CN101250553A - Human thrombopoietin expression vector and constructing method therefor - Google Patents
Human thrombopoietin expression vector and constructing method therefor Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering and transgenic engineering and in particular relates to a method for building mammary gland specific expression vectors of human thrombopoietin gene and goat beta-casein gene promoter. The vectors which are built are transferred to COS-7 and HC-11 cells and expressed on eukaryotic cell level verification hTPO. The built mammary gland specific expression vectors of the human thrombopoietin gene and the goat beta-casein gene promoter are applied to prepare a mammary gland bioreactor of transgenic animals and are expressed with high level in the milk of transgenic mammalian, and the vectors provide useful drug protein-hTPO for clinic. The mammary gland specific expression vectors of human thrombopoietin gene and goat beta-casein gene promoter which are provided by the invention provide a foundation for further research and development.
Description
The application is that application number is 02136029.4, and the applying date is on July 13rd, 2002, and denomination of invention is divided an application for " a kind of preparation method's of thrombopoietin ".
Technical field
The invention belongs to genetically engineered and gene engineering technique field, relate to a kind of preparation method of thrombopoietin.Thrombopoietin (hTPO) genomic dna and the structure of cDNA expression vector and the method for producing hTPO that are specifically related to utilize goat-β casein promoter to instruct by these carrier cells transfected and transgenic animal.
Background technology
Thrombopoietin (Thrombopoietin, TPO) be an important Hemopoietic factor, its acceptor c-mpl is mainly at megalokaryocyte, thrombocyte and hemopoietic forebody cell, therefore the main biological function of TPO is a stimulating megakaryocyte propagation, differentiation and Thrombopoiesis, and the acceleration hemopoietic forebody cell enters the cell cycle.Experimentation on animals and clinical study show that recombinant human thrombopoietin (rhTPO) can effectively promote platelet recovery, alleviate the thrombocytopenia that causes owing to radiation and chemotherapy.
HTPO genome total length 6.2kb is included to give by six exons and five and forms, and can detect an extra exon in first exon upstream, called after exon0 (exon 0).Southern trace and fluorescence in situ hybridization show: hTPO is a single copy gene, is positioned at No. 3 karyomit(e) of people (3q26-27).HTPO cDNA is made up of the additional polyA tail of 1774 Nucleotide.Initiator codon is positioned at Nucleotide 216-218 place, its open reading frame is made up of 1059 Nucleotide, is responsible for coding 353 amino acid whose hTPO molecules, and wherein to hold preceding 21 amino acid be signal peptide sequence to N, people's mature T PO molecule is made up of 332 amino acid, and theoretical molecular is 36KD.
HTPO genome sequence and cDNA sequence are announced by GeneBank, in the patent application of U.S. Zymogenetics, Inc. (PCT/US95/14932), the method that discloses DNA construct useful in the production of TPO and produced the TPO polypeptide by the eukaryotic cell of cultivating.But Shang Weijian hTPO gene (Genomic DNA, cDNA) make up the report of mammary gland-specific expression vector with goat-β casein promoter, do not see that (Genomic DNA, cDNA) carrier reaches the report of producing hTPO by transgene mammal with the hTPO gene yet.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of thrombopoietin.Specifically be to utilize thrombopoietin (hTPO) genomic dna, the cDNA that goat-β casein promoter instructs and the structure of the hTPO cDNA expression vector that contains intron V and the method for producing hTPO by described carrier construction cells transfected and transgenic animal technology.
The present invention starts the thrombopoietin expression vector that gives guidance with the goat-β casein that makes up and is applied to prepare mammary gland bioreactor of transgenic animals, make it high expression level in the milk of transgene mammal, be the clinical pharmaceutical protein-hTPO that provides usefulness.HTPO gene provided by the present invention (Genomic DNA, cDNA) and the mammary gland-specific expression vector that makes up with goat-β casein promoter, for further research and development provide the foundation.
The present invention realizes by following technical proposals and step.
