CN106699875A - Human recombination tissue factor, and gene, expression carrier, host bacterium and expression method of human recombination tissue factor - Google Patents
Human recombination tissue factor, and gene, expression carrier, host bacterium and expression method of human recombination tissue factor Download PDFInfo
- Publication number
- CN106699875A CN106699875A CN201710101492.7A CN201710101492A CN106699875A CN 106699875 A CN106699875 A CN 106699875A CN 201710101492 A CN201710101492 A CN 201710101492A CN 106699875 A CN106699875 A CN 106699875A
- Authority
- CN
- China
- Prior art keywords
- expression
- tissue factor
- gene
- human recombination
- factor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a human recombination tissue factor, and a gene, an expression carrier, a host bacterium and an expression method of the human recombination tissue factor, and belongs to the technical field of protein engineering. The provided human recombination tissue factor is better in biological activity; the expression carrier is connected with a target gene expressing the tissue factor to express the tissue factor in a host bacterium strain, a large quantity of tissue factors are rapidly and efficiently acquired, the expression efficiency of the human recombination tissue factor is higher, and the activity is higher. The provided expression method of the tissue factor is simple in operation step, convenient in operation and mild in reaction conditions, is convenient and concise, and is favorable for acquiring a large quantity of the human recombination tissue factors.
Description
Technical field
The present invention relates to protein engineering field, in particular to a kind of human recombination factor and its gene,
Expression vector, Host Strains and expression.
Background technology
Prothrombin time (PT) is that extrinsic coagulation system often uses one of screening test, refers to lack hematoblastic blood
Excessive tissue factor and Ca is added in slurry2+Afterwards, make prothombin for fibrin ferment, the latter makes fibrinogen be changed into fibre
Fibrillarin, the time required for the clotting of plasma is PT.
Tissue factor (TF), is a kind of complete membrane glycoprotein.TF as non-enzyme albumen confactor, can be with blood
Liquid coagulation factor FVIIa (blood-coagulation factor VIIa) interaction forms TF-FVIIa complexs and starts external source
Property coagulation process.The TF albumen of people is made up of 263 amino acid, comprising three domains：Ectodomain (1-219), across
Spanning domain (220-242) and intracellular domain (243-263).The function of ectodomain mainly in conjunction with and activate FVIIa
Start external source coagulation process.The interaction of TF and FVIIa is that calcium ion is relied on, and TF-FVIIa complexs to FIX and
The proteolysis process of FX is that film is relied on, and membrane spaning domain is exactly mainly by TF albumen anchored cells films, to tissue factor
Bioactivity it is extremely important, and intracellular domain primarily serves the effect of signal transduction.The extracellular structure of TF albumen in theory
Just there is the repertoire for starting extrinsic coagulation process in both domain and membrane spaning domain.
It is complicated to there is production process in the human tissue factor of existing technological expression, activity less than normal glycosylated tissue because
Son；And there is codon preference and influence the expression of albumen in heterologous expression system；The activity of albumen can similarly be influenceed.
The content of the invention
The first object of the present invention is to provide a kind of human recombination factor, with bioactivity higher.
The second object of the present invention is the gene for providing the above-mentioned human recombination factor of coding.
The third object of the present invention is to provide the expression vector containing said gene.
The fourth object of the present invention is to provide the Host Strains containing said gene.
The fifth object of the present invention is to provide a kind of expression of human recombination factor, can be rapidly and efficiently obtain
There must be the human recombination factor compared with high bioactivity.
In order to realize above-mentioned purpose of the invention, spy uses following technical scheme：
A kind of human recombination factor, the amino acid sequence of the tissue factor is as shown in SEQ ID NO.1.
The gene of the above-mentioned human recombination factor of coding.
Expression vector containing said gene.
Host Strains containing said gene.
A kind of expression of human recombination factor, it includes：
By above-mentioned gene cloning to first vector, the first cloning vector is obtained；
The cloning vector of digestion first is simultaneously transformed into host cell, obtains expression bacterial strain；
Culture expression bacterial strain and with the derivant induced expression bacterial strain expression tissue factor.
