CN100460507C - Human tissue factor pathway inhibitory factor mutation gene m0TFPI, recombination carrier and recombination microzyme including the same - Google Patents
Human tissue factor pathway inhibitory factor mutation gene m0TFPI, recombination carrier and recombination microzyme including the same Download PDFInfo
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- CN100460507C CN100460507C CNB2006100156528A CN200610015652A CN100460507C CN 100460507 C CN100460507 C CN 100460507C CN B2006100156528 A CNB2006100156528 A CN B2006100156528A CN 200610015652 A CN200610015652 A CN 200610015652A CN 100460507 C CN100460507 C CN 100460507C
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Abstract
The invention discloses the m0TFPI, comprising the recombination carrier and host cell. The m0TFPI has nucleic acid sequence said by SEQ ID No.1 in sequence table. The recombination carrier m0TFPI-pPIC9 is made by slaking fragment of m0TFPI DNA and pPIC9 carrier with XhoI and EcoRIand, adding T4 ligases, and carrying out coupled reaction. On the base of the invention, the metabolism time of the TFPI recombination protein in blood circulation can be extended, the administer drug frequencies of TFPI can be reduced, and the therapeutic expense can be reduced.
Description
Technical field
The invention belongs to gene engineering technology field, the recombination microzyme and the expressed proteins thereof that relate in particular to the tissue factor approach inhibition factor mutator gene, contain the recombinant vectors of this gene, transform with this carrier.
Background technology
(Tissue Factor Pathway Inhibitor TFPI) is the important supressor of tissue factor approach to human tissue factor approach inhibition factor, as the physiological inhibitor of TF:FVIIa mixture coagulation process is had the significant effects effect.TFPI can suppress thrombosis and vascular smooth muscle cell proliferation etc., therefore, the generation and the development of vascular restenosis, atherosclerosis and myocardial infarction etc. is had the effect of slowing down.In addition, experimentation on animals proves that human TFPI recombinant protein (hrTFPI) has preventive and therapeutic effect to thrombosis, septic shock, vascular restenosis, endotoxemia and DIC (disseminated inravascular coagulation) etc.Therefore, TFPI has huge pharmaceutical use, has wide practical use clinically.
At present, existing a plurality of companies utilize different expression systems to carry out the preparation of TFPI recombinant protein with the laboratory both at home and abroad, and China Medicine Academy Sciences China Xiehe Medicine University Biology Medicine Engineering Research Institute genetically engineered laboratory goes out lower-cost high specific acitivity TFPI recombinant protein with pichia yeast expression system stably express.
But, because TFPI is removed in circulation of blood fast, need heavy dose (about 20mg/kg/d) just can reach the clinical treatment purpose, medication inconvenience and high expense have greatly limited its application and result of treatment (Palmier MO clinically, Hall LJ, Reisch CM, et al.Clearance of recombinant tissue factor pathway inhibitor (TFPI) in rabbi ts.Thromb Haemost, 1992.68 (1): 33-36.Warshawsky l, Bu G, Mast A, et al.The carboxy terminus of tissue factor pathway inhibi tor is required forinteracting with hepatoma cells in vitro and in vivo.J Clin Invest, 1995.95 (4): 1773-1781.Ho G, Narita M, Broze GJ, Jr., et al.Recombi nantfull-length tissuefactor pathway inhibitor fails to bind to the cell surface:implications forcatabolism in vitro and in vivo.Blood, 2000.95 (6): 1973-1978.Bai H, MaD, ZhangYG, et al.Molecular design and characterization of recombinant long half-lifemutants of humanti ssue factor pathway inhibitor.Thromb Haemost, 2005.93 (6): 1055-1060.).
Therefore prolong the metabolism time of TFPI recombinant protein in circulation of blood, will reduce medical expense greatly, have great importance promoting its application clinically.
Summary of the invention
The objective of the invention is provides a kind of m of human tissue factor pathway inhibitory factor mutation gene for prolonging the TFPI metabolism time of recombinant protein in circulation of blood
0TFPI.
Second purpose of the present invention provides a kind of by the m of human tissue factor pathway inhibitory factor mutation gene
0The TFPI encoded polypeptides.
