CN102199607B - Stable cell strains of recombinant human blood coagulation factor IX minigene and premature termination codon (PTC) mutant thereof - Google Patents
Stable cell strains of recombinant human blood coagulation factor IX minigene and premature termination codon (PTC) mutant thereof Download PDFInfo
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- CN102199607B CN102199607B CN2011100850252A CN201110085025A CN102199607B CN 102199607 B CN102199607 B CN 102199607B CN 2011100850252 A CN2011100850252 A CN 2011100850252A CN 201110085025 A CN201110085025 A CN 201110085025A CN 102199607 B CN102199607 B CN 102199607B
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Abstract
The invention discloses a method for establishing a stable cell strain containing a recombinant human blood coagulation factor IX (F9) minigene and a stable cell strain containing a premature termination codon (PTC) nonsense mutant of the recombinant human F9 minigene, and the two stable cell strains which are established by the method and contain the recombinant human F9 minigene and the PTC mutant of the recombinant human F9 minigene, and belongs to the field of animal cell strains. The method for establishing the cell strains comprises the following steps of: importing a human blood coagulation factor IX minigene (mini hF9A) and a PTC mutant (mini hF9A-N) thereof into HepG2 cells respectively, and screening by using G418 to obtain the stable cell strains. The stable cell strains, which are established by the method, of the recombinant human blood coagulation factor IX minigene and the PTC mutant thereof are named HepG2-WT and HepG2-N, and the stable cell strains are collected in Institute of Biotechnology, Shanxi University. The stable cell strains are characterized in that: a HepG2-WT genome is integrated with the mini hF9A minigene; and a HepG2-N genome is integrated with the mini hF9A-N minigene which contains PTC mutation and is subjected to a nonsense-mediated mRNA decay (NMD) pathway. The HepG2-WT and HepG2-N cell strains established by the method can be used for screening medicaments for treating hemophilia caused by nonsense mutation, screening PTC read-through medicaments and the like.
Description
Technical field:
The present invention relates to molecular biology and cytobiology; The genetic and cell engineering technology is specifically related to the establishment method of a kind of recombinant human blood coagulation factor IX minigene and PTC two mutants stable cell line thereof and recombinant human blood coagulation factor IX minigene and the PTC two mutants stable cell line of setting up according to this method thereof.
Background technology:
Hemophilia (haemophilia) is a kind of X-linkage recessive inheritance property disease.Being divided into HA, HB amphitypy, is respectively because the congenital disturbances of blood coagulation property disease that the activity of two factors reduces or the content minimizing causes in the blood that coding blood coagulation factor VIII (F8) and plasma thromboplastin component (F9) transgenation cause.Social man crowd's sickness rate 5-10/10 ten thousand, baby's sickness rate is about 1/5000, belongs to characteristic of disease genetic diseases occurred frequently.In the haemophiliac, have 80% approximately for heavy.Hemophilia does not still have the ideal regimen at present; With alternative medicine is main; Infusion fresh plasma, thrombin that from blood plasma, extract or the genetically engineered preparation; Medical expense is expensive, and might produce antibody behind the infusion, has caused heavy economical load and psychological pressure for haemophiliac and family thereof.
To hemophilia data website (http://europium.csc.mrc.ac.uk; HAMSTeRS database and http://www.kcl.ac.uk) the mutator gene The sequencing results of registration shows, haemophiliachemophiliac thrombin defective mainly is missense or nonsense, deletion or inserts sudden change and cause.For the F8 factor gene; In 26 exons, have 781 mutational sites, wherein nonsense mutation has reached 145, accounts for 18.6% of total sudden change; The hot spot region that nonsense mutation takes place is positioned at the 719-1709 of the 14th exon, has 56 nonsense codons and occurs; For the F9 factor gene, in 630 point mutation reported, 70 are nonsense mutation, account for 11.1% of sudden change sum.Owing to produce PTC, probably cause the generation of body mRNA detection system NMD approach after the gene nonsense mutation, thus the mRNA of this PTC of containing that degrades, and then cause its protein level very low.