Compared with prior art, beneficial effects of the present invention are：The present invention provides human recombination factor, with preferable
Bioactivity；Expressed in host's strain by the genes of interest of expression vector connection table intelligent's recombinant tissue factor, can be fast
Speed, efficiently obtain substantial amounts of tissue factor, the expression efficiency of human recombination factor is higher, activity it is also higher.There is provided
The expression of tissue factor, operating procedure is simple, easy to operate, and reaction condition is gentle, convenient succinct；Be conducive to substantial amounts of obtaining
Obtain human recombination factor.
Brief description of the drawings
Technical scheme in order to illustrate more clearly the embodiments of the present invention, below will be attached to what is used needed for embodiment
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, thus be not construed as it is right
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 is the pPIC9K carrier schematic diagrames that the embodiment of the present invention 3 is provided；
Fig. 2 is the standard curve schematic diagram that experimental example of the present invention 2 is provided.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are
The conventional products that can be obtained by commercially available purchase.
A kind of human recombination factor and its gene below to the embodiment of the present invention, expression vector, Host Strains and expression
Method is specifically described.
Early in the twentieth century, Schmid and Moranitz are found that the startup factor of blood clotting in tissue first, and by its
It is named as tissue factor (Tissue factor, TF).To the eighties in 20th century, tissue factor is proved to be physiological blood coagulation
Most important startup factor in journey.People are by modern molecular biology skills such as recombinant DNA, artificial mutagenesis and gene knockouts
Art, further in molecular level and gene level research TF, finds TF and systemic inflammatory response syndrome and shock, DIC, thrombus
Property disease, graft-rejection, malignant tumour etc. generation, development close relation, research finds that TF also has again in recent years
There are the different physiological roles such as the reparation of participation callus, new vessels formation and embryonic development.Therefore, tissue factor is made in biology
There is good application development prospect in medicine field.
A kind of human recombination factor, the amino acid sequence of the tissue factor is as shown in SEQ ID NO.1.
Common human tissue factor is made up of 263 amino acid, comprising three domains：Ectodomain (1-219), across
Spanning domain (220-242) and intracellular domain (243-263).One section is merged by 6 group ammonia in the C-terminal of common tissue factor
His-tag protein tags of acid composition, and eliminate the signal peptide sequence of tissue factor, obtain the tissue of present invention offer because
Son.
Usually, after protein sequence synthesis, extracellular protein can be transported under the guiding of signal peptide；Then, believe
Number peptide can be cut out, and be also not include signal peptide sequence when actually occurring effect；So, when expressing protein
Signal peptide is removed, is the function of not interfering with albumen.C-terminal addition one in albumen simultaneously is made up of 6 histidines
His-tag protein tags, also will not produce influence to the space structure of albumen and biological function, also not interfere with the work(of albumen
Energy.
The gene of the above-mentioned human recombination factor of coding.
Further, the base sequence of said gene is as shown in SEQ ID NO.2.
Above-mentioned base sequence expression human recombination factor protogene sequence is done on the basis of removing signal peptide sequence
Codon optimization.It is excellent that the gene order is specific to that pichia yeast expression system carries out to the codon of human tissue factor
Change, the expressing gene sequence for obtaining；And introduce 6 nucleotide sequences of histidine of coding in the end of sequence：
CATCACCATCACCACCAC, then along with terminator codon：TAA.
6 nucleotide sequences of histidine of coding are introduced in sequence：CATCACCATCACCACCAC；Can be very easily
After the completion of protein expression, purification process is carried out to albumen.
One of histidine-tagged (Histidine-tag), most current mark of purification tag that genetic engineering builds, be
The aminoterminal of protein adds His-tag labels, and it is special to occur with many kinds of metal ions under general or Denaturing
Mutual chelation, these metal ions include Ca2+、Mg2+、Ni2+And Co2+Deng wherein especially being used with nickel ion the widest
It is general, because nickel ion and histidine-tagged joint efficiency highest.After generally by thalline broken wall, human recombination factor will be contained
Solution by Ni adsorption columns, the nickel ion in Ni adsorption columns will reach with the chelating of the His-tag protein tags on destination protein
The purpose that destination protein is separated, then elutes Ni adsorption columns with the imidazole solution of high concentration, can just wash destination protein
Take off.
