCTG CTGGTCCTCC AGGTGAAGCA GGCAAACCTG GTGAACAGGG TGTTCCTGGA 1200
GACCTTGGCG CCCCTGGCCC CTCTGGAGCA AGAGGCGAGA GAGGTTTCCC TGGCGAGCGT 1260
GGTGTGCAAG GTCCCCCTGG TCCTGCTGGT CCCCGAGGGG CCAACGGTGC TCCCGGCAAC 1320
GATGGTGCTA AGGGTGATGC TGGTGCCCCT GGAGCTCCCG GTAGCCAGGG CGCCCCTGGC 1380
CTTCAGGGAA TGCCTGGTGA ACGTGGTGCA GCTGGTCTTC CAGGGCCTAA GGGTGACAGA 1440
GGTGATGCTG GTCCCAAAGG TGCTGATGGC TCTCCTGGCA AAGATGGCGT CCGTGGTCTG 1500
ACTGGCCCCA TTGGTCCTCC TGGCCCTGCT GGTGCCCCTG GTGACAAGGG TGAAAGTGGT 1560
CCCAGCGGCC CTGCTGGTCC CACTGGAGCT CGTGGTGCCC CCGGAGACCG TGGTGAGCCT 1620
GGTCCCCCCG GCCCTGCTGG CTTTGCTGGC CCCCCTGGTG CTGACGGCCA ACCTGGTGCT 1680
AAAGGCGAAC CTGGTGATGC TGGTGCTAAA GGCGATGCTG GTCCCCCTGG CCCTGCCGGA 1740
CCCGCTGGAC CCCCTGGCCC CATTGGTAAT GTTGGTGCTC CTGGAGCCAA AGGTGCTCGC 1800
GGCAGCGCTG GTCCCCCTGG TGCTACTGGT TTCCCTGGTG CTGCTGGCCG AGTCGGTCCT 1860
CCTGGCCCCT CTGGAAATGC TGGACCCCCT GGCCCTCCTG GTCCTGCTGG CAAAGAAGGC 1920
GGCAAAGGTC CCCGTGGTGA GACTGGCCCT GCTGGACGTC CTGGTGAAGT TGGTCCCCCT 1980
GGTCCCCCTG GCCCTGCTGG CGAGAAAGGA TCCCCTGGTG CTGATGGTCC TGCTGGTGCT 2040
CCTGGTACTC CCGGGCCTCA AGGTATTGCT GGACAGCGTG GTGTGGTCGG CCTGCCTGGT 2100
CAGAGAGGAG AGAGAGGCTT CCCTGGTCTT CCTGGCCCCT CTGGTGAACC TGGCAAACAA 2160
GGTCCCTCTG GAGCAAGTGG TGAACGTGGT CCCCCTGGTC CCATGGGCCC CCCTGGATTG 2220
GCTGGACCCC CTGGTGAATC TGGACGTGAG GGGGCTCCTG GTGCCGAAGG TTCCCCTGGA 2280
CGAGACGGTT CTCCTGGCGC CAAGGGTGAC CGTGGTGAGA CCGGCCCCGC TGGACCCCCT 2340
GGTGCTCCTG GTGCTCCTGG TGCCCCTGGC CCCGTTGGCC CTGCTGGCAA GAGTGGTGAT 2400
CGTGGTGAGA CTGGTCCTGC TGGTCCCGCC GGTCCTGTCG GCCCTGTTGG CGCCCGTGGC 2460
CCCGCCGGAC CCCAAGGCCC CCGTGGTGAC AAGGGTGAGA CAGGCGAACA GGGCGAC 2517
(2), prepare recombinant plasmid pPIC9K-co3a1-co1a1 in a large number
The carrier bacterial strain containing cultivation about 20 hours on the substratum of kalamycin resistance, centrifugal 10 minutes of 8000r/min, is removed supernatant, extract plasmid DNA with plasmid extraction kit.