At present in the gene PTC read over that research is one of focus of molecular cytobiology with the NMD pathway mechanisms, the treatment of the genetic diseases that causes is thus had potential using value.Research is illustrated in external; Aminoglycosides (gentamicine and geneticine) compound can be attached to ribosomal identification center (decoding center); Reduce codon and anticodon paired accuracy; Cause mispronouncing of codon among the mRNA, in cultured animals cell and human body cell, all obtained the confirmation of experiment.The inhibition of NMD approach and the short PTC of medicine are readed over the mechanism research that expression has been used for cancer, tumour and part genetic diseases that the gene nonsense mutation causes, and obtained considerable progress, obtained a series of significant molecules data and test-results.Domestic and international at present none cell strain still can be used for because the haemophiliachemophiliac drug screening that nonsense mutation causes; Therefore be badly in need of setting up the outer screening of corresponding cell strain donor because the haemophiliachemophiliac potential drug that nonsense mutation causes more can be used for screening because the clinical treatment medicine of the heredopathia that nonsense mutation causes.
Summary of the invention:
The object of the present invention is to provide the stable cell line of a kind of recombinant human blood coagulation factor IX minigene and PTC two mutants thereof, this stable cell line can be used for in-vitro screening because the hemophilia that nonsense mutation causes and the clinical treatment medicine of other heredopathias.
A kind of recombinant human blood coagulation factor IX minigene provided by the invention contains exons 1+part introne 1 (699bp), part intron 5+ exon 6+ part intron 6 (2115bp), part intron 6+ exon 7+intron 7+ exon 8+HA label (1536bp); Wherein 7～124,1599～1801,3059～3173,3842～4371 is exon, and 4372～4401 is the HA sequence label; Its nucleotides sequence is classified SEQ ID NO:1 as.
A kind of recombinant human blood coagulation factor IX minigene PTC two mutants provided by the invention, its be by described recombinant human blood coagulation factor IX minigene through the PCR site-directed mutagenesis technique with in its nucleotide sequence 3092 sport t by g.
A kind of stable cell line provided by the invention, it contains above-mentioned recombinant human blood coagulation factor IX minigene.
A kind of stable cell line provided by the invention, it contains above-mentioned recombinant human blood coagulation factor IX minigene PTC two mutants.
The establishment method of a kind of stable cell line provided by the invention comprises the steps:
1) utilize recombinant gene that human blood coagulation IX minigene (mini hF9A) is cloned among the mammalian expression vector pCMV-Tag3B, be built into pCMV-Tag3B-mini hF9A, recombinant plasmid is cut evaluation through enzyme;
2) utilize the quick rite-directed mutagenesis method of PCR, obtain 3092 and sport t, the PTC mutant plasmid pCMV-Tag3B-mini hF9A-N of human blood coagulation IX minigene by g;
3) culturing human liver cancer cell HepG2, nutrient solution is DMEM, adds 10% foetal calf serum, does not add any microbiotic and cultivates, trypsin digestion cell is by 2 * 10
5Cell/mL density kind is in 6 orifice plates;
4) utilize liposome LipofectamineTM 2000 with plasmid pCMV-Tag3B-mini hF9A and pCMV-Tag3B-minihF9A-N difference transfection HepG 2 cell; After the transfection 24 hours; Trypsin digestion cell; Be inoculated in 6 orifice plates by dilution in 1: 10, add G418 (500 μ g/mL) screening, replacings in per 3～5 days once contain the screening and culturing liquid of G418;
5) about 3 weeks of screening, positive cell bunch appears in 6 orifice plates, and microscopically picking cell cluster adopts limiting dilution assay in 96 orifice plates, carry out mono-clonalization;
6) in 96 orifice plates in the unicellular breeding, dispel with trysinization and to make its adherent growth again, wait to grow to more than 80%, the cultivation of going down to posterity, and identify through RT-PCR;
7) identify positive colony for RT-PCR, enlarged culturing is extracted genome, carries out PCR and identifies, finally obtains two kinds of cytotostatic strain HepG2-WT and HepG2-N;
8) two kinds of cytotostatic strain HepG2-WT and HepG2-N are frozen preservation in liquid nitrogen.
The human blood coagulation IX minigene of the stable cell line HepG2-WT genome conformity wild-type that the present invention sets up; The HepG2-N genome conformity contains the PTC sudden change and the human blood coagulation IX minigene of NMD approach takes place.HepG2-WT, HepG2-N cell strain that the present invention sets up can be used for the screening that nonsense mutation causes haemophiliachemophiliac medicine, the screening that PTC reads over medicine etc.The stable cell line set up of the present invention also can be used for the inhibition of NMD approach is readed over expression with the short PTC of medicine and then is used for the mechanism research of cancer, tumour and part genetic diseases that the gene nonsense mutation causes in addition.