(1) makes up the hTPO expression vector that goat-β casein promoter instructs.
1, obtain hTPO full length gene nucleotide sequence by following method:
DNA is a template with Chinese han population tire liver gene group, is that primer-oligonucleotide A1:5 '-AGG TACCTGATGACCTGCTGCTGT-3 ' is a forward primer with a pair of Nucleotide; Oligonucleotide A2:5 '-CGGCGCTCC CATTTATTCCTTATAC-3 ' is a reverse primer, adopts Long-PCR Kit (Bao Bio-Engineering Company) to carry out pcr amplification.The PCR reaction conditions is: 95 ℃ of 1min; (98 ℃, 20sec; 65 ℃, 10min) * 30; 72 ℃, 10min.Electrophoresis detection purpose fragment then.
2, the hTPO full length sequence that amplification, transfection obtain:
The pcr amplification product that is obtained is connected with pGEM-T carrier (Promega company) respectively, ordinary method transfection Escherichia coli Top10F ' host bacterium competence cell, plasmid increases in a small amount, the alkaline lysis method of extracting plasmid.The plasmid that takes a morsel and extract is cut preliminary evaluation with PstI and BamHI enzyme.AB1370 automatic sequencer (Perkin-Elemer, company) order-checking, the hTPO full-length gene of acquisition 6340bp comprises exons 1-6 and whole introns.Complete sequence analysis shows: in the hTPO genome sequence of above-mentioned amplification, all encoding sequence, whole intron/exon montage recognition sequence are compared consistent with the gene order of known hTPO; But at 349 and 1490 G → A and G → T sudden change take place; 4940,5887, A → G, T → C, A → T sudden change takes place respectively, all be positioned at non-coding region.
3, obtain hTPO cDNA nucleotide fragments:
MRNA is a template with people's tire liver, at first adopts RT-PCR Kit (STRATA GENE), carries out reverse transcription; The cDNA that obtains with reverse transcription is a template then, adopt two to be primer to (B1/B2, C1/C2) Nucleotide: oligonucleotide B1:5 '-GCGTCGACAGAATGGAGCTGA-CTGAATT-3 ' is a forward primer, and oligonucleotide B2:5 '-GGCATTGGGATCCTT-GTGAGCTGTGG-3 ' is a reverse primer; Oligonucleotide C1:5 '-CAGCTCACAA-GGATCCCAATGCCATCTTC-3 ' is a forward primer, and C2:5 '-CGCAGCTG-ACACGGCAGTGTCTGAGAACCTTA-3 ' is a reverse primer, carries out pcr amplification.The reaction conditions of PCR is: 85-95 ℃ of 3-5min; 48-54 ℃ of 3-5min; (85-94 ℃ of 1-5min; 58-62 ℃ of 1-5min; 65-72 ℃ of 50-90sec) * 35; 68-72 ℃, 5-10min.Electrophoresis detection purpose fragment then obtains two TPO cDNA fragments of 450bp and 660bp respectively.
4, obtain hTPO cDNA molecule complete sequence:
Two cDNA fragments through pcr amplification are cut with Sal I and BamHI enzyme respectively, order-checking plasmid pBSK Sal I single endonuclease digestion linearizing, reclaim endonuclease bamhi, then three fragments are connected with T4 ligase enzyme (NewEngland Bio company), and transform JM109 competence bacterium, plasmid increases on a small quantity, alkaline lysis extracts plasmid, cut evaluation through electrophoresis and enzyme, select the recon that inserts hTPO cDNA, called after pCTPO.
After the ABI370 order-checkingGet the full molecular core nucleotide sequence of hTPO cDNA of 1062bp.
