CN108623672A - The combination mutant of new EGF-A and B prepares and its application in bio-pharmaceutical - Google Patents
The combination mutant of new EGF-A and B prepares and its application in bio-pharmaceutical Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Abstract
The present invention provides the preparation methods of new human ldl receptor EGF (AB) mutant and mutant and human immunoglobulin(HIg) Fc segment compositions.EGF (AB) mutant ratio EGF A mutant of the present invention has better biological activity;EGF (AB) mutant ratio EGF (AB) mutant for merging Fc segments has longer half-life period, can be used for treating the angiocardiopathies such as hypercholesterolemia, atherosclerosis and benefited Other diseases by reducing blood plasma PCSK9 levels.
Description
Technical field
The invention belongs to biotechnologies, are related to LDL receptor (low-density lipoprotein
Receptor, LDLR) extracellular domain epidermal growth factor precursor homeodomain A and B (Epidermal growth factor
Precursor homology domain A and B, EGF-AB) mutant and its fusion protein, preparation method and treatment
The angiocardiopathies such as hypercholesterolemia, atherosclerosis and benefited other diseases by reducing blood plasma PCSK9 levels
The application of disease.
Background technology
Ldl receptor is a kind of transmembrane cell surface glycoprotein, including 5 functional domains:Ligand binding domain, epidermal growth
Factor precursor homeodomain, the sugared domain of O- connections, membrane-spanning domain and cytosolic domain.The ligand binding domain of LDLR and epidermal growth factor precursor are same
Source domain is respectively positioned on extracellular, the former can be with aPoA poB, apolipoprotein E, low-density lipoprotein (LDL) and β extra-low densities
Lipoprotein (β-VLDL) combines, and is formed in the complex of ligand and receptor and swallow born of the same parents, thus during cholesterol metabolic from
Key effect.Epidermal growth factor precursor homeodomain because with epidermal growth factor (epithelial growth factor,
EGF) precursor extracellular domain has the structural homology of 33-35% and gains the name, the areas EGF-A and proprotein convertases subtilisin 9
The catalytic activity of (proprotein convertase subtilisin/kexin 9, PCSK9) is related, and PCSK9 is urged by it
Change structural domain to be combined with EGF-A, and LDLR is caused to degrade or reduce the ability that it absorbs LDL.And there is calcium to combine spy in the areas EGF-B
Property, contribute to the stabilization between functional domain.So far, the defect type of nearly more than 700 kind LDLR and familial are high in global range
Correlation occurs for cholesterolemia (FH), wherein the defect (http that EGF-A or EGF-B that 10% defect is LDLR occur://
www.ucl.ac.uk/fh)。EGF-A is to influencePCSK9The important segment of catalytic activity,External functional study is found, to EGF-A The site mutation in area can make it combine PCSK9Reduced capability or enhancing, such as to the Leu of EGF-A318Implement the ammonia of different charges
Base acid is replaced, and compared with wild type EGF-A, only arginine displacement significantly increases EGF-A mutant and the binding ability of PCSK9
(Gu etc., Journal of Lipid Research.2013;54:3345-3357);The areas EGF-A Cys308Mutation (C308Y)
It is mutation type (Zhang etc., J Lipid the Res.2003 Oct in China and Polish hypercholesterolemiapatients patients;44
(10):1850-8;- Miarka etc., Mol Biol Rep.2012 May;39(5):5181-6.), have and combine
The ability of PCSK9.Chinese patent discloses the cascade of C308Y and other mutational sites, and solid preparing the high courage for the treatment of
Purposes in alcoholemia.But we are recently, it has been found that the areas EGF-A Cys308Another mutation type (C308T) also have notable combination
The ability of PCSK9;We it has further been found that, EGF-B or EGF-B mutant can be significantly increased comprising (C308T) or other prominent
Become the ability of the EGF-A mutant combinations PCSK9 in site, the LDLR degradations for inhibiting PCSK9 to mediate restore surface of hepatocytes LDLR
To the intake ability of LDL;And EGF-AB mutant is substantially better than wild type EGF-AB.
Invention content
The object of the present invention is to provide new EGF (AB) mutant and its with the soluble fusion protein of Fc, provided
Mutant is the 295th asparagine (Asn) in ldl receptor extracellular domain by serine (Ser) substitution (or displacement), the 308th
Position cysteine acid (Cys) is replaced by threonine (Thr) substitution (or displacement), the 318th leucine (Leu) by arginine (Arg)
(or displacement), the 334th isoleucine (Ile) replace (or displacement), the 346th leucine (Leu) by group by valine (Val)
The EGF-AB segments of substitution (or displacement) occur with upper amino acid for any one position of propylhomoserin (His) substitution (or displacement).