(3), the linearizing of pPIC9K-co3a1-co1a1 plasmid
Get restriction enzyme SalI 10 μ L, about pPIC9K-co3a1-co1a1 plasmid 20 μ g, add 100 μ L buffer H, add water and supply mixing behind the 1000 μ L, 37 ℃ of water-bath digestion 5h take out 5 μ L reaction solutions earlier before the termination reaction, detect with 0.7% agarose gel electrophoresis whether enzyme cuts entirely, after enzyme cuts entirely, with plasmid extraction kit linearization plasmid is carried out plasmid and extract.When extraction was finished, the volume of linearization plasmid will be more than 10 μ L, and concentration is more than the 1.0 μ g/ μ L.Use deionized water dissolving, the electricity that is used for next step transforms.
(4), change the pichia spp method
1. the preparation of pichia spp competent cell
The single bacterium colony of picking pichia spp (SMD1168) is seeded to and contains in the 5mL YPD substratum test tube, and 30 ℃, 250rpm～300rpm overnight incubation; The culture of getting 50 μ L is seeded in the 300mL triangular flask that contains the fresh YPD substratum of 50mL, and 28 ℃～30 ℃, 250rpm～300rpm overnight incubation reaches 1.1～1.3 to the OD600 value; With cell culture in 4 ℃, the centrifugal 5min of 1500g, with the sterilized water of the ice precooling of 50mL that bacterial sediment is resuspended; Use after the repeated centrifugation 1mol/L Sorbitol Solution USP of ice precooling of 20mL that bacterial sediment is resuspended by the same way, centrifugal, the 1mol/L Sorbitol Solution USP of the ice precooling of usefulness 0.3mL is resuspended with bacterial sediment, and its final volume is about 0.5mL, be packed as 80 μ L portions ,-70 ℃ of preservations.
2. the electricity of pichia spp transforms
Competent cell is thawed and ice bath, with the pichia spp competent cell mixing of linearizing DNA about 10 μ g and 80 μ L, the electricity that goes to the precooling of 0.2cm ice transforms in the cup, is 1.5kV at voltage, electric capacity is 25 μ F, and resistance is the 4msec～10msec that shocks by electricity under the 200 Ω conditions; After electric shock finished, the 1mol/L Sorbitol Solution USP that adds the precooling of 1mL ice formed bacteria suspension with the thalline mixing, goes in the 1.5mL centrifuge tube; Bacteria suspension is coated on the MD flat board, and per 100 μ L～200 μ L are coated with one flat plate, place 30 ℃ of incubators to be inverted then and cultivate.
3. multiple copied inserts the screening of recon
(1) there is the MD planar surface of transformant to add 2mL sterilization distilled water in growth, is coated with gently on His+ transformant surface with aseptic triangle spreader then and scrapes, and transfer in the 50mL centrifuge tube;
(2) in centrifuge tube described in the step (1), add the dilution of 20mL sterilization distilled water, measure OD600 value (1OD600=5 * 10 behind the mixing
7Cells/mL);
(3) get 10
5Individual transformant is coated on the YPD flat board that contains 0.5mg/mL G418, is inverted, and cultivates 72h～96h for 30 ℃;
(4) every hole adds 200 μ L YPD liquid nutrient mediums in aseptic 96 orifice plates;
(5) insert the transformant of cultivating acquisition in the step (3) with connecing the bacterium toothpick respectively in 96 orifice plates described in the step (4), mixing is cultivated 48h in 30 ℃;
(6) get new aseptic 96 orifice plates, every hole adds 190 μ L YPD liquid nutrient mediums, adds the culture of 96 orifice plate gained in the 10 μ L steps (5) then in respective aperture, cultivates 24h in 30 ℃;
(7) get new aseptic 96 orifice plates again, every hole adds 190 μ L YPD liquid nutrient mediums, adds the culture of 96 orifice plate gained in the 10 μ L steps (6) in respective aperture, cultivates 24h in 30 ℃;
(8) take out 1 μ L nutrient solution in 96 orifice plates from step (7) respectively, point is on the YPD flat board that contains 1.0mg/mL and 4.0mg/mL G418, cultivate 96h～120h in 30 ℃, pichia spp SMD1168 transformant is if can grow containing on the substratum of high density G418 more, illustrate that this transformant contains the goal gene of multiple copied more, promptly have a plurality of pPIC9K-co3a1-co1a1 segments to transform into pichia spp SMD1168 and be incorporated on the karyomit(e) of pichia spp SMD1168, the recombinant yeast pichia pastoris of the height copy that obtains through this step screening will more likely be realized efficiently expressing of target protein.
