CN1920029A - Human tissue factor pathway inhibitory factor mutation gene mTFPI, recombination carrier including the same and host cell thereof - Google Patents

Abstract

The invention discloses the mTFPI gene, comprising the recombination carrier and host cell. The recombination protein content expressed by mTFPI in pichia is improved, and the cost is reduced.

Description

The mTFPI of human tissue factor pathway inhibitory factor mutation gene, the recombinant vectors that comprises this gene and host cell thereof

Technical field:

The invention belongs to gene engineering technology field, relate in particular to the tissue factor approach inhibition factor mutator gene, contain the recombinant vectors of this gene, and with this carrier transformed host cells.

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.

Human TFPI plasma concentration low (54-124ng/ml), directly purification difficult from blood plasma can't be obtained in a large number.Be used for functional study and clinical application for obtaining capacity TFPI, existing both at home and abroad many companies and the engineered method of laboratory applications prepare TFPI recombinant protein (rTFPI).Yet the TFPI complex structure all fails to realize the expression of high yield in many expression systems.It is very low to utilize the yeast saccharomyces cerevisiae expression system to express TFPI output, about 20ug/L.The recombinant protein that utilizes the mammalian cell expression system to be obtained, the about 2～6mg/L of output, biological activity is better, but cost is too high, is unfavorable for suitability for industrialized production.

Pichia yeast expression system is the novel thermophilic methanol yeast expression system of extremely paying attention in recent years, it expresses foreign protein output height, and is simple to operate, is beneficial to large scale fermentation and cultivates, compare with yeast saccharomyces cerevisiae, it more is similar to mammalian cell to the modification of foreign protein.In the Pichia anomala expression system, the expression of exogenous gene amount mainly is subjected to the regulation and control of transcriptional level.Some sequence of foreign gene may cause premature transcription termination, as TTTTTATA, ATTATTTTATAAA (Pichia Expression Kit).Human TFPI-cDNA 337-346 bit base sequence is TATTTTTATA, makes TFPI recombinant protein expression amount in pichia spp low.Therefore, this sequence of suddenling change becomes the prerequisite that improves TFPI recombinant protein output in pichia spp, large scale fermentation is cultivated a large amount of TFPI recombinant proteins of acquisition have great importance.

Summary of the invention

The objective of the invention is provides a kind of mTFPI of human tissue factor pathway inhibitory factor mutation gene for improving the output of TFPI recombinant protein.

Second purpose of the present invention provides the recombinant vectors of a kind of TFPI of including mutator gene mTFPI.

The 3rd purpose of the present invention provides the transformed host cell of a kind of TFPI of containing mutator gene mTFPI.

Technical scheme of the present invention is summarized as follows:

A kind of TFPI mutator gene mTFPI, it has the described nucleotide sequence of SEQ ID No.1 in the sequence table.

A kind of recombinant vectors that comprises TFPI mutator gene mTFPI is with the described Nucleotide of SEQ ID No.1 in the sequence table

Sequence is handled back and pPic9 carrier (American I nvitrogen company) with XhoI and EcoRI, adds T ₄Ligase enzyme carries out ligation, makes mTFPI-pPic9.

A kind of transformed host cell that contains TFPI mutator gene mTFPI, it is that mTFPI-pPic9 is imported to host's Pichia yeast GS115, becomes GS115/mTFPI-pPic9.

TFPI mutator gene mTFPI characteristics among the present invention are: carried out the change of partial nucleotide sequence on the basis of the TFPI polynucleotide sequence that other have been reported; But their amino acid sequence coded is identical, promptly is the TFPI of wild-type.

Experimental results show that, compare with the recombinant protein amount that wild-type TFPI gene is expressed in pichia spp, TFPI mutator gene mTFPI of the present invention expressed recombinant protein amount in pichia spp improves a lot (seeing that Fig. 5 mTFPI recombinant protein and TFPI Recombinant Protein Expression amount are relatively), cultivates the production cost that obtains this recombinant protein thereby can reduce large scale fermentation greatly.

Description of drawings

Fig. 1 deletes wild-type TFPI-cDNA secretory signal sequence policy map;

Fig. 2 mTFPI-cDNA construction strategy figure;

A kind of mTFPI-cDNA of containing of Fig. 3 reorganization (clone) carrier and contain the construction strategy figure of the host cell of this recombinant vectors;

The polyacrylamide gel electrophoresis collection of illustrative plates of Fig. 4 mTFPI recombinant protein;

Fig. 5 mTFPI recombinant protein and TFPI Recombinant Protein Expression amount are relatively.

Embodiment

The method for preparing a kind of TFPI mutator gene mTFPI of the present invention, recombinant vectors and host cell thereof comprises the steps:

1. the secretion signal peptide sequence deletion of one section 78 base will be contained by pcr amplification in the natural TFPI-cDNA sequence, through the restriction enzyme partial hydrolysis, obtain dna fragmentation, be connected to pPic9 carrier (American I nvitrogen company) and go up and be transformed among the intestinal bacteria TB1.Extract the TFPI-pPic9 plasmid DNA, suddenly change with the partial dna sequence of PCR method to TFPI.Behind the purified pcr product, after handling with restriction enzyme, be connected on the pPic9 carrier and be transformed into intestinal bacteria TB1, the recombinant plasmid of formation is mTFPI-pPic9.