5, obtain intron V in the hTPO genome:
DNA is a template with people's tire liver gene group, and be primer with a pair of Nucleotide: oligonucleotide D1:5 '-ATAAGGCTCCCCACCCGCTTTTAG-3 ' is a forward primer; Oligonucleotide D2:5 '-CGGCGCTCCCATTTATTCCTTATAC-3 ' is a reverse primer, adopts Long-PCRKit (Bao Bio-Engineering Company) to carry out pcr amplification.The PCR reaction conditions is: 95 ' C 1min; (98 ℃, 20sec; 65 ℃, 10min) * 30; 72 ℃, 10min.Electrophoresis detection purpose fragment then.The pcr amplification product that obtains above is connected with pGEM-T carrier (Promega company) respectively, ordinary method transfection Escherichia coli Top10F ' host bacterium competence cell, plasmid increases in a small amount, the alkaline lysis method of extracting plasmid.Complete sequence analysis shows: in the hTPO of above-mentioned amplification genome sequence, and the gene order unanimity of whole encoding sequences, whole intron/hTPO that exon montage recognition sequence has been delivered together; 5887,5925 sudden changes that T → C, A → T take place respectively.
6, utilize above-mentioned gained hTPO genomic dna, cDNA, intron V, make up the hTPO expression vector:
The hTPO cDNA that utilizes the pCMV of pcDNA3.1 (+) and hTPO genomic dna, hTPO cDNA and contain intron V, the pCMV of construction of expression vector: (1) pcDNA3.1 (-) starts the hTPO genomic dna expression vector of expressing; (2) pCMV of pcDNA3.1 (-) starts the hTPO cDNA expression vector that contains intron V of the pCMV startup expression of the hTPO cDNA expression vector of expressing: (3) pcDNA3.1 (-).
7, utilize above-mentioned gained hTPO genomic dna, cDNA, intron V, combine, make up hTPO gene mammary gland expression vector with the mammary gland expression plasmid:
Utilization contains the mammary gland expression vector of goat beta-casein gene promoter region nucleotide sequence SEQ ID No.3, in conjunction with above-mentioned hTPO genomic dna, cDNA, intron V, makes up three-type-person TPO gene mammary gland expression vector.As starting hTPO genomic dna expression vector, the hTPO cDNA expression vector of expression jointly by the pCMV of pcDNA3.1 (-) and goat beta-casein gene 5 ' end 6606bp sequence, and the hTPO cDNA expression vector etc. that contains intron V.
(2) validity and the practicality of three kinds of carriers of above-mentioned hTPO of checking and structure:
1, the expression of checking hTPO gene in eukaryotic cell:
The carrier that makes up is adopted the liposome transfection method, carry out (1) transfection Africa green hair monkey-kidney cells COS-7, (2) second trimester mouse mammary epithelial cell HC-11.Through cell cultures, the collecting cell nutrient solution adopts hTPO Kit (R﹠amp; D) content of detection hTPO proves that hTPO has obtained expression.It is the highest wherein to start the hTPO expression amount of the hTPO cDNA expression vector that contains intron V of expression jointly by the pCMV of pcDNA3.1 (-) and goat beta-casein gene 5 ' end 6609bp sequence.
2, set up the hTPO transgenic mice, verify the expression of hTPO in mouse milk in individual level:
PCMV and goat beta-casein gene 5 ' the end 6609bp sequence set up by pcDNA3.1 (-) by the microinjection transgenic technology start hTPO genomic dna expression vector, the hTPO cDNA expression vector of expression jointly, and the transgenic mice that contains the hTPO cDNA expression vector integration of intron V.Detect confirmation through PCR and Southern.Blot blot hybridization, the integration of hTPO gene is arranged in the transgenic mice body; Adopt hTPO Kit (R﹠amp; D) content of hTPO in the detection mouse milk proves the expression that hTPO is arranged in the mouse milk.Wherein, the hTPO expression amount of holding the 6606bp sequence to start the hTPO cDNA expression vector that contains intron V of expression jointly by pCMV and the goat beta-casein gene 5 ' of pcDNA3.1 (-) is up to 122.8 μ g/ml.
Description of drawings
Fig. 1 is the design of graphics that is started the hTPO genomic dna expression vector of expression by the pCMV of pcDNA3.1 (-) and goat beta-casein gene 5 ' end 6609bp sequence jointly.