4. bacterium colony PCR identifies recombinant yeast pichia pastoris
Picking pichia spp transformant to be measured, carry out the prefabricated of pcr template as follows:
(1) single bacterium colony of the high copy of picking transformant places the 1.5mL centrifuge tube that 200 μ L deionized waters are housed, and the centrifugal 5min of 2000g abandons supernatant;
(2) centrifuge tube that thalline will be housed places the middle-grade heating of microwave oven 2min, transfers to-80 ℃ of freezing 5min then;
(3) transfer to-80 ℃ of freezing 5min after placing the middle-grade heating of microwave oven 2min again, move to the middle-grade heating of microwave oven 2min at last again;
(4) in step (3), add 30 μ L deionized waters in the centrifuge tube after the heating,, draw the template of supernatant as the PCR reaction in the centrifugal 1min of 10000g;
(5) use 9k-col1 primers designed F (SEQ ID NO.7), R (SEQ ID NO.8), carry out pcr amplification; Wherein 9k-col1 primers designed sequence is:
F：5’-GCTGAAGCTG TCATCGGTTA CTCA-3’；
R：5’-TCGCTCTCCA GCCTTGCCG-3’。
(6) after PCR finishes, carry out agarose electrophoresis and detect, band about 1155bp, occurs, can tentatively be defined as positive transformant.
(5), protein expression analysis
To screen from high resistant panel (the YPD flat board that contains G418) and the positive strain process checking carries out the abduction delivering experiment according to following program:
Positive strain is inoculated in 5mL YPD substratum, cultivate 12h for 30 ℃, switching is gone in the 20mLBMGY substratum then, and 30 ℃, 250rpm cultivates 12h, outwell supernatant behind the centrifugal 10min of 4000rpm, use the sterile water wash thalline, the centrifugal 10min of 4000rpm, adding an amount of BMMY substratum, to make the OD600 of bacterium liquid be 1.0, at 30 ℃, under the 250rpm condition, methanol induction 120h (adding 1% methyl alcohol of substratum cumulative volume every 24h) gets bacterium liquid sample respectively by time point, sampling amount is 1.0mL, place the 1.5mL centrifuge tube, normal temperature, the centrifugal 5min of 12000rpm, collect supernatant, time point is generally got: 12h, 24h, 36h, 48h, 60h and 72h.
(6), recombination human collagen N end order-checking
Analyze carrying out SDS-PAGE (polyacrylamide gel electrophoresis) behind the supernatant purifying of collecting, and with Xylene Brilliant Cyanine G G-250 dye single band, as seen molecular weight is at 100KD, bigger than theoretical molecular, go to recombinant protein on the pvdf membrane and cut and carry out N end order-checking, the result that the N terminal amino acid is measured is A-G-N-T-G, and is consistent with gene design.
(7), recombination human collagen mass spectroscopy
With the supernatant AXIMA-CFR behind the purifying
TMThe Human-like Collagen molecular weight that the substance assistant laser desorpted attached ionization flight mass spectrometer of plus records is 74019.02, is-0.26% with calculating molecular weight 74213.9 relative errors, accuracy＜± 0.5%.
(8), dna sequence analysis
Extract the positive strain genome, as template, adopt PCR method amplification recombination human collagen gene, amplified production carries out the dna sequencing analysis through purifying, the recombinant DNA measurement result of PCR method amplification is consistent with gene design, determine that this positive strain is pichia yeast genetic engineering bacteria (pichia pastoris phaff Pichiapastoris C13, deposit number are CGMCC No.4437).