When expression due to doing protein hetero, different expression systems, the Preference that can there is codon；With multiple
The amino acid of codon, a single transhipment rRNA can be used when translation；If one kind in 20 kinds of amino acid or
The transhipment rRNA of several amino acids can influence the synthesis of albumen using excessive, the termination in advance that albumen may be caused to synthesize, and make egg
White amino acid chain is imperfect, causes the processes such as processing and folding that protein is follow-up to be also affected, and then influences the work(of albumen
Energy.In addition even some expression systems also in the presence of the rare codon that some are rarely employed, once in the sequence of heterogenous expression
Occur excessive rare codon occur in rare codon, or the sequence of heterogenous expression, both of which may be led
Cause protein expression failure.So needing to optimize gene order.Successful expression is also also needed to further after going out destination protein
Checking albumen whether be with corresponding bioactivity, because sometimes giving expression to albumen can have no bioactivity
Situation occurs.
In addition, adding terminator codon at the end of sequence, terminate to use termination codon in the gene order expression of expression
Son is by sequence ends, it is to avoid expression vector sequence；If giving expression to the amino acid of carrier sequence, cause the primary structure of albumen
Change, and then be likely to result in two structures, tertiary structure and the spatial orientation and avtive spot of albumen and be changed；Or it is living
Property position is covered, causes the forfeiture of function.So needing, plus termination codon, the expression of albumen to be terminated in time.
Carrier containing said gene.
Being connected by expression vector can express the gene order of above-mentioned albumen, can be by the method for heterogenous expression, largely
Express express target protein；By great expression destination protein, then can apply on a large scale.
Host Strains containing said gene.
A kind of expression of human recombination factor, it includes：
By above-mentioned gene cloning to first vector, the first cloning vector is obtained；
The cloning vector of digestion first is simultaneously transformed into host cell, obtains expression bacterial strain；
Culture expression bacterial strain and with the derivant induced expression bacterial strain expression tissue factor.
When the expression tissue factor, with SalI restriction enzymes by the first cloning vector linearization for enzyme restriction, then
The first cloning vector for linearizing is transformed into competent yeast cells by electroporation.
Using homologous recombination machinery in eucaryote, recombinated by homology arm, expressed sequence is incorporated into yeast chromosomal
On, positive expression bacterial strain is obtained by screening, conveniently carry out the expression of tissue factor.
Further, first vector is pPIC9K carriers.
Further, said gene is located between pPIC9K carrier EcoRI and NotI sites.
Further, host cell is yeast cells.
The normal glycosylated to the active extremely important of tissue factor of tissue factor is shown by existing research, so this is heavy
The active and normal glycosylated tissue factor of group tissue factor protein is still present gap.Protokaryon table is carried out using Escherichia coli
Reach, causing the tissue factor of expression in escherichia coli can not be glycosylated, and then influence the function of tissue factor；It is simultaneously heterologous
Memebrane protein expression in prokaryotic system is usually present the low problem for forming inclusion body because of incorrect folding with albumen of expression quantity, increases
The difficulty of expression and purification of recombinant proteins is added.Again because Yeast protein expression systems belong to eukaryotic protein system, can be certain
Evade these problems in degree, while having cycle is short, low cost, operation letter compared to mammalian cell and insect expression system
Single advantage.So carrying out the expression of tissue factor from yeast expression system.
Meanwhile, tissue factor is carried out into codon optimization for yeast expression system, and select yeast expression system energy
Improve the bioactivity of the tissue factor of expression.
Further, derivant is methyl alcohol.
When Yeast expression, in the case of no environmental stimuli, the expression quantity of albumen can be very low；But, if
By adding derivant, just energy stimulation of host cell fast and efficiently gives expression to substantial amounts of destination protein.PPIC9K carriers have
Strong alcohol oxidase (Alochol Oxidase, AOX1) gene promoter, can strictly regulate and control the expression of foreign protein；
In the present invention, derivant is preferably methyl alcohol, and the expression of destination protein is largely induced using methyl alcohol.