2. extract the mTFPI-pPic9 plasmid DNA, with restriction enzyme with its linearizing after, be transformed among the yeast competent cell GS115 (American I nvitrogen company), obtain recombination microzyme GS115/mTFPI-pPic9.

Under the condition that TFPI mutator gene mTFPI is expressed, cultivate this reorganization bacterium, measure the proteic activity of TFPI with substrate color development method; And, prove TFPI albumen great expression through the SDS-PAGE analysis.

TFPI mutator gene mTFPI nucleotide sequence SEQ ID No.1.The TFPI genetic comparison of TFPI mutator gene mTFPI total length 828bp and other wild-types, sudden change has taken place in its 337-346 position nucleotide sequence.

According to nucleotide sequence SEQ ID No.1, infer that the proteic aminoacid sequence of TFPI of expression is seen SEQ ID No.2.The aminoacid sequence of this TFPI is made up of 276 amino acid, and molecular weight is 42KDa.By TFPI mutator gene mTFPI deduced amino acid and other wild-types TFPI Argine Monohydrochloride sequence that the present invention relates to compared, the result shows that no any difference is (referring to Thomas JG, Roger E, Robin LW, et al.1991.Structure of the humanlipoprotein-associated coagulation inhibitor gene.The Journal of Biological Chemistry.226 (8) 5036-5041.)

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.

Embodiment 1 makes up the carrier that contains TFPI mutator gene mTFPI

With plasmid DNA TFPI-pFastBac (biomedical engineering institute of China Concord Medical Science University of Chinese Academy of Medical Sciences genetically engineered laboratory makes up) is template, XhoI (gtc agt ctc gag aaa aga gat tct gag gaa gat gaag; Contain the XhoI restriction enzyme site), EcoI (gct ata acg aat tca cat att ttt aac; Contain the EcoRI restriction enzyme site) carry out pcr amplification (seeing that Fig. 1 deletes wild-type TFPI secretory signal sequence policy map) for positive anti-primer.Through agarose gel electrophoresis, reclaim test kit (Beijing ancient cooking vessel state biotech development center) purified pcr product with dna gel.Handle PCR product and pPic9 carrier with restriction enzyme XhoI and EcoRI, through agarose gel electrophoresis, after above-mentioned test kit recovery, get 200ng pPIC9 and reclaim product, PCR product with the 200ng recovery, in 20 μ L linked systems, spend the night and carry out ligation (linked system contains 2ul10x and connects damping fluid, 1ulT4DNA ligase enzyme) in 16 ℃.Get connection product Transformed E .coli (intestinal bacteria) TB1, be coated on the LB culture medium flat plate.Extract the TFPI-pPic9 plasmid DNA: picking one single colony inoculation 37 ℃ of 250rpm incubated overnight in 10ml LB substratum; Centrifugal collection thalline, (pH8.0) resuspended, recentrifuge is collected thalline for Tris-HCl 10mmol/L, EDTA 1mmol/L, and adding 15ul concentration with the resuspended back of GTE damping fluid is the N,O-Diacetylmuramidase of 10mg/ml, 30 ℃ of water-bath 30min behind the mixing with 1ml TE damping fluid; Add 300ul lysate (0.2N NaOH/1%SDS, prepared fresh) then, leave standstill 5min after gently putting upside down mixing; The potassium acetate solution (pH 5.5 for 3M potassium acetate, 2M acetate) that adds 225ul 5M, ice bath 10min behind the mixing; The centrifugal 15min of 13000rpm gets supernatant, adds RNaseA (ribonuclease A) solution of 2ul 10mg/ml, 37 ℃ of water-bath 20min behind the mixing; (phenol: chloroform=24: 1) extracting once for isopyknic phenol-chloroform mixed solution; Add isopyknic PEG (polyoxyethylene glycol) solution (13%PEG 8000,0.8M NaCl) mixing, 4 ℃ leave standstill 30min, and the centrifugal 20min of 13000rpm abandons supernatant.Precipitation is dissolved in the TE damping fluid of proper volume with ice-cold 70% ethanol rinsing twice.Using the OE-PCR fixed-point mutation method, is template with plasmid DNA TFPI-pPic9, with 5 ' Aox (GCA AAT GGC ATT CTGACA TCC) and mutant primer m2 (tgt ctg att gtt gta gaa gta cct ggt aat ata ac); 3 ' Aox (GAC TGG TTC CAA TTG ACA AGC) and mutant primer m1 (tat att acc agg tac ttc tacaac aat cag aca aaa c) are primer, carry out twice PCR amplification (seeing that Fig. 2 deletes the wild-type TFPI site-directed point mutation policy map of secretory signal sequence).With aforesaid method purified pcr product (mTFPI), enzyme is cut the PCR product and is connected on the pPic9 carrier, and transforms mTFPI-pPic9 to intestinal bacteria TB1 with aforesaid method.