Two, the fermentation culture of pichia yeast genetic engineering bacteria and inducing
Picking pichia yeast genetic engineering bacteria list bacterium colony places the 150mL triangular flask that 30mL BMGY substratum is housed, and being cultured to OD600 in 28 ℃～30 ℃, 250rpm～300rpm is 2～6; The centrifugal 5min of 1500g～3000g under the room temperature collects thalline, and with the resuspended thalline of BMMY substratum, making the thalline OD600 after resuspended is 1.0; The bacterium liquid of resuspended thalline gained is placed the triangular flask of 500mL, seal,, continue on the shaking table of 250rpm～300rpm to cultivate in 28 ℃～30 ℃ with four layers of gauze; Every 24h adds anhydrous methanol to substratum in substratum, the addition of anhydrous methanol is 1% of a substratum cumulative volume; Get bacterium liquid sample respectively by time point, sampling amount is 1.0mL, place the 1.5mL centrifuge tube, normal temperature, the centrifugal 5min of 12000rpm collects supernatant, the best harvest time of proteic expression amount of analysis purposes and bacterium liquid, time point is generally got: 12h, 24h, 36h, 48h, 60h and 72h, detected sample is standby in-70 ℃ of preservations.
Three, the purification of recombination human collagen
Separation purifying technique
The pichia yeast genetic engineering bacteria bacterium liquid of cultivating is collected supernatant liquor through after the centrifugation, and supernatant liquor is removed colour substance and fishy smell through chromatography column S100, removes unnecessary salinity through desalting column G75 again, and ultrafiltration and concentration after the desalination is collected the protein frozen drying.
The present invention is selected water-soluble, nucleotide sequence that stability is strong from the helical region of the human collagen protein gene I type of known array and III type, this two fragment gene is inserted in the Pichia anomala expression plasmid, transform the pichia spp screening and obtain the high expression level pichia yeast genetic engineering bacteria, jar large scale fermentation secreting, expressing and a series of purification step obtain highly purified recombination human collagen by fermentation.Experimental results show that the recombination human collagen that this method is produced has good hydrophilicity and stability, because its structure is identical with natural collagen protein gene order corresponding section 100%, so be applied to can not cause immunological rejection in the middle of the human body, can be widely used in fields such as bio-medical material, makeup.The Human-like Collagen expression amount height that this invention obtains, purifying is convenient, expression amount in fermentor tank can reach 5.0g/L, single batch of 50 liters of jars can be produced about crude protein 150g, because the expression amount height, crude protein purity is more than 80%, and foreign protein seldom, have only common by product of pichia spp and fishy smell etc. to need to remove, bring great convenience to purifying like this.What the present invention adopted is secreted expression carrier; successfully realized the secretion type expression of recombination human collagen, expression product is secreted in supernatant liquor, makes things convenient for purifying; have other recombination human collagens and produce not available advantage, be convenient to the large-scale production operation.Because the carrier electricity changes over to after the pichia spp, gene integration is on the pichia spp genome, so good stability of reorganization bacterium, be not easy to run off and can keep the proterties that efficiently expresses through the gene that repeatedly goes down to posterity, can be good at realizing stably manufactured, the pichia spp production method is an aerobic fermentation, the cell density height, and expression amount also has very big room for promotion.
Fig. 1 is that the structure route map of carrier pPIC9K-co3a1-co1a1: co3a1 represents III collagen type helical region portion gene, and co1a1 represents type i collagen albumen helical region portion gene, and pPIC9k represents yeast expression vector.Co3a1 and pPIC9k after double digestion (Xho I and EcoR I are two to be cut) connect into pPIC9k-co3a1, and pPIC9k-co3a1 and co1a1 connect into pPIC9k-co3a1-co1a1 with after the EcoR1 single endonuclease digestion, are final expression vector.