Feature of the invention and performance are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of human recombination factor, the amino acid sequence such as SEQ ID NO.1 institutes of the tissue factor
Show.
The tissue factor is made up of 269 amino acid, comprising three domains：Ectodomain (1-219), transmembrane structure
Domain (220-242) and intracellular domain (243-263)；And merge one section of His-tag albumen being made up of 6 histidines in C-terminal
Label.
The tissue factor can form TF-FVIIa complexs and start extrinsic coagulation with blood clotting factor FVIIa interactions
Process.
Embodiment 2
The present embodiment provides the gene of coding human recombination factor, the base sequence such as SEQ ID NO.2 institutes of the gene
Show.
The sequence is the former expressed sequence that human tissue factor is encoded in NCBI accounting databases, by bioinformatics
Method carries out codon optimization for pichia yeast expression system；Enable the gene order of optimization it is efficient in Pichia pastoris,
Stable gives expression to human recombination factor albumen, and keeps its bioactivity.
It is excellent that the base sequence is that expression tissue factor protogene sequence removes the codon done on the basis of signal peptide sequence
Change, and 6 nucleotide sequences of histidine of coding are introduced in the end of sequence：CATCACCATCACCACCAC, then adds
Terminator codon：TAA.
6 His-tag protein tags of histidine composition of addition coding, facilitate follow-up protein purification to test, and end adds
Plus terminator codon, it is consistent with natural expression status, it is to avoid the function of albumen is influenceed with the presence of unnecessary amino acid.
Embodiment 3
The present embodiment provide containing shown in embodiment 2 coding human recombination factor gene order expression vector and
Host Strains.
Using pPIC9K carriers as expression vector in the present embodiment, carrier schematic diagram is shown in Fig. 1.By to optimize tissue because
The analysis of the sequence of the gene of sublist and the MCS of combination pPIC9K carrier sequences, from MCS EcoRI
With NotI sites as the expressing gene of tissue factor the insertion point on carrier, be built into the first cloning vector pPIC9K-TF.
PPIC9K-TF carrier construction methods, detailed process is as follows：
1.1 are mixed in the centrifuge tube of 200 μ L with linearizing the μ L of pEASY-Blunt carriers 3 and the μ L of purpose fragment 1, quickly
Mixed reaction solution is put after centrifugation 30min is reacted under the conditions of 25 DEG C, then ice bath；
1.2 take TOP10 competent cells is placed in ice bath, after the TOP10 impressions for after competent cell thawing, taking 501 μ L
State cell, and add the connection product reaction solution in 1.1, pipettor gently to blow and beat mixing, ice bath 30min；
1.3 under 42 DEG C of parts heat shock 45s, rapidly by centrifuge tube transfer ice bath 2-3min；
The aseptic LB culture mediums without antibiotic of 500 μ L are added in 1.4 centrifuge tubes crossed to ice bath, at 37 DEG C after mixing
Shaking table on 150rpm concussion and cultivates 45min；
The 1.5 TOP10 competent cells for going appropriate conversion, are spread evenly across LB solid mediums (Amp+) on, wait to train
Culture dish is covered after supporting base table ground drying, 37 DEG C are inverted culture 8h；
1.6 after flat board grows single bacterium colony, and picking single bacterium colony simultaneously identifies positive colony, then positive gram of Amplification Culture
It is grand；
1.7 plasmids for extracting positive colony；
The 1.8 positive colony plasmids and pPIC9K vector plasmids extracted with EcoRI and NotI double digestions, and recovery purifying enzyme
Section section；
The pPIC9K carrier segments that 1.9 plus 2 μ L are reclaimed, 6 μ L genes of interest fragments, μ L, the T4 ligases of T4 ligases 1
Buffer2 μ L, use ddH2O is supplied to 15 μ L, and 16 DEG C connect overnight, obtain pPIC9K-TF vector plasmids；
Connection product is converted TOP10 competent cells by 1.10, and coats LB solid mediums (Kan+) on, 37 DEG C are fallen
Culture 8h is put, 1.6 and 1.7 operation is repeated；
The 1.11 pPIC9K-TF vector plasmids extracted with restriction enzyme SalI digestions 1.10, and by 1% agar
The pPIC9K-TF carrier segments of sugared gel electrophoresis recovery purifying linearisation；
The 1.12 pPIC9K-TF carrier segments that will be linearized are converted with after aseptic ultrapure water dissolves by electroporation
GS115 competent yeast cells；
1.13 after MD flat screens select positive colony, and choose 10 clones carries out further positive verification using PCR,
Will confirm that the yeast of the positive accesses fluid nutrient medium and carries out protein induced expression using methyl alcohol.