The 3rd purpose of the present invention provides a kind of m of human tissue factor pathway inhibitory factor mutation gene that includes
0The recombinant vectors of TFPI.
Last purpose of the present invention provides a kind of m of human tissue factor pathway inhibitory factor mutation gene that utilizes
0The recombination microzyme that the recombinant vectors of TFPI transforms.
Technical scheme of the present invention is summarized as follows:
A kind of m of human tissue factor pathway inhibitory factor mutation gene
0TFPI, it has the described nucleotide sequence of SEQ ID No.1 in the sequence table.
A kind of by the m of human tissue factor pathway inhibitory factor mutation gene
0The TFPI encoded polypeptides, it has the described aminoacid sequence of SEQ ID No.2.
A kind of m of human tissue factor pathway inhibitory factor mutation gene that comprises
0The recombinant vectors of TFPI, it is with the m of human tissue factor pathway inhibitory factor mutation gene
0TFPI DNA adds T with the fragment and the pPIC9 carrier of XhoI and EcoRI digestion
4Ligase enzyme carries out ligation, makes m
0TFPI-pPIC9.
A kind of m of human tissue factor pathway inhibitory factor mutation gene that utilizes
0The recombination microzyme that the recombinant vectors of TFPI transforms, it is to contain the m of human tissue factor pathway inhibitory factor mutation gene
0The recombinant vectors of TFPI imports host's Pichia yeast GS115, becomes GS115/m
0TFPI-pPIC9.
The m of human tissue factor pathway inhibitory factor mutation gene among the present invention
0The TFPI characteristics are: carried out the change of partial nucleotide sequence on the basis of the TFPI polynucleotide sequence that other have been reported; The part carboxyl terminal aminoacid sequence of its coding is different with the TFPI of wild-type; Long 1.5 times of metabolism time ratio wild-type TFPI (mTFPI) in circulation of blood, and its activity at external, interior inhibition FXa, do not occur obviously weakening with the cooperative ability of heparin.
On the basis that the present invention finishes, can prolong the metabolism time of TFPI recombinant protein in circulation of blood, simultaneously, keep its biological characteristics, as anticoagulant active, with binding ability of heparin etc. as far as possible.Thereby can significantly reduce the administration number of times of TFPI, reduce medical expense, promote the application clinically of TFPI recombinant protein.
Description of drawings
Fig. 1 is m
0TFPI-cDNA construction strategy figure;
Fig. 2 is a kind of m that contains
0TFPI-cDNA reorganization (clone) carrier and contain the construction strategy figure of the host cell of this recombinant vectors;
Fig. 3 is m
0The polyacrylamide gel electrophoresis collection of illustrative plates of TFPI recombinant protein;
Fig. 4 is different concns m
0The TFPI recombinant protein is to the restraining effect of FXa;
Fig. 5 is different incubation time m
0TFPI is to the restraining effect of FXa;
Fig. 6 is different incubation time m
0TFPI is to the restraining effect (when heparin sodium exists) of FXa;
Fig. 7 is that heparin is to m
0The TFPI recombinant protein suppresses the influence of FXa effect;
Fig. 8 is that the different concns heparin is to m
0The TFPI recombinant protein suppresses the influence of FXa;
Fig. 9 is m
0The metabolism content specific activity of TFPI recombinant protein in blood plasma.
Embodiment
Prepare a kind of m of human tissue factor pathway inhibitory factor mutation gene of the present invention
0TFPI, recombinant vectors, recombinant host bacterium and recombinant protein mutant (m
0TFPI) method comprises the steps:
1. be template with mTFPI-pPIC9 plasmid DNA (China Medicine Academy Sciences China Xiehe Medicine University Biology Medicine Engineering Research Institute genetically engineered laboratory makes up), suddenly change with the partial dna sequence of PCR method, reconnect on the pPIC9 carrier and form m mTFPI
0TFPI-pPIC9, and be transformed into intestinal bacteria TB1.
2. extract m
0The TFPI-pPIC9 plasmid DNA, with restriction enzyme with its linearizing after, be transformed among the yeast competent cell GS115, obtain recombination microzyme GS115/m
0TFPI-pPIC9.