Fig. 2 is the electrophorogram that bacterium colony PCR identifies recombinant yeast pichia pastoris: through the repeated screening of G418 different concns, finally under high density 4.0mg/mL condition, screen 9 high copy recons, and it is carried out bacterium colony PCR identify, the result shows, 9 recons can both amplify the fragment about about 1155bp, illustrate that the recombinant screen result is correct.
Fig. 3 is the electrophorogram behind the pPIC9K-co3a1-co1a1 recombinant yeast pichia pastoris abduction delivering: select high copy recon bacterial strain shake flask fermentation, after the methanol induction fermentation, respectively in 24h, 36h, 48h, 60h and 72h sampling, the SDS-PAGE electrophoretic analysis, the expressed proteic apparent molecular weight of the co3a1-co1a1 gene of demonstration and prediction is 100KDa, and is bigger than theoretical molecular, ferment and reached the highest to the 60h expression amount, and band is single, does not have other foreign protein substantially, is convenient to the purifying in later stage.
Claims (6)
1. a recombination human collagen is characterized in that, its sequence is as follows:
AGNTGAPGSP GVSGPKGDAG QPGEKGSPGA QGPPGAPGPL GIAGITGARG LAGPPGMPGP 60
RGSPGPQGVK GESGKPGANG LSGERGPPGP QGLPGLAGTA GEPGRDGNPG SDGLPGRDGS 120
PGGKGDRGEN GSPGAPGAPG HPGPPGPVGP AGKSGDRGES GPAGPAGAPG PAGSRGAPGP 180
QGPRGDKGET GERGAAGIKG HRGFPGNPGA PGSPGPAGQQ GAIGSPGPAE FGADGVAGPK 240
GPAGERGSPG PAGPKGSPGE AGRPGEAGLP GAKGLTGSPG SPGPDGKTGP PGPAGQDGRP 300
GPPGPPGARG QAGVMGFPGP KGAAGEPGKA GERGVPGPPG AVGPAGKDGE AGAQGPPGPA 360
GPAGERGEQG PAGSPGFQGL PGPAGPPGEA GKPGEQGVPG DLGAPGPSGA RGERGFPGER 420
GVQGPPGPAG PRGANGAPGN DGAKGDAGAP GAPGSQGAPG LQGMPGERGA AGLPGPKGDR 480
GDAGPKGADG SPGKDGVRGL TGPIGPPGPA GAPGDKGESG PSGPAGPTGA RGAPGDRGEP 540
GPPGPAGFAG PPGADGQPGA KGEPGDAGAK GDAGPPGPAG PAGPPGPIGN VGAPGAKGAR 600
GSAGPPGATG FPGAAGRVGP PGPSGNAGPP GPPGPAGKEG GKGPRGETGP AGRPGEVGPP 660
GPPGPAGEKG SPGADGPAGA PGTPGPQGIA GQRGVVGLPG QRGERGFPGL PGPSGEPGKQ 720
GPSGASGERG PPGPMGPPGL AGPPGESGRE GAPGAEGSPG RDGSPGAKGD RGETGPAGPP 780
GAPGAPGAPG PVGPAGKSGD RGETGPAGPA GPVGPVGARG PAGPQGPRGD KGETGEQGD 839
The structure of described recombination human collagen is strand, single coil configuration, and its amino acid is 839, and wherein 1-241 is an III type peptide section, and 242-839 is an I type peptide section.
2. a method of producing recombination human collagen as claimed in claim 1 is characterized in that, this method may further comprise the steps:
(1) structure of pichia yeast genetic engineering bacteria;
(2) fermentation culture of pichia yeast genetic engineering bacteria;
(3) recombination human collagen inducing and expressing;
(4) purifying of recombination human collagen.
3. the production method of a kind of recombination human collagen according to claim 2 is characterized in that, the structure of pichia yeast genetic engineering bacteria is as follows described in the step (1):
A. the fragment gene with known I type of human body and III collagen type gene helical region extracts respectively, sews up recombinant DNA then;
B. recombinant DNA is changed over to and is incorporated in the genome of pichia spp, screening obtains pichia yeast genetic engineering bacteria.