The present invention provides the amino acid sequence table of EGF-AB mutant such as SEQ ID NO:1、3、5、7、9、11、13、15、
17, described in 19,21;
EGF (AB) mutant fusion protein provided by the invention, be the EGF (AB) mutant in the following way with people
IgG Fc segment compositions:EGF (AB)-connection peptide-Fc or EGF (AB)-Fc;EGF (AB)-connection peptide-Fc-connection peptides-
EGF (AB) or EGF (AB)-Fc-EGF (AB);(EGF (AB)-connections peptide) n-Fc-(connection peptide-EGF (AB)) n or EGF
(AB) n-Fc-EGF (AB) n, wherein n takes 1~3 integer, connects peptide sequence and is:GGGSGGGSGGGSGGGS.Its particular sequence
It is as follows:
The amino acid of IgG1 Fc such as the SEQ ID NO in sequence table:Described in 23.
The amino acid of IgG2 Fc such as the SEQ ID NO in sequence table:Described in 25.
The amino acid of IgG3 Fc such as the SEQ ID NO in sequence table:Described in 27.
The amino acid of IgG4 Fc such as the SEQ ID NO in sequence table:Described in 29.
The amino acid of mEGF (AB) 15-Fc such as the SEQ ID NO in sequence table:Described in 31;
The amino acid of mEGF (AB) 16-Fc such as the SEQ ID NO in sequence table:Described in 33;
The amino acid of mEGF (AB) 19-Fc such as the SEQ ID NO in sequence table:Described in 35;
The amino acid of mEGF (AB) 52-Fc such as the SEQ ID NO in sequence table:Described in 37;
The amino acid of mEGF (AB) 55-Fc such as the SEQ ID NO in sequence table:Described in 39;
The amino acid of 2mEGF (AB) 55-Fc such as the SEQ ID NO in sequence table:Described in 41;
The amino acid of 2mEGF (AB) 56-Fc such as the SEQ ID NO in sequence table:Described in 43;
It is as follows to encode the above-mentioned fusion protein nucleotide sequence:
The nucleotide of IgG1 Fc such as the SEQ ID NO in sequence table:Described in 24.
The nucleotide of IgG2 Fc such as the SEQ ID NO in sequence table:Described in 26.
The nucleotide of IgG3 Fc such as the SEQ ID NO in sequence table:Described in 28.
The nucleotide of IgG4 Fc such as the SEQ ID NO in sequence table:Described in 30.
The nucleotide of mEGF (AB) 15-Fc such as the SEQ ID NO in sequence table:Described in 32;
The nucleotide of mEGF (AB) 16-Fc such as the SEQ ID NO in sequence table:Described in 34;
The nucleotide of mEGF (AB) 19-Fc such as the SEQ ID NO in sequence table:Described in 36;
The nucleotide of mEGF (AB) 52-Fc such as the SEQ ID NO in sequence table:Described in 38;
The nucleotide of mEGF (AB) 55-Fc such as the SEQ ID NO in sequence table:Described in 40;
The nucleotide of 2mEGF (AB) 55-Fc such as the SEQ ID NO in sequence table:Described in 42;
The nucleotide of 2mEGF (AB) 56-Fc such as the SEQ ID NO in sequence table:Described in 44;
Invention also provides the expression vector of above-mentioned nucleic acid sequence, can in the host cell transfected duplication table
It reaches.
Can be Chinese hamster ovary (CHO) cell containing the host cell for expressing above-mentioned mutant or its fusion protein
And its subbreed or human embryo kidney 293 cells and its subbreed.
The present invention also provides above-mentioned EGF-AB mutant or its fusion protein and prepare for treating hypercholesterolemia,
And the purposes by reducing the Other diseases be benefited by blood plasma PCSK9 is horizontal.
Description of the drawings
Fig. 1 EGF of the present invention (AB) polypeptides or fusion protein mediate PCSK9 the influence of LDLR degradations.1 is EGF (AB) wt
Group, 2 be EGF (AB) wt-Fc groups, and 3 be mEGF (AB) 55-Fc groups, and 4 be 2mEGF (AB) 55-Fc groups, and 5 be 2mEGF (AB) 56-Fc
Group.