3. can make TFPI mutator gene (m
0TFPI) cultivate this reorganization bacterium under the condition of Biao Daing, measure the proteic activity of TFPI with substrate color development method; And, prove TFPI albumen great expression through the SDS-PAGE analysis.
4. with after the isotopic labeling, measure TFPI protein mutant m
1TFPI metabolism situation in animal body proves TFPI protein mutant m
0The metabolism time ratio wild-type TFPI of TFPI in circulation of blood prolongs 1.5 times.
5. measure TFPI protein mutant m with substrate color development method
0The activity of TFPI proves that its activity compares with wild-type TFPI, occurs obviously reducing.
TFPI mutator gene m
0The TFPI nucleotide sequence is seen SEQ ID No.1.TFPI mutator gene m
0The partial sequence sudden change except that with the mTFPI gene has identical mutational site, has also taken place at its carboxyl terminal in the TFPI genetic comparison of TFPI total length 828bp and other wild-types.
According to nucleotide sequence SEQ ID No.1, infer the m that expresses
1The proteic aminoacid sequence SEQ of TFPI ID No.2.This m
0The aminoacid sequence of TFPI is made up of 276 amino acid, and molecular weight is 42Da.By mTFPI mutator gene m to the present invention relates to
0TFPI deduced amino acid and other wild-types TFPI Argine Monohydrochloride sequence compare, the result shows that its carboxyl terminal partial amino-acid series sudden change is (referring to Thomas JG, Roger E, Robin LW, et al.Structure ofthe human lipoprotein-associated coagulation inhibitor gene.The Journal ofBiological Chemistry.1991.226 (8) 5036-5041.).Thereby make it in basic maintenance biological characteristics, prolonged the blood plasma metabolism time.
Below by embodiment the present invention is further elaborated.It should be understood that described embodiment is only used for explanation rather than restriction the present invention.
The extraction of embodiment 1 mTFPI-pPIC9 plasmid DNA
The fresh bacterium liquid of 100ul TB1 (containing the mTFPI-pPIC9 plasmid) is inoculated in the 10ml LB substratum 37 ℃, the 250rpm incubated overnight; Get the triangular flask of 10 250ml, every bottle adds the 100ml substratum, and the above-mentioned bacterium liquid of inoculation 1ml, and 37 ℃, the 250rpm incubated overnight; The centrifuging and taking precipitation with the resuspended precipitation of 120ml GTE damping fluid, and adds 240mg lytic enzyme, concuss mixing, 30 ℃ of water-bath 30min; Add 240ml lysate (0.2N NaOH 1% SDS prepared fresh) and softly put upside down mixing, leave standstill 5min; (pH 5.5 for the potassium acetate solution of adding 180ml 5M; The 3M potassium acetate, 2M acetate), put upside down mixing, ice bath 10min.The centrifugal 15min of 8000rpm uses two-layer filtered through gauze, gets supernatant, and adds isopyknic Virahol, and the centrifugal 20min of 8000rpm gets precipitation behind the mixing; With the resuspended precipitation of 4ml TE damping fluid, (phenol: the extracting of chloroform=24:1) once for isopyknic phenol-chloroform mixed solution; The 7.5M ammonium acetate solution that adds 1/2 volume, ice bath 20min behind the mixing gets supernatant after centrifugal, adds the ice-cold ethanol of 2.5 times of volumes, and mixing-20 ℃ leaves standstill more than the 2h; The centrifugal 20min of 4000rpm gets precipitation, and resuspended with 0.6ml TNE (STE) damping fluid, adds the RNaseA solution of 10ul 10mg/ml, 37 ℃ of water-bath 30min behind the mixing; Use phenol successively, the phenol-chloroform mixed solution, each extracting of chloroform is once; Add isopyknic PEG solution (13% PEG 8000,0.8M NaCl) mixing, 4 ℃ leave standstill 30min, the centrifugal 20min of 13000rpm, precipitation is dissolved in (Tris-HCl 10mmol/L in an amount of TE damping fluid with ice-cold 70% ethanol rinsing twice, EDTA1mmol/L, pH8.0).