Certain the present embodiment provides a kind of method of pPIC9K-TF vector constructions, still also has other structure sides
Method can obtain pPIC9K-TF expression vectors.
Experimental example 1
This experimental example provides dividing for the purity of the human recombination factor of the carrier and Host Strains expression provided embodiment 3
Analysis.
The purity analysis of human recombination factor, specific method is as follows：
1.1 will confirm that the yeast of the positive accesses fluid nutrient medium and carries out protein induced expression using methyl alcohol；
Bacterium solution is by taking supernatant after 1.2 expression after centrifugation, and Dot-Blot (Anti-His antibody) is carried out to supernatant enters
Row protein expression verifies, checking expressing quantity 3 plants of bacterial strains higher further by SDS-PAG proteins gel electrophoresis and
Western-Blot is further confirmed that, and identifies expressing quantity highest bacterial strain；
After 1.3 by culture in expression inoculation the most obvious to BMGY fluid nutrient mediums 24 hours, renewed vaccination is arrived
Fiber differentiation in BMMY fluid nutrient mediums, 5% methyl alcohol was added every 24 hours；
1.4 collect supernatant nutrient solution after 72 hours, and the zymotic fluid of 72 hours is by utilizing fusion protein after concentration
His-tag protein tags are purified；
1.5 albumen after purification is put into bag filter, and removing purifying protein using PBS (PH=7.4) dialysed overnight remains
Salt ion；
Human recombination factor albumen after 1.6 expression is passed through by BCA determination of protein concentration kit measurement concentration
SDS-PAGE proteins gel electrophoresis detect purity.
Identified by above step, the human recombination factor purity of protein of monitoring result display purifying is more than 95%.
Experimental example 2
This experimental example provides living to the biology of the human recombination factor of carrier and the Host Strains expression provided embodiment 3
The measure of property.
The specific method of the measure of the bioactivity of human recombination factor is as follows：
The preparation of 1.1 Human Factor Ⅸ Complex's solution：0.1mol/L Tris-HCl buffer solutions 10ml is taken (to contain
0.012mol/LCaCl2, PH=7.4), add Lyophilized Prothrombin Complex Contrates 10U, Human Factor Ⅸ Complex's solution it is dense
It is 1U/ml to spend；
The definition of normal structure APTT stoste is diluted 10 by 1.25It is again 1U, dilution 104It is 10U, and so on；
1.3 add the different μ L of dilution factor tissue thromboplastin 100 to each enzyme mark hole in ELISA Plate, are subsequently adding 100 μ l
Human Factor Ⅸ Complex's solution (1U/ml) and 100 μ l S2222 (2.0mg/ml), 37 DEG C incubation 30min, use ELIASA
(405nm) mensuration absorbance；
The human recombination factor stoste of our expression and purifications is carried out Chromogenic assay experiment by 1.4 measures absorbancemA405
=2786.
The chromophoric substrate of table 1 hair determines TFA's data
| PCA(U) | Extension rate | lgPCA | mA405(standard TF) |
| 105 | 0 | 5 | 1983 |
| 104 | 10 | 4 | 749 |
| 103 | 102 | 3 | 248 |
| 102 | 103 | 2 | 89 |
| 10 | 104 | 1 | 31 |
Experimental result as shown in table 1 and Fig. 2, the APTT of different extension rates and the linear pass of the logarithm of absorbance
System, obtains the calibration curve formula y=10.972e of APTT and absorbance1.0447x；The calibration curve formula of gained is obtained
The active PCA=199526U of the human recombination factor of expression, because as can be seen that the human recombination factor of the expression of embodiment 2
There is activity very high.