Fig. 2 is the design of graphics that is started the hTPO cDNA expression vector of expression by the pCMV of pcDNA3.1 (-) and goat beta-casein gene 5 ' end 6609bp sequence jointly.
Fig. 3 is the design of graphics that is started the people TPO cDNA expression vector that contains intron V of expression by the pCMV of pcDNA3.1 (-) and goat beta-casein gene 5 ' terminal sequence jointly.
Embodiment
Among the of the present invention and following embodiment, employed technology, comprise gene sequencing, pcr amplification and detection, cell transfecting, vector construction, transgenosis equimolecular biology techniques, and cell cultures, detection technique etc., unless stated otherwise, be the known routine techniques of those skilled in the art; Employed plant and instrument, reagent, plasmid and carrier, cell strain etc., only dated especially in this specification sheets, be that the research of this area and technician can obtain by public approach.
The present invention adopts the transgenic experiments animal to comprise all non-human mammals.
Below in conjunction with specific embodiment, the present invention is further elaborated.Described embodiment only is used for explanation rather than limitation of the present invention that the present invention is done.
Embodiment 1: the clone of hTPO genomic dna sequence and mensuration
1.PCR amplification
DNA is a template with people's tire liver gene group, is primer with a pair of Nucleotide;
Oligonucleotide A1:5 '-AGGTACCTGATGACCTGCTGCTGT-3 ' is a forward primer,
Oligonucleotide A2:5 '-CGGCGCTCCCATTTATTCCTTATAC-3 ' is a reverse primer.
Adopt Long-PCR Kit (Bao Bio-Engineering Company) to carry out pcr amplification.The PCR reaction conditions is: 95 ℃ of 1min; (98 ℃, 20sec; 65 ℃, 10min) * 30; 72 ℃, 10min.Electrophoresis detection purpose fragment then.
2.PCR the order-checking of product
The above-mentioned pcr amplification product that obtains is connected with pGEM-T carrier (Promega company) respectively, ordinary method transfection Escherichia coli Top 10F ' host bacterium competence cell, plasmid increases in a small amount, the alkaline lysis method of extracting plasmid.Get the plasmid that extracts in a small amount, cut preliminary evaluation with PstI and BamHI enzyme.AD1370 automatic sequencer (Perkin-Elemer company) order-checking, obtain the hTPO full-length gene of 6340bp, comprise that (see sequence table 1.SEQ ID No.1, wherein: the 121-135 position is the binding site of nucleotide primer A1 for the full sequence of exons 1-6 and 5 intron;
The 6326-6350 position is the binding site of nucleotide primer A2;
2046 is initiator codon;
5792 is terminator codon;
The 4891-5127 position is intron V;
4871 is the restriction enzyme site of Pst I;
5159 is the restriction enzyme site of BamHI.)。
Embodiment 2: the clone and the order-checking of hTPO cDNA sequence
1、RT-PCR
MRNA is a template with people's tire liver, at first adopts RT-PCR Kit (STRATA GENE), and reversing the cDNA that obtains with reverse transcription then is template, adopts two to be primer to (B1/B2, C1/C2) Nucleotide
B1:5 '-GCGTCGACAGAATGGAGCTGACTGAATT-3 ' is a forward primer
B2:5 '-GGCATTGGGATCCTTGTGAGCTGTGG-3 ' is a reverse primer;
C1:5 '-CAGCTCACAAGGATCCCAATGCCATCTTC-3 ' is a forward primer,
C2:5 '-CGCAGCTGACACGGCAGTGTCTGAGAACCTTA-3 ' is a reverse primer, carries out pcr amplification.
The reaction conditions of PCR is: 95 ℃ of 5min; 54 ℃ of 5min; (94 ℃ of 1min; 62 ℃ of 1min; 72 ℃ of 90sec) * 35; 72 ℃, 10min.Electrophoresis detection purpose fragment then obtains two hTPO cDNA fragments of 450bp and 660bp respectively.