With plasmid DNA mTFPI-pPIC9 (biomedical engineering institute of China Concord Medical Science University of Chinese Academy of Medical Sciences genetically engineered laboratory makes up) is template, with mutant primer m
0(5 ' AAA GGA GGC CTA ATT
GAT GAC GAT GACAAA AGA AAG AAG CAG AGA 3 '), 3 ' Aox (5 ' GAC TGG TTC CAA TTG ACAAGC 3 '); For positive anti-primer carries out the pcr amplification first time.Through agarose gel electrophoresis, reclaim test kit (Beijing ancient cooking vessel state biotech development center) purified pcr product with dna gel.Being template with plasmid DNA mTFPI-pPIC9 still, is that primer carries out the pcr amplification second time and (sees Fig. 1, m among the figure with above-mentioned PCR product and 5 ' Aox (5 ' GCA AAT GGC ATT CTG ACA TCC 3 ')
0, m
1, m
2Be mutant primer, contain the mutational site.5 ' Aox and 3 ' Aox are the universal primer of yeast expression vector pPIC9.■ represents the mutational site).Use the aforesaid method purified pcr product.With restriction enzyme XhoI and EcoRI incomplete digestion PCR product and pPIC9 carrier, through agarose gel electrophoresis, after above-mentioned test kit recovery, get 200ng pPIC9 and reclaim product, the PCR product that reclaims with 200ng spends the night under 16 ℃ of conditions in 20 μ L linked systems and to carry out ligation.Linked system contains 1ul and connects damping fluid, 1ulT4DNA ligase enzyme, 17ul water.Get and connect product C aCl
2Be transformed among E.coli (intestinal bacteria) TB1, be coated on the LB culture medium flat plate.
Get the plasmid DNA (m that 50ug extracts
0TFPI-pPIC9) cut 1 hour with 50u Sac1 enzyme.With plasmid dehydrated alcohol post precipitation, be dissolved in the 20ul sterilized water.Be equipped with the yeast competent cell with the PEG1000 legal system.The 50ug dna direct added still be in the competent cell of freezing state,, add 1.2ml PEG solution (40%PEG 1000,0.2M Bicine, pH 8.35) then, 30 ℃ of water-bath 1h in 37 ℃ of water-baths 5 minutes.Centrifugal, with the resuspended thalline of 1.5ml NaCl solution (pH 8.35 for 0.15M NaCl, 10mM Bicine).Recentrifuge is selected to hatch 4-6 day (see figure 2) for 30 ℃ on the flat board with coating RDB behind the resuspended thalline of 0.2ml NaCl solution (pH 8.35 for 0.15M NaCl, 10mMBicine).Select dull and stereotypedly to go up the picking clone and be inoculated in the 10ml YPD substratum from RDB, 30 ℃, 250rpm were cultivated 18-24 hour.Extract pastoris genomic dna with the granulated glass sphere cracking process.Get the 5ul pastoris genomic dna as touching plate, 5 ｀ Aox and 3 ｀ Aox. are as just, and anti-primer carries out pcr amplification.Agarose gel electrophoresis is analyzed the PCR product, and judges m according to the size of amplified fragments
0Whether the TFPI-pPIC9 plasmid inserts in the yeast host bacterium genome.Screening m
0The recombination microzyme GS115 that the TFPI-pPIC9 plasmid has inserted.
Embodiment 4 m
0The abduction delivering of TFPI recombinant protein
Positive colony is inoculated in the 10ml YPD substratum, and 30 ℃ of 250rpm cultivate 18h; Get 100ul bacterium liquid and be inoculated in the 10mlBMGY substratum, 30 ℃ of 250rpm are cultured to OD600 ≈ 3 (about 16-18h); Centrifugal collecting cell is resuspended in the 30ml BMMY substratum.30 ℃ of 250rpm add 100% methyl alcohol to final concentration and are 0.5% and cultivate 36h, and to add methyl alcohol to final concentration every 12h be 0.5%.And get that supernatant carries out the biological activity analysis and the SDS-PAGE electrophoresis (is seen Fig. 3,1. molecular weight of albumen standard 2.mTFPI recombinant protein 3.m among the figure
0TFPI recombinant protein 4.m
1TFPI recombinant protein 5.m
2The blank plasmid albumen of TFPI recombinant protein 6.pPIC9).