Description of drawings:
Fig. 1 is that recombinant human blood coagulation factor IX minigene is formed synoptic diagram.This minigene contains exons 1+part introne 1 (699bp), part intron 5+ exon 6+ part intron 6 (2115bp), part intron 6+ exon 7+intron 7+ exon 8+HA label (1536bp).
Fig. 2 is recombinant human blood coagulation factor IX minigene carrier for expression of eukaryon evaluation figure and synoptic diagram.A is for identifying figure, and swimming lane 1 is a recombinant plasmid; Swimming lane 2 is identified for PCR; Swimming lane 3 is identified for double digestion.B is a synoptic diagram.
Fig. 3 is the RT-PCR evaluation figure of recombinant human blood coagulation factor IX minigene transfection HepG 2 cell.Swimming lane 1 is the non-transfected cells negative control; Swimming lane 2 is a recombinant human blood coagulation factor IX minigene transfectional cell.
Fig. 4 is that collection of illustrative plates is identified in the order-checking of recombinant human blood coagulation factor IX minigene PTC two mutants.
Fig. 5 is recombinant human blood coagulation factor IX minigene and the real-time fluorescence quantitative PCR figure of PTC transfection with mutant HepG2 cell after 24 hours thereof.A, Real-time PCR detected result; B, Real-time pcr gene amplification curve; C, Real-timePCR gene solubility curve, yellow is a goal gene recombinant human blood coagulation factor IX minigene, pink colour is the internal reference gene.
Fig. 6 is stable cell line HepG2-WT of the present invention cellular form photo of (200 *) under inverted microscope.
Fig. 7 is stable cell line HepG2-N of the present invention cellular form photo of (200 *) under inverted microscope.
Embodiment
1, recombinant human blood coagulation factor IX minigene Construction of eukaryotic
Utilize BamH I and HindIII that human blood coagulation IX minigene is carried out the gene fragment that double digestion obtains 4.4kb, electrophoresis reclaims.Mammals efficient expression vector pCMV-Tag3B also uses BamH I and the linear carrier of HindIII double digestion, and electrophoresis reclaims.Use the T4DNA ligase enzyme to be connected itself and the human blood coagulation IX minigene fragment that reclaims, obtain recombinant plasmid pCMV-Tag3B-mini hF9A, recombinant plasmid is cut evaluation through enzyme.
2, the structure of recombinant human blood coagulation factor IX minigene PTC two mutants
Design a pair of complete complementary mutant primer; Make 3092 bit bases sport t by g, use the high-fidelity enzyme to carry out pcr amplification with recombinant plasmid pCMV-Tag3B-mini hF9A as template, product is through Dpn I digestion template; Get 10 μ L product transform bacterias; The picking clone extracts plasmid, and order-checking is identified, obtained mutant plasmid pCMV-Tag3B-mini hF9A-N.
3, the cultivation of mammalian cell HepG2 with go down to posterity
Cell derives from biotechnology research institute of University Of Shanxi, and recovery cell cultures well is in 25cm
2In the Tissue Culture Flask, 37 ℃, 5%CO
2, nutrient solution is DMEM, adding foetal calf serum to final concentration is 10%, does not add any microbiotic.Treating that cell degree of converging reaches more than 70% can go down to posterity, and discards nutrient solution, washes 2 times with PBS; Discard PBS, add 0.25% trysinization liquid 1mL, culturing bottle is placed 37 ℃ of incubator preheatings; Treat cell rounding but do not discard pancreatin as yet during fall in flakes; Add the DMEM contain 10% foetal calf serum and stop digestion, and blow and beat cell gently and make it to come off, then add an amount of nutrient solution in 3 Tissue Culture Flasks and cultivate according to being sub-packed at 1: 3.
4, the transfection of HepG2 cell and screening
(1) transfection is first day, and the HepG2 cell is pressed 2 * 10
5Cell/mL density kind is in 6 well culture plates, and incubated overnight makes that next day, cell degree of converging reached 90-95%.
(2) transfection is second day, and following plasmid of configuration and liposome mixed solution in aseptic centrifuge tube are used for the HepG2 cell of cultivating in transfection 6 well culture plates.Solution A: 4 μ g DNAs are dissolved in the 250 μ L serum free mediums.Solution B: 10 μ LLipofectamine
TM2000 are dissolved in the 250 μ L serum free mediums, and room temperature was placed 5-15 minute.Mixed solution A and solution B, mixing gently, room temperature is placed more than 20 minutes.The solution 500 μ L of mixing are added in the cell culture fluid of 6 well culture plates mixing gently, 37 ℃, 5%CO
2Cultivated 24 hours.