2, two segmental connections of hTPO and order-checking
Two cDNA fragments through pcr amplification are cut with SalI and BamH I enzyme respectively, use T4 ligase enzyme (New EnglandBio company) that two fragments are inserted order-checking plasmid pBSK then.Through the AB1370 order-checking, find 15 base pairs of disappearance.In order to obtain complete hTPO cDNA molecule, at first with the hTPO molecular cloning in carrier for expression of eukaryon pcDNA3.1/zeo (-); Secondly, according to the hTPO cDNA sequence of having delivered, synthetic comprises the dna fragmentation of above-mentioned disappearance nucleotide sequence, utilize Pst I and BamHI that the synthetic gene is inserted among the pCTPOd, utilize T7 primer and synthetic sequencing primer to finish complete sequence determination at last to TPO molecule among the pCTPO, obtain 1062bp hTPO cDNA molecule (see sequence table 2.SEQID No.2, wherein:
The 1-17 position is the binding site that nucleotide primer is lain prone;
The 416-441 position is the binding site of nucleotide primer B2;
The 419-447 position is the binding site of nucleotide primer C1;
The 1060-1062 position is the binding site of nucleotide primer C2;
377 is the point of contact of Pst I;
429 is the point of contact of BamHI.)。
Embodiment 3, mammary gland-specific expression vector structure
1, hold the 6606bp sequence to start the structure of the hTPO genomic dna expression vector of expression jointly by pCMV and the goat beta-casein gene 5 ' of pcDNA3.1 (-)
The PCR product of hTPO genomic dna is connected with the T carrier, and cloning vector pBSK packs into behind Sal 1 and Sma I (NEB company) double digestion; Cut through NotI and KpnI enzyme again, downcut the hTPO genomic dna, same enzyme is cut eukaryotic gene expression vector pcDNA3.1 (-), and electrophoresis reclaims the purpose fragment, with T4 ligase enzyme (NEB company) above-mentioned fragment is had successively sequentially to be loaded in pcDNA3.1 (-) expression vector; With the NotI single endonuclease digestion goat beta-casein gene 5 ' end 6609bp sequence is inserted into 5 ' end of hTPO genomic dna at last, cut evaluation through enzyme and select clone in the right direction, and to cloning the junction order-checking of son, result's correct (seeing sequence table 3.SEQ ID No.3).
2, hold the 6606bp sequence to start the structure of the hTPO cDNA expression vector of expression jointly by pCMV and the goat beta-casein gene 5 ' of pcDNA3.1 (-)
The N of hTPO cDNA end and C hold the PCR product at first respectively with
The T carrierConnect, then with SalI and SmaI double digestion, downcut two fragments, be connected with the cloning vector pBSK that cuts through the SalI enzyme, order-checking finds that the hTPO cDNA molecule that obtains has lacked 15 Nucleotide; With Kpn I and EcoR V with the hTPOcDNA molecular cloning in carrier for expression of eukaryon pcDNA3.1 (-); Synthetic contains the nucleic acid molecule of deletion fragment, with PstI and BamHI deletion fragment is inserted among the hTPOcDNA, obtains complete hTPOcDNA molecule.With Not I single endonuclease digestion goat beta-casein gene 5 ' terminal sequence is inserted into 5 ' of hTPOcDNA at last and holds, cut evaluation through enzyme and select clone in the right direction, and to cloning the junction order-checking of son, result's correct (seeing sequence table 3.SEQ ID No.3).
3, the structure of the hTPO cDNA expression vector that contains intron V that starts expression jointly by pCMV and the goat beta-casein gene 5 ' terminal sequence of pcDNA3.1 (-)
The PCR product of hTPO intron V at first T carrier connects, and with PstI and BamHI intron V is inserted into pcDNA3.1 (-) carrier and gets among the hTPO cDNA, obtains containing the hTPO cDNA carrier for expression of eukaryon of intron 5; The WAP promotor of rat is inserted into the 5 ' end of hTPO cDNA with Not I and Kpn I; The BamHI single endonuclease digestion must hold the 6609bp sequence start the hTPO cDNA expression vector that contains intron V of expression jointly by pCMV and the goat beta-casein gene 5 ' of pcDNA3.1 (-) with the WAP promotor of goat beta-casein gene 5 ' terminal sequence replacement rat.Cut evaluation through enzyme and select clone in the right direction, and to cloning the junction order-checking of son, the result is correct.