Embodiment 5 m
0The metabolism in animal body of TFPI recombinant protein
M with purifying
0The TFPI recombinant protein is used
125Behind the I mark, be injected into the SD rat tail vein, in injection back 0.5,1.0,2.0,4.0,6.0,8.0,10.0min from the eye socket venous plexus get blood centrifugal (1000g, 15min) blood plasma, respectively get blood plasma 100ul and carry out
125M is measured in the I radiocounting
0The metabolism content (see Table 1) of TFPI recombinant protein in blood plasma.
Embodiment 6 m
0The external determination of activity of TFPI recombinant protein
FXa is made into the solution of 2nM with TBSA (0.5% BSA, 100mM NaCl, 50mM Tris-HCl PH 7.8); With pPIC9 (blank), mTFPI (positive control), m
0TFPI is diluted to the 0.5-2.0nM different concns with TBS (100mM NaCl, 50mM Tris-HClPH 7.8), gets its each 100ul and mixes with 100ul FXa, places 37 ℃ of water-bath 30min of 96 orifice plates; Add then behind the 15ul 1mM S-2222 mixing in 37 ℃ of water-bath 30min, measure OD with microplate reader therebetween
405Value.Proof is than the TFPI albumen (mTFPI) of wild-type, m
0The TFPI recombinant protein obvious reduction do not occur in external activity, has excellent activity (seeing Fig. 4,5).
Embodiment 7 heparin are to m
0The mensuration of the external activity influence of TFPI recombinant protein
Get 1.0nM pPIC9, mTFPI, m
0Each 100ul of TFPI mixes with 100ul FXa, and to add heparin sodium to final concentration be 0.05-0.5U/ml, 37 ℃ of water-bath 30min; Add then behind the 15ul 1mM S-2222 mixing in 37 ℃ of water-bath 30min, measure OD with microplate reader therebetween
405Value.Proof is than the TFPI albumen (mTFPI) of wild-type, m
0The bonding force of TFPI recombinant protein and heparin does not obviously reduce yet, and heparin still obviously improves m
0The TFPI recombinant protein is to the inhibition ability (seeing Fig. 6-8) of FXa.
Embodiment 8 m
0Determination of activity in the TFPI recombinant protein body
24 SD rats (180-220g) are divided into four groups (6 every group) at random, injection mTFPI, m
0TFPI goes into the SD rat tail vein; Before injection, injection afterwards 0.5,1.0,2.0,4.0,6.0,8.0,10.0min gets blood from the eye socket venous plexus; Blood sample is centrifugal, and (1000g 15min) gets blood plasma, measures activity in vivo with the method for said determination external activity.Proof is than the TFPI albumen (mTFPI) of wild-type, m
0Obvious reduction does not appear in TFPI recombinant protein activity in vivo, still keeps the excellent activity (see figure 9) in vivo in the metabolic process.
In the table 1 rat body
125I-rTFPI blood plasma metabolism content (%)
SEQ?ID?No.1?828
gattctgagg?aagatgaaga?acacacaatt?atcacagata?cggagttgcc?accactgaaa?60
cttatgcatt?cattttgtgc?attcaaggcg?gatgatggcc?catgtaaagc?aatcatgaaa?120
agatttttct?tcaatatttt?cactcgacag?tgcgaagaat?ttatatatgg?gggatgtgaa?180
ggaaatcaga?atcgatttga?aagtctggaa?gagtgcaaaa?aaatgtgtac?aagagataat?240
gcaaacagga?ttataaagac?aacattgcaa?caagaaaagc?cagatttctg?ctttttggaa?300
gaagatcctg?gaatatgtcg?aggttatatt?accaggtact?tctacaacaa?tcagacaaaa?360
cagtgtgaac?gtttcaagta?tggtggatgc?ctgggcaata?tgaacaattt?tgagacactg?420
gaagaatgca?agaacatttg?tgaagatggt?ccgaatggtt?tccaggtgga?taattatgga?480