The influence of Fig. 2 EGF of the present invention (AB) polypeptides or fusion protein to HepG2 cellular uptakes LDL.
Specific implementation mode
Following embodiment is provided to be further explained the present invention.It should be appreciated that these embodiments are merely to illustrate this hair
It is bright rather than have any restrictions to the present invention.Those skilled in the art under the enlightenment of this specification to the present invention implement in made by
Any variation will all be fallen within the scope of the appended claims.
The preparation of 1 polypeptide of embodiment and its fusion protein
1. plasmid construction
1.1 genes and primer synthesis
Expressed sequence is recombinated by the gene and primer of following synthesis to expression vector.Gene and primer are given birth to by Shanghai life work
Object Engineering Co., Ltd synthesizes, and synthetic gene sequence recombinates into plasmid vector pUC19, be named as pUC19-mEGF (A) 16,
pUC19-EGF(AB)wt、pUC19-mEGF(AB)15、pUC19-mEGF(AB)16、pUC19-mEGF(AB)19、pUC19-mEGF
(AB)52、pUC19-mEGF(AB)55、pUC19-2mEGF(AB)55、pUC19-2mEGF(AB)56。
Synthesize fusion protein gene fraction:
EGF(A)wt:
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAG
TCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTGCAATGACCTTAAGATCGGCTAC
GAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCCACCTCCTCCACCACCTCTGATTAATTAA
GCG
mEGF(A)16:
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAG
TCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTACAATGACCTTAAGATCGGCTAC
GAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGC
EGF(AB)wt:
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAG
TCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTGCAATGACCTTAAGATCGGCTAC
GAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATATCGATGAGTGTCAGGATCCCGA
CACCTGCAGCCAGCTCTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCC
ACACGAAGGCCTGCAAGCCACCTCCTCCACCACCTCTGATTAATTAAGCG
mEGF(AB)15:
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAG
TCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCACCAATGACCTTAAGATCGGCTAC
GAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATGTCGATGAGTGTCAGGATCCCGA
CACCTGCAGCCAGCACTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCC
ACACGAAGGCCTGCAAG
mEGF(AB)16:
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAG
TCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTACAATGACCTTAAGATCGGCTAC
GAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATGTCGATGAGTGTCAGGATCCCGA
CACCTGCAGCCAGCACTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCC
ACACGAAGGCCTGCAAG
mEGF(AB)19:
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAG
TCGACTCTGGGACCAGCGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCACCAATGACCTTAAGATCGGCTAC
GAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATGTCGATGAGTGTCAGGATCCCGA
CACCTGCAGCCAGCACTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCC
ACACGAAGGCCTGCAAG
mEGF(AB)52:
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAG
TCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCACCAATGACCTTAAGATCGGCTAC
GAGTGCCGGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATGTCGATGAGTGTCAGGATCCCGA
CACCTGCAGCCAGCACTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCC
ACACGAAGGCCTGCAAG
mEGF(AB)55:
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAG
TCGACTCTGGGACCAGCGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCACCAATGACCTTAAGATCGGCTAC
GAGTGCCGGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATGTCGATGAGTGTCAGGATCCCGA
CACCTGCAGCCAGCACTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCC
ACACGAAGGCCTGCAAG
Synthetic primer:
P367F:GCAGATCTCCTAGGGCCACCATGGCCTGGATGAT
P367R:TACCGCCACCTCCGGACCCTCCGCCACCCTTGCAGGCCTT
P369F:AGGAGGCGGATCCGGTGGAGGCGGATCCGGGACCAGCGAATGCTT
P369R:ATCCGCCTCCTCCGGAGCCGCCACCGCCCTTGCAGGCCTTCGT
P370F:TCCGGAGGTGGCGGTAGCGGCGGTGGGGGATCCGCTGAGTCCAAGTATG
P370R1:CGCTTAATTAATCAGCCCAGGCTCAGGGA
P370R2:GATCCGCCTCCTCCGGAGCCGCCACCGCCGCCCAGGCTCAGGGACA
P371R:CGCTTAATTAATCACTTGCAGGCCTTCGT
The amplification of 1.2 genetic fragments
It is expanded by PCR method using special primer and obtains partial gene fragments.PCR reaction systems 1 (50 μ l of total volume):
0.5 μ l of Phusion enzymes, 5 × Buffer, 10 μ l, 1 dNTP μ l, 1 μ l of primer, 1 μ l of template, are finally mended with distilled water to 50 μ l;
Reaction condition:98 DEG C of pre-degeneration 30s, 98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 30s, 30 cycles, 72 DEG C of extension 5min.Gene outcome
It is detected with agarose gel electrophoresis.