Embodiment 4: the expression of hTPO gene in eukaryotic cell
1, transfection Cos-7 cell strain:
Cell cultures routinely.PcDNA3.1 (-)/hTPO cDNA and pcDNA3.1 (-)/hTPOcDNA-Intron V plasmid transfection Cos-7 cell is to adopt DOTAP (DM company) liposome transfection method, transfection Cos-7 cell 48 hours, and the collecting cell supernatant liquor adopts hTPO Kit (R﹠amp; D) content of detection hTPO proves that hTPO has obtained expression.
2, transfection HC-11 cell strain
PcDNA3.1 (-)/hTPO cDNA and pcDNA3.1 (-)/hTPO cDNA-htron V plasmid transfection HC-11 cell (available from Switzerland Friedrich Miescher institute) is to adopt DOTAP (DM company) liposome transfection method, according to cultivating grown cultures phase, shock incubation period and inductive phase, the HC-11 cell is cultivated in three kinds of requirements, transfection HC-11 cell 48 hours, the collecting cell supernatant liquor adopts people TPOKit (R﹠amp; D) content of detection hTPO proves that hTPO has obtained expression.
Embodiment 5: set up transgenic mice
Extract pcDNA3.1 (-)/hTPO cDNA and pcDNA3.1 (-)/hTPOcDNA-Intron V plasmid with QIAquick Kit, the purifying quantitative assay is for microinjection.Carry out the preparation of transgenic mice according to conventional procedure.Detect the integration that confirms to have in its body of transgenic mice the hTPO gene through PCR and Southern blot blot hybridization, adopt hTPO Kit (R﹠amp; D) detect the expression amount of hTPO in its milk, the highest 122.8 μ g/ml that reach.
Sequence table
Claims (9)
1, a kind of thrombopoietin hTPO expression vector is characterized in that, described expression vector comprises the hTPO cDNA that contains intron V, and utilizes goat beta-casein promotor to instruct the described expression that contains the hTPO cDNA of intron V.
2, expression vector as claimed in claim 1 is characterized in that, described intron V has the nucleotide sequence of 4891-5127 position shown in the SEQID NO:1.
3, expression vector as claimed in claim 1 is characterized in that, described hTPO cDNA has the nucleotide sequence shown in the SEQ ID NO:2.
4, expression vector as claimed in claim 1 is characterized in that, described goat beta-casein promotor has the nucleotide sequence shown in the SEQ ID NO:3.
5, expression vector as claimed in claim 1 is characterized in that, described expression vector also comprises the CMV promotor.
6, a kind of construction of carrier according to claim 1 is characterized in that, may further comprise the steps:
A, pcr amplification hTPO cDNA fragment, and, be built into the hTPO cDNA carrier for expression of eukaryon that contains intron V with among the hTPO intron V fragment insertion hTPO cDNA;
B, goat beta-casein promotor is inserted the 5 ' end of hTPO cDNA, be built into expression vector by CMV promotor, the common hTPO cDNA that contains intron V that starts of goat beta-casein promotor.
7, method as claimed in claim 6 is characterized in that, the hTPO cDNA fragment of described steps A has the nucleotide sequence shown in the SEQ ID NO:2.
8, method as claimed in claim 6 is characterized in that, the hTPO intron V fragment of described steps A has the nucleotide sequence of 4891-5127 position shown in the SEQ ID NO:1.
9, method as claimed in claim 6 is characterized in that, the goat beta-casein promotor of described step B has the nucleotide sequence shown in the SEQ ID NO:3.
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