Embodiment 2 makes up and identifies recombination microzyme GS115, (yeast GS115, American I nvitrogen company)

Getting the plasmid DNA (mTFPI-pPic9) of 50ug extraction 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 1.2mlPEG 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 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, as just, anti-primer carries out pcr amplification with 5`Aox and 3`Aox..Agarose gel electrophoresis is analyzed the PCR product, and judges according to the size of amplified fragments whether the mTFPI-pPIC9 plasmid inserts in the yeast host bacterium genome.The recombination microzyme GS115 that screening mTFPI-pPic9 plasmid has inserted (a kind of mTFPI-cDNA of containing reorganization (clone) carrier of Fig. 3 and contain the construction strategy figure of the host cell of this recombinant vectors).

The abduction delivering of embodiment 3TFPI 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 10ml BMGY substratum, 30 ℃ of 250rpm are cultured to OD600 ≈ 3 (about 16-18h); Centrifugal collecting cell is resuspended in the 30ml BMMY substratum.Adding 100% methyl alcohol to final concentration is that 0.5%, 30 ℃ of 250rpm cultivates 36h, and to add methyl alcohol to final concentration every 12h be 0.5%.And get supernatant and carry out biological activity analysis and SDS-PAGE electrophoresis (see that Fig. 4 SDS-PAGE analyzes the mTFPI Recombinant Protein Expression, lane1 is the mTFPI-pPic9-GS115 culture supernatant among the figure; Lane2 is the TFPI-pPic9-GS115 culture supernatant; Lane3 is the pPic9-GS115 culture supernatant; Lane 4 is the molecular weight of albumen standard).

The expression amount of proof mTFPI recombinant protein in pichia spp is than improve a lot (the seeing the expression of the different TFPI of Fig. 5 in Pichia yeast) of TFPI recombinant protein.

SEQ?ID?No.1

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?ggcctaatta?aaaccaaaag?aaaaagaaag?780

aagcagagag?tgaaaatagc?atatgaagaa?atttttgtta?aaaatatg 828

SEQ?ID?No.2

AspSerGluGluAspGluGluHisThrIleIleThrAspThrGluLeuProProLeuLys

1 5 10 15 20

LeuMetHisSerPheCysAlaPheLysAlaAspAspGlyProCysLysAlaIleMetLys

25 30 35 40

ArgPhePhePheAsnIlePheThrArgGlnCysGluGluPheIleTyrGlyGlyCysGlu

45 50 55 60

GlyAsnGlnAsnArgPheGluSerLeuGluGluCysLysLysMetCysThrArgAspAsn

65 70 75 80

AlaAsnArgIleIleLysThrThrLeuGlnGlnGluLysProAspPheCysPheLeuGlu

85 90 95 100

GluAspProGlyIleCysArgGlyTyrIleThrArgTyrPheTyrAsnAsnGlnThrLys

105 110 115 120

GlnCysGluArgPheLysTyrGlyGlyCysLeuGlyAsnMetAsnAsnPheGluThrLeu

125 130 135 140

GluGluCysLysAsnIleCysGluAspGlyProAsnGlyPheGlnVlaAspAsnTyrGly

145 150 155 160

ThrGlnLeuAsnAlaVlaAsnAsnSerLeuThrProGlnSerThrLysVlaProSerLeu

165 170 175 180

PheGluPheHisGlyProSerTrpCysLeuThrProAlaAspArgGlyLeuCysArgAla

185 190 195 200

AsnGluAsnArgPheTyrTyrAsnSerVlaIleGlyLysCysArgProPheLysTyrSer

205 210 215 220

GlyCysGlyGlyAsnGluAsnAsnPheThrSerLysGlnGluCysLeuArgAlaCysLys

225 230 235 240

LysGlyPheIleGlnArgIleSerLysGlyGlyLeuIleLysThrLysArgLysArgLys

245 250 255 260

LysGlnArgVlaLysIleAlaTyrGluGluI?lePheVlaLysAsnMet

265 270 275

Claims (3)

1. the mTFPI of human tissue factor pathway inhibitory factor mutation gene is characterized in that it has the described nucleotide sequence of SEQ IDNo.1 in the sequence table.

2. a recombinant vectors that comprises claim 1 mutator gene is characterized in that the described nucleotide sequence of SEQ ID No.1 in the sequence table is handled back and pPic9 carrier with XhoI and EcoRI, adds T ₄Ligase enzyme carries out ligation, makes mTFPI-pPic9.

3. transformed host cell that contains the TFPI mutator gene mTFPI of claim 1, it is that mTFPI-pPic9 is imported to host's Pichia yeast GS115, becomes GS115/mTFPI-pPic9.

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