1 primer of table and Template Information 1
It is expanded by PCR method using special primer and obtains target gene fragment.PCR reaction systems 2 (50 μ l of total volume):
0.5 μ l of Phusion enzymes, 5 × Buffer, 10 μ l, 1 dNTP μ l, 1 μ l of primer, 1 μ l of template, are finally mended with distilled water to 50 μ l;
Reaction condition:98 DEG C of pre-degeneration 30s, 98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 60s, 30 cycles, 72 DEG C of extension 5min.Gene outcome
It is detected with agarose gel electrophoresis.
2 primer of table and Template Information 2
The digestion of 1.3 carriers and genetic fragment is handled
To pUC19-mEGF (A) wt plasmids, pUC19-mEGF (AB) wt plasmids, pCHO1.0 plasmids and mEGF (A) 16-Fc,
mEGF(AB)wt-Fc、mEGF(AB)15-Fc、mEGF(AB)16-Fc、mEGF(AB)19-Fc、mEGF(AB)52-Fc、mEGF
(AB) 55-Fc, 2mEGF (AB) 55-Fc and 2mEGF (AB) 56-Fc target gene fragments carry out double digestion processing, digestion body respectively
It ties up in 1.5ml centrifuge tubes and establishes following ingredient:40 μ l of above-mentioned plasmid or target gene fragment, 10 Cutsmart μ l, Avr II
With each 5 μ l of Pac I (A/P), 50 μ l of aqua sterilisa, after mixing 37 DEG C of reactions overnight, utilize the recycling of QIAGEN Product Purification Kits.
The connection of 1.4 recombinant plasmids converts
Under the action of T4 ligases, plasmid pCHO1.0 (Avr II and Pac I, A/ that above-mentioned digestion recycling is obtained
P) respectively with the mEGF of digestion (A) wt (A/P), mEGF (AB) wt (A/P), mEGF (A) 16-Fc (A/P), mEGF (AB) wt-Fc
(A/P)、mEGF(AB)15-Fc(A/P)、mEGF(AB)16-Fc(A/P)、mEGF(AB)19-Fc(A/P)、mEGF(AB)52-Fc
(A/P), mEGF (AB) 55-Fc (A/P), 2mEGF (AB) 55-Fc (A/P) and 2mEGF (AB) 56-Fc (A/P) genetic fragment connect
It connects.Following system is established in connection reaction in 1.5mlEP pipes:1 μ l of pCHO1.0 (A/P), digestion target gene fragment distinguish 7 μ l,
10 × T4 Buffer, 1 μ l, 1 μ l of T4 DNA ligase react 4h or more, connection production after mixing under room temperature (20 DEG C or so)
Object is converted into Top10 competent escherichia coli cells, is applied on 2YT (AMP) plating medium, 37 DEG C of constant incubators are static
It is incubated overnight, tablet number is as follows:
pCHO-EGF(A)wt、pCHO-EGF(AB)wt、pCHO-mEGF(A)16-Fc、pCHO-mEGF(AB)wt-Fc、
pCHO-mEGF(AB)15-Fc、pCHO-mEGF(AB)16-Fc、pCHO-mEGF(AB)19-Fc、pCHO-mEGF(AB)52-Fc、
PCHO-mEGF (AB) 55-Fc, pCHO-2mEGF (AB) 55-Fc and pCHO-2mEGF (AB) 56-Fc.
The bacterium colony PCR screenings of 1.5 recombinant plasmids
The several recombination single bacterium colonies of picking carry out PCR identifications respectively after culture from above-mentioned bacterium colony.Bacterium solution PCR reactants
It is (20 μ l of total volume):It is 2 × Taq HS, 10 μ l, 3 μ l of bacterium solution template, each 0.5 μ l of upstream and downstream primer (P367F and 367R), last
It is mended to 20 μ L, reaction condition with distilled water:95 DEG C of 3min, 94 DEG C of 60s, 53 DEG C of 60s, 72 DEG C of 30s, 30 cycles;72 DEG C of extensions
5min.Agarose gel electrophoresis identification and analysis result.