acccagctca?atgctgtgaa?taactccctg?actccgcaat?caaccaaggt?tcccagcctt?540
tttgaatttc?acggtccctc?atggtgtctc?actccagcag?acagaggatt?gtgtcgtgcc?600
aatgagaaca?gattctacta?caattcagtc?attgggaaat?gccgcccatt?taagtacagt?660
ggatgtgggg?gaaatgaaaa?caattttact?tccaaacaag?aatgtctgag?ggcatgtaaa?720
aaaggtttca?tccaaagaat?atcaaaagga?ggcctaattg?atgacgatga?caaaagaaag?780
aagcagagag?tgaaaatagc?atatgaagaa?atttttgtta?aaaatatg?828
SEQ?ID?No.2?276
AspSerGluGluAsp?GluGluHisThrIle?IleThrAspThrGlu?LeuProProLeuLys
1 5 10 15 20
LeuMetHisSerPhe?CysAlaPheLysAla?AspAspGlyProCys?LysAlaIleMetLys
25 30 35 40
ArgPhePhePheAsn?IlePheThrArgGln?CysGluGluPheIle?TyrGlyGlyCysGlu
45 50 55 60
GlyAsnGlnAsnArg?PheGluSerLeuGlu?GluCysLysLysMet?CysThrArgAspAsn
65 70 75 80
AlaAsnArgIleIle?LysThrThrLeuGln?GlnGluLysProAsp?PheCysPheLeuGlu
85 90 95 100
GluAspProGlyIle?CysArgGlyTyrIle?ThrArgTyrPheTyr?AsnAsnGlnThrLys
105 110 115 120
GlnCysGluArgPhe?LysTyrGlyGlyCys?LeuGlyAsnMetAsn?AsnPheGluThrLeu
125 130 135 140
GluGluCysLysAsn?IleCysGluAspGly?ProAsnGlyPheGln?VlaAspAsnTyrGly
145 150 155 160
ThrGlnLeuAsnAla?VlaAsnAsnSerLeu?ThrProGlnSerThr?LysVlaProSerLeu
165 170 175 180
PheGluPheHisGly?ProSerTrpCysLeu?ThrProAlaAspArg?GlyLeuCysArgAla
185 190 195 200
AsnGluAsnArgPhe?TyrTyrAsnSerVla?IleGlyLysCysArg?ProPheLysTyrSer
205 210 215 220
GlyCysGlyGlyAsn?GluAsnAsnPheThr?SerLysGlnGluCys?LeuArgAlaCysLys
225 230 235 240
LysGlyPheIleGln?ArgIleSerLysGly?GlyLeuIleAspAsp?AspAspLysArgLys
245 250 255 260
LysGlnArgVlaLys?IleAlaTyrGluGlu?IlePheVlaLysAsn?Met
265 270 275?276
Claims (4)
1. m of human tissue factor pathway inhibitory factor mutation gene
0TFPI is characterized in that it has the described nucleotide sequence of SEQ ID No.1 in the sequence table.
2. one kind by the described m of human tissue factor pathway inhibitory factor mutation gene of claim 1
0The TFPI encoded polypeptides is characterized in that it has the described aminoacid sequence of SEQ ID No.2 in the sequence table.
3. one kind comprises the described m of human tissue factor pathway inhibitory factor mutation gene of claim 1
0The recombinant vectors of TFPI, it is with the described m of human tissue factor pathway inhibitory factor mutation gene
0TFPI DNA adds T with the fragment and the pPIC9 carrier of XhoI and EcoRI digestion
4Ligase enzyme carries out ligation, makes m
0TFPI-pPIC9.
4. recombination microzyme that utilizes the described recombinant vectors of claim 3 to transform, it is to contain the described m of human tissue factor pathway inhibitory factor mutation gene
0The recombinant vectors of TFPI imports host's Pichia yeast GS115, becomes GS115/m
0TFPI-pPIC9.
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| CN1528894A (en) * | 2003-09-25 | 2004-09-15 | 复旦大学 | Long-acting reconbinant tissue factor channel inhibitor and preparing method thereof |
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| CN1528894A (en) * | 2003-09-25 | 2004-09-15 | 复旦大学 | Long-acting reconbinant tissue factor channel inhibitor and preparing method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 人类组织因子途径抑制因子的研究进展. 余波等.国际生物医学工程杂志，第29卷第1期. 2006 * |
| 人组织因子途径抑制因子重组蛋白表达菌株的建立. 冷希岗等.生物医学工程学杂志，第20卷第2期. 2003 * |
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