4. the production method of a kind of recombination human collagen according to claim 2, it is characterized in that, the fermentation culture process of pichia yeast genetic engineering bacteria is as follows described in the step (2): picking pichia yeast genetic engineering bacteria list bacterium colony, place the 150mL triangular flask that 30mL BMGY substratum is housed, in 28 ℃～30 ℃, it is 2.0～6.0 that 250rpm～300rpm is cultured to OD600, the centrifugal 5min of 1500g～3000g under the room temperature, collect thalline, with the resuspended thalline of BMMY substratum, making the thalline OD600 after resuspended is 1.0; The bacterium liquid of resuspended thalline gained is placed the triangular flask of 500mL, seal with four layers of gauze, continue to cultivate on 28 ℃～30 ℃, the shaking table of 250rpm～300rpm, every 24h adds anhydrous methanol in substratum; The addition of described anhydrous methanol is 1% of a substratum cumulative volume.
5. the production method of a kind of recombination human collagen according to claim 2, it is characterized in that, the purge process of the described recombination human collagen of step (4) is: the pichia yeast genetic engineering bacteria of cultivation is collected supernatant liquor through after the centrifugation, the supernatant liquor of collecting is removed colour substance and fishy smell through chromatography column S100, remove unnecessary salinity through desalting column G75 again, ultrafiltration and concentration after the desalination is collected the protein frozen drying.
6. the production method of a kind of recombination human collagen according to claim 3 is characterized in that, the sequence of described recombinant DNA is as follows:
GCGGGTAACA CTGGTGCTCC TGGCAGCCCT GGAGTGTCTG GACCAAAAGG TGATGCTGGC 60
CAACCAGGAG AGAAGGGATC GCCTGGTGCC CAGGGCCCAC CAGGAGCTCC AGGCCCACTT 120
GGGATTGCTG GGATCACTGG AGCACGGGGT CTTGCAGGAC CACCAGGCAT GCCAGGTCCT 180
AGGGGAAGCC CTGGCCCTCA GGGTGTCAAG GGTGAAAGTG GGAAACCAGG AGCTAACGGT 240
CTCAGTGGAG AACGTGGTCC CCCTGGACCC CAGGGTCTTC CTGGTCTGGC TGGTACAGCT 300
GGTGAACCTG GAAGAGATGG AAACCCTGGA TCAGATGGTC TTCCAGGCCG AGATGGATCT 360
CCTGGTGGCA AGGGTGATCG TGGTGAAAAT GGCTCTCCTG GTGCCCCTGG CGCTCCTGGT 420
CATCCAGGCC CACCTGGTCC TGTCGGTCCA GCTGGAAAGA GTGGTGACAG AGGAGAAAGT 480
GGCCCTGCTG GCCCTGCTGG TGCTCCCGGT CCTGCTGGTT CCCGAGGTGC TCCTGGTCCT 540