The digestion of 1.6 recombinant plasmids is identified
By carrying out digestion identification again after bacterium colony PCR identifications correctly.The plasmid extraction for carrying out recombinant bacterium first, then carries out
Digestion, digestion system are that following ingredient is added in 1.5ml EP pipes:7 μ l of plasmid, 1 Cutsmart μ l, Avr II and Pac I
(A/P) each 0.5 μ l, mend aqua sterilisa to 10 μ l, react 4h at 37 DEG C after mixing.Agarose gel electrophoresis analysis result.
The sequencing of 1.7 recombinant plasmids is identified
It will identify that correct recombinant plasmid is sent again to genome company by bacterium colony PCR and digestion and carry out sequencing identification.Through mirror
After sequencing row are correct, expression plasmid is largely extracted using QIAGEN Plasmid Midi Kit, and save backup.
1.8 plasmid transfections and cell screening
Using host cell CHO-S or DG44, according to FreedomTM CHO-STMKit kit specifications carry out respectively
Plasmid transfection.The cell for being transferred to plasmid is respectively placed in shaking flask culture (37 DEG C, 5%CO2, 110rpm/min) to 48h, cell count
Instrument detects Cell viability.
Gradient pressurization screening is carried out after transfecting 48h:10P/100M, 20P/200M (P=10 μ g/mL Puromycin, M=
nM MTX);30P/500M, 50P/1000M obtain initial screening cell;Monoclonal screening, testing goal are carried out with limiting dilution assay again
Protein expression level screening obtains preferred clone, and expands culture.
1.9 Protein expression and purification
1.9.1 fusion protein expression purifies
1) recombination fusion protein affinity capture:Recombination fusion protein is captured using Mabselect SuRe filled columns.Specifically
Step is:Optimization clone is enlarged culture, cell conditioned medium is collected by centrifugation, and removal of impurities is filtered off through 0.45 μm of bellows filter
Matter.Chromatographic column is connected with AKTA tomographic systems, mobile phase A is:20mM phosphate, sodium chloride containing 150mM, pH 7.2;Flowing
Phase B is:200mM glycine hydrochlorides, pH 3.2;Flow velocity 10ml/min, Detection wavelength 214nm and 280nm.It is first flat with mobile phase A
Weigh affinity column 5-10CV, then carries out loading, and destination protein is incorporated on affinity media,.After end of the sample, using mobile phase A
Cleaning chromatographic column 3-5CV is carried out, finally its destination protein is eluted with Mobile phase B, collects eluent 1M Tris (pH
9.0) pH value is adjusted to neutrality;Elution samples concentration is changed into liquid to 20mM Tris-HCl buffer solutions, pH7.4.
2) recombination fusion protein polishing purification:Using the consummate recombination of Sepharose High Performance Q filled columns
Fusion protein.The specific steps are:Chromatographic column is connected with AKTA tomographic systems.Mobile phase A is 20mM Tris-HCl, pH7.4;
Mobile phase B is A+0.035M sodium chloride, pH7.4;Mobile phase C is A+0.215M sodium chloride, pH7.4;Flow velocity 10ml/min, detection
Wavelength 214nm and 280nm.Chromatographic column 5-10CV first is balanced with mobile phase A, is rushed by affinity elution sample loading, then with buffer solution B
3CV is washed, buffer solution C is eluted, and elution fraction is recombination fusion protein.
1.9.2 recombinant polypeptide expression and purification
1) the affine capture of recombinant polypeptide:Recombination fusion protein is captured using Ni Sepharose 6FF filled columns.Specific step
Suddenly it is:Optimization clone is enlarged culture, cell supernatant is collected by centrifugation and is filtered with 0.45 μm of bellows filter.By chromatographic column
It is connected with AKTA tomographic systems;Mobile phase A is:20mM phosphate, sodium chloride containing 350mM, 20mM imidazoles, pH 7.4;Mobile phase B
For:A+500mM imidazoles;Flow velocity 10mL/min, Detection wavelength 214nm and 280nm.Affinity column 5-10CV first is balanced with mobile phase A,
Loading is carried out to the cell supernatant collected, destination protein is incorporated on medium,.After end of the sample, carried out using mobile phase A
Chromatographic column 3-5CV is cleaned, finally its destination protein is eluted with Mobile phase B.Liquid is changed into the concentration of collected elution samples
To 20mM Tris-HCl buffer solutions, pH7.4.