(3) screening of transfectional cell
After the transfection 24 hours, trypsin digestion cell is inoculated in 6 well culture plates by dilution in 1: 10, adds G418 (500 μ g/mL) screening, changes the screening and culturing liquid that once contains G418 in per 3～5 days, screens about 3 weeks visible positive cell bunch.
(4) mono-clonalization of cell strain and enlarged culturing
Screen about 3 weeks, positive cell bunch appears in 6 orifice plates, and microscopically picking cell cluster adopts limiting dilution assay in 96 well culture plates, carry out mono-clonalization.In 96 orifice plates in the unicellular breeding, dispel with trysinization and to make its adherent growth again, wait to grow to more than 80%, the cultivation of going down to posterity, and identify through RT-PCR.Identify positive colony for RT-PCR, enlarged culturing is extracted genome, and PCR identifies, finally obtains two kinds of cytotostatic strain HepG2-WT and HepG2-N, and frozen in liquid nitrogen.
5, human blood coagulation IX minigene and the expression of PTC two mutants in stable cell line thereof
(1) PCR of human blood coagulation IX minigene
Be with myc, HA label respectively according to human blood coagulation IX minigene sequence two ends, design a pair of special label primer, amplified fragments should be the 4449bp size.PCMV-Tag3B-mini hF9A plasmid is as positive control; The HepG2 cell of untransfected is as negative control; Genomic dna with stable cell line HepG2-WT and HepG2-N is a template amplification, and through 30 circulations, the PCR product carries out 1% agarose gel electrophoresis analysis.It is thus clear that two samples have a specific band at the place of bp more than 4000 respectively, and are identical with the positive control stripe size, and do not have amplified band in the HepG2 cell genomic dna of untransfected.Explain that plasma thromboplastin component minigene and two mutants thereof distinguished in the successful transfered cell.
(2) RT-PCR of human blood coagulation IX minigene
Be with myc, HA label respectively according to human blood coagulation IX minigene sequence two ends, design a pair of special label primer, amplified fragments should be the 1041bp size.The HepG2 cell of untransfected is as negative control, and the cDNA that becomes with the mRNA reverse transcription of stable cell line HepG2-WT and HepG2-N is a template, and through 30 circulations, the PCR product carries out 1% agarose gel electrophoresis analysis.It is thus clear that two samples have a specific band at the place of bp more than 1000 respectively, and do not have amplified band in the HepG2 cell genomic dna of untransfected.Explain that stable cell line HepG2-WT and HepG2-N exist plasma thromboplastin component minigene and the expression of its two mutants at transcriptional level respectively.
Claims (4)
1. recombinant human blood coagulation factor IX minigene, its nucleotides sequence is classified SEQ IDNO:1 as.
2. recombinant human blood coagulation factor IX minigene PTC two mutants, its be by the described recombinant human blood coagulation factor IX of claim 1 minigene through the PCR site-directed mutagenesis technique with in its nucleotide sequence 3092 sport t by g.
3. stable cell line, it contains the described recombinant human blood coagulation factor IX of claim 1 minigene.
4. stable cell line, it contains the described recombinant human blood coagulation factor IX of claim 2 minigene PTC two mutants.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001070968A2 (en) * | 2000-03-22 | 2001-09-27 | Octagene Gmbh | Production of recombinant blood clotting factors in human cell lines |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2001070968A2 (en) * | 2000-03-22 | 2001-09-27 | Octagene Gmbh | Production of recombinant blood clotting factors in human cell lines |
Non-Patent Citations (4)
| Title |
|---|
| Armentano D等.Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: potential for gene therapy of hemophilia B.《Proceedings of the National Academy of Sciences of the United States of America》.1990,第87卷(第16期),6141-6145. |
| Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: potential for gene therapy of hemophilia B;Armentano D等;《Proceedings of the National Academy of Sciences of the United States of America》;1990;第87卷(第16期);6141-6145 * |
| 杨晓青等.通过启动子同源重组研究人凝血因子Ⅸ的组成型表达.《中国科学C辑》.2000,第30卷(第05期),535-540. |
| 通过启动子同源重组研究人凝血因子Ⅸ的组成型表达;杨晓青等;《中国科学C辑》;20001025;第30卷(第05期);535-540 * |
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