Embodiments described above is a part of embodiment of the invention, rather than whole embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiment in the present invention, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
SEQUENCE LISTING
<110>Qingdao Gu Gao Bioisystech Co., Ltd
<120>A kind of human recombination factor and its gene, expression vector, Host Strains and expression
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 269
<212> PRT
<213> Homo sapiens
<400> 1
Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser
1 5 10 15
Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln
20 25 30
Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys
35 40 45
Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val
50 55 60
Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala
65 70 75 80
Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn
85 90 95
Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr
100 105 110
Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu
115 120 125
Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg
130 135 140
Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser
145 150 155 160
Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu
165 170 175
Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val
180 185 190
Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu
195 200 205
Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Ile Phe Tyr Ile Ile
210 215 220
Gly Ala Val Val Phe Val Val Ile Ile Leu Val Ile Ile Leu Ala Ile
225 230 235 240
Ser Leu His Lys Cys Arg Lys Ala Gly Val Gly Gln Ser Trp Lys Glu
245 250 255
Asn Ser Pro Leu Asn Val Ser His His His His His His
260 265
<210> 2
<211> 810
<212> DNA
<213> Homo sapiens
<400> 2
tccggtacta ccaacactgt tgctgcctac aacttgactt ggaagtccac caacttcaag 60
accatcttgg aatgggagcc aaagccagtt aaccaggttt acactgttca gatctccacc 120
aagtctggtg actggaagtc taagtgtttc tacactaccg acaccgagtg tgacttgact 180
gacgaaatcg ttaaggacgt caagcagacc tacttggcca gagttttttc ttaccctgcc 240
ggtaacgttg agtctactgg ttctgctggt gaaccactgt acgaaaactc tccagagttc 300
accccatact tggagactaa cttgggtcag ccaactatcc aatccttcga gcaggttggt 360
actaaggtta acgttactgt cgaggacgag agaaccctgg tcagaagaaa caacaccttc 420
ttgtccctga gggacgtttt cggtaaggac ttgatctaca ccctgtacta ctggaaatcc 480
agctcctccg gtaaaaagac tgctaagact aacaccaacg agttcttgat cgacgtggac 540
aagggtgaga actactgttt ctccgttcag gctgttatcc catccagaac cgttaacaga 600
aagtccactg actccccagt tgagtgtatg ggtcaagaaa agggtgagtt cagagagatc 660
ttctacatca tcggtgccgt cgttttcgtc gtcatcatct tggttattat cctggccatc 720
tccttgcaca agtgcagaaa agctggtgtt ggtcagtcct ggaaagagaa ctctccattg 780
aacgtttctc atcaccatca ccaccactaa 810
Claims (10)
1. a kind of human recombination factor, it is characterised in that the amino acid sequence of the tissue factor such as SEQ ID NO.1 institutes
Show.
2. the gene of human recombination factor as claimed in claim 1 is encoded.
3. gene according to claim 2, it is characterised in that the base sequence of the gene is as shown in SEQ ID NO.2.
4. the expression vector of gene described in Claims 2 or 3 is contained.
5. the Host Strains of gene described in Claims 2 or 3 are contained.
6. a kind of expression of human recombination factor, it is characterised in that it includes：
By the gene cloning described in Claims 2 or 3 to first vector, the first cloning vector is obtained；
First cloning vector described in digestion is simultaneously transformed into host cell, obtains expression bacterial strain；
The culture expression bacterial strain simultaneously induces the expression bacterial strain to express the tissue factor with derivant.
7. expression according to claim 6, it is characterised in that the first vector is pPIC9K carriers.
8. expression according to claim 7, it is characterised in that the gene is located at the pPIC9K carriers
Between EcoRI and NotI sites.
9. expression according to claim 6, it is characterised in that the host cell is yeast cells.