CAAGGCCCAC GTGGTGACAA AGGTGAAACA GGTGAACGTG GAGCTGCTGG CATCAAAGGA 600
CATCGAGGAT TCCCTGGTAA TCCAGGTGCC CCAGGTTCTC CAGGCCCTGC TGGTCAGCAG 660
GGTGCAATCG GCAGTCCAGG ACCTGCAGAA TTCGGCGCAG ATGGTGTTGC TGGTCCCAAG 720
GGTCCCGCTG GTGAACGTGG TTCTCCTGGC CCTGCTGGCC CCAAAGGATC TCCTGGTGAA 780
GCTGGTCGTC CCGGTGAAGC TGGTCTGCCT GGTGCCAAGG GTCTGACTGG AAGCCCTGGC 840
AGCCCTGGTC CTGATGGCAA AACTGGCCCC CCTGGTCCCG CCGGTCAAGA TGGTCGCCCC 900
GGACCCCCAG GCCCACCTGG TGCCCGTGGT CAGGCTGGTG TGATGGGATT CCCTGGACCT 960
AAAGGTGCTG CTGGAGAGCC CGGCAAGGCT GGAGAGCGAG GTGTTCCCGG ACCCCCTGGC 1020
GCTGTCGGTC CTGCTGGCAA AGATGGAGAG GCTGGAGCTC AGGGACCCCC TGGCCCTGCT 1080
GGTCCCGCTG GCGAGAGAGG TGAACAAGGC CCTGCTGGCT CCCCCGGATT CCAGGGTCTC 1140
CCTGGTCCTG CTGGTCCTCC AGGTGAAGCA GGCAAACCTG GTGAACAGGG TGTTCCTGGA 1200
GACCTTGGCG CCCCTGGCCC CTCTGGAGCA AGAGGCGAGA GAGGTTTCCC TGGCGAGCGT 1260
GGTGTGCAAG GTCCCCCTGG TCCTGCTGGT CCCCGAGGGG CCAACGGTGC TCCCGGCAAC 1320
GATGGTGCTA AGGGTGATGC TGGTGCCCCT GGAGCTCCCG GTAGCCAGGG CGCCCCTGGC 1380
CTTCAGGGAA TGCCTGGTGA ACGTGGTGCA GCTGGTCTTC CAGGGCCTAA GGGTGACAGA 1440
GGTGATGCTG GTCCCAAAGG TGCTGATGGC TCTCCTGGCA AAGATGGCGT CCGTGGTCTG 1500
ACTGGCCCCA TTGGTCCTCC TGGCCCTGCT GGTGCCCCTG GTGACAAGGG TGAAAGTGGT 1560
CCCAGCGGCC CTGCTGGTCC CACTGGAGCT CGTGGTGCCC CCGGAGACCG TGGTGAGCCT 1620
GGTCCCCCCG GCCCTGCTGG CTTTGCTGGC CCCCCTGGTG CTGACGGCCA ACCTGGTGCT 1680
AAAGGCGAAC CTGGTGATGC TGGTGCTAAA GGCGATGCTG GTCCCCCTGG CCCTGCCGGA 1740
CCCGCTGGAC CCCCTGGCCC CATTGGTAAT GTTGGTGCTC CTGGAGCCAA AGGTGCTCGC 1800
GGCAGCGCTG GTCCCCCTGG TGCTACTGGT TTCCCTGGTG CTGCTGGCCG AGTCGGTCCT 1860
CCTGGCCCCT CTGGAAATGC TGGACCCCCT GGCCCTCCTG GTCCTGCTGG CAAAGAAGGC 1920
GGCAAAGGTC CCCGTGGTGA GACTGGCCCT GCTGGACGTC CTGGTGAAGT TGGTCCCCCT 1980
GGTCCCCCTG GCCCTGCTGG CGAGAAAGGA TCCCCTGGTG CTGATGGTCC TGCTGGTGCT 2040
CCTGGTACTC CCGGGCCTCA AGGTATTGCT GGACAGCGTG GTGTGGTCGG CCTGCCTGGT 2100
CAGAGAGGAG AGAGAGGCTT CCCTGGTCTT CCTGGCCCCT CTGGTGAACC TGGCAAACAA 2160
GGTCCCTCTG GAGCAAGTGG TGAACGTGGT CCCCCTGGTC CCATGGGCCC CCCTGGATTG 2220
GCTGGACCCC CTGGTGAATC TGGACGTGAG GGGGCTCCTG GTGCCGAAGG TTCCCCTGGA 2280
CGAGACGGTT CTCCTGGCGC CAAGGGTGAC CGTGGTGAGA CCGGCCCCGC TGGACCCCCT 2340
GGTGCTCCTG GTGCTCCTGG TGCCCCTGGC CCCGTTGGCC CTGCTGGCAA GAGTGGTGAT 2400
CGTGGTGAGA CTGGTCCTGC TGGTCCCGCC GGTCCTGTCG GCCCTGTTGG CGCCCGTGGC 2460
CCCGCCGGAC CCCAAGGCCC CCGTGGTGAC AAGGGTGAGA CAGGCGAACA GGGCGAC 2517
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