10. expression according to claim 6, it is characterised in that the derivant is methyl alcohol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710101492.7A CN106699875A (en) | 2017-02-23 | 2017-02-23 | Human recombination tissue factor, and gene, expression carrier, host bacterium and expression method of human recombination tissue factor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710101492.7A CN106699875A (en) | 2017-02-23 | 2017-02-23 | Human recombination tissue factor, and gene, expression carrier, host bacterium and expression method of human recombination tissue factor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106699875A true CN106699875A (en) | 2017-05-24 |
Family
ID=58917051
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710101492.7A Pending CN106699875A (en) | 2017-02-23 | 2017-02-23 | Human recombination tissue factor, and gene, expression carrier, host bacterium and expression method of human recombination tissue factor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106699875A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112341533A (en) * | 2020-11-20 | 2021-02-09 | 东北师范大学 | Non-label human galectin 13 and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1251532A (en) * | 1997-01-22 | 2000-04-26 | 德克萨斯州立大学董事会 | Tissue factor methods and compositions for coagulation and tumor treatment |
-
2017
- 2017-02-23 CN CN201710101492.7A patent/CN106699875A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1251532A (en) * | 1997-01-22 | 2000-04-26 | 德克萨斯州立大学董事会 | Tissue factor methods and compositions for coagulation and tumor treatment |
Non-Patent Citations (3)
| Title |
|---|
| INVITROGEN: "《pPIC9K A Pichia Vector for Multicopy Integration and Secreted Expression. Catalog no. V175-20》", 31 December 2002, INVITROGEN产品手册 * |
| 研小弟: "密码子的简并性及偏好性", 《乐研生物》 * |
| 阮建兵，等: "多聚组氨酸融合标签在蛋白药物开发中的应用", 《生物技术通报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112341533A (en) * | 2020-11-20 | 2021-02-09 | 东北师范大学 | Non-label human galectin 13 and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110845603B (en) | Human collagen 17-type polypeptide, production method and use thereof | |
| CN108912221A (en) | For producing auxilin, encoding gene, recombination fusion protein, recombinant expression carrier and the preparation method of recombination fusion protein | |
| CN112209995B (en) | Preparation method of SARS-CoV-2 surface protein receptor binding region | |
| JPH04507346A (en) | Alkaline proteolytic enzyme and its production method | |
| JPH03505527A (en) | Purified ubiquitin hydrolase, the DNA sequence encoding it, and its use in polypeptide recovery | |
| CN107098979A (en) | PPR virus H F fusion proteins and its relevant biological material and application | |
| CN109486800A (en) | A kind of novel lysyl endopeptidase and preparation method thereof | |
| CN106701813A (en) | Expression vector as well as construction method and application thereof | |
| CN109439643A (en) | A kind of novel lysine specificity restriction endonuclease and preparation method thereof | |
| CN106699875A (en) | Human recombination tissue factor, and gene, expression carrier, host bacterium and expression method of human recombination tissue factor | |
| CN111088241B (en) | Genetically engineered human lysozyme | |
| CN110484516A (en) | It is a kind of with CRISPR system by the quantum dot-labeled methods and applications to viral nucleic acid | |
| JPH03503843A (en) | Method for purifying phospholipase A2 and method for producing phospholipase A2-like polypeptide | |
| Gwynne et al. | Development of an expression system in Aspergillus nidulans | |
| CN109295041A (en) | With active polypeptide of serrapeptase and preparation method thereof | |
| CN108265042A (en) | A kind of preparation method of recombinant enterokinase | |
| CN101240284B (en) | Recombination staphylokinase and highly effective secretion expression method thereof | |
| Satoh et al. | A chimeric inorganic pyrophosphatase derived from Escherichia coli and Thermus thermophilus has an increased thermostability | |
| EP0718403B1 (en) | Process for producing human matrilysin by means of recombinant DNA | |
| CN106636019A (en) | Synthesis of (S)-1-Phenyl-1,2-ethanediol through efficient catalysis of sortase A mediated (S)-carbonyl reductase oligomer | |
| CN109371003A (en) | The beta-glucosidase that a kind of pair of pepsin resistance improves | |
| CN117466992B (en) | Fibronectin mutant and preparation and application thereof | |
| CN107400665A (en) | A kind of mannase of rite-directed mutagenesis and its application | |
| CN111172139B (en) | Calf abomasum rennin, gene, strain and application | |
| JPH09173083A (en) | End-beta-n-acetylglucosaminidase gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170524 |
|
| RJ01 | Rejection of invention patent application after publication |