2) recombinant polypeptide polishing purification:Using the consummate recombination fusion of Sepharose High Performance Q filled columns
Albumen.The specific steps are:Chromatographic column is connected with AKTA tomographic systems.Mobile phase A is 20mM Tris-HCl, pH7.4;Flowing
Phase B is A+0.02M sodium chloride, pH7.4;Mobile phase C is A+0.185M sodium chloride, pH7.4;Flow velocity 10mL/min, Detection wavelength
214nm and 280nm.Chromatographic column 5-10CV first is balanced with mobile phase A, 3CV is rinsed by affine sample loading, then with buffer solution B, delays
Fliud flushing C is eluted, and elution fraction is recombinant polypeptide.
Embodiment 2 recombinates EGF (AB) mutant and its fusion protein and PCSK9 affinity
The binding affinity of recombinant polypeptide or fusion protein and PCSK9 is measured by biomembrane interferometry in Otect QK
(Fortebio) it is measured on.SA sensor sensors (Fortebio, article No. 18-5063) are cured in containing 0.5%BSA and 1mM
CaCl27.4 buffer solutions of TrisHCl pH in PCSK9, washed in same buffer, and be transferred into and contain
In the hole for having a concentration of 0~1 μM of EGF (A/AB) polypeptides and its fusion protein in same buffer.It will be directed to and only contain
The signal in the reference hole of buffer solution is subtracted from all combination data.Affinity KD uses Octet by being fitted to steady-state algorithm
Software obtains, and data are indicated with mean ± SD.
The binding kinetics parameter being summarised in table 3 is shown:Compared with EGF (A) wt polypeptides, mEGF (A) 16-Fc, EGF
(AB) wt, EGF (AB) wt-Fc and PCS and the binding affinity of EGF (AB) mutant fusion proteins and PCSK9 obviously increase;
Wherein mEGF (AB) 55-Fc, 2mEGF (AB) 55-Fc, 2mEGF (AB) 56-Fc are opposite with the affinity of PCSK9 best.Illustrate this
There is provided EGF (AB) mutant fusion protein of invention can increase the affinity of EGF (A) and PCSK9 to some extent.
3 EGF of table (A/AB) polypeptides and EGF (A/AB) mutant fusion proteins are detected with PCSK9 affinity
Embodiment 3 recombinates EGF (AB) mutant and its combination of fusion protein Competitive assays PCSK9 and LDLR
The activity that recombinant polypeptide or fusion protein inhibit PCSK9 to be combined with LDLR is detected by competitive ELISA.Specifically,
The recombined human LDLR (R&D companies) of 1 μ g/ml is coated in 96 hole elisa Plates with carbonate buffer solution (pH9.6), 4 DEG C were incubated
Night;By the PCSK9 of 10nM biotin labelings and the EGF polypeptide mutants of different molar concentrations or fusion protein (0.046-
8100nM) isometric mixing is incubated at room temperature 1h;Then it adds in the ELISA Plate of coating recombined human LDLR in advance, 37 DEG C of incubation 2h;
The streptavidin that lotus root is associated with horseradish peroxidase detects the biotin labeling PCSK9 for being incorporated into LDLR;Calculate the mutation of EGF polypeptides
Medium effective concentration (the IC that body or fusion protein Competitive assays PCSK9 are combined with LDLR50).Test result is shown in Table 4, and the present invention carries
EGF (AB) mutant fusion protein of confession can obviously inhibit the combination of PCSK9 and LDLR, wherein mEGF (AB) 55-Fc, 2mEGF
(AB) 55-Fc, 2mEGF (AB) 56-Fc effects are preferable.
The combination of 4 EGF mutant of table and its fusion protein Competitive assays PCSK9 and LDLR detect
3 EGF of table (A/AB) polypeptides and EGF (A/AB) mutant fusion protein inhibit the combination of PCSK9 and LDLR
Embodiment 3 recombinates the influence of EGF (AB) mutant and its fusion protein to PCSK9 degradation LDLR effects
Human liver cancer cell HepG2 is cultivated to exponential phase, and it is primary to wash cell with PBS, and it is outstanding that cell is made through pancreatin digestion
Liquid and adjust cell density be 1 × 105Cells/ml, inoculating cell in 48 well culture plates (500 holes μ l/),.In 37 DEG C, 5%CO2
Continue culture for 24 hours.Cells and supernatant is removed, 500 μ l serum free mediums are added per hole, continues to cultivate 16h;It discards supernatant,
Be added PCSK9 (25 μ g/ml of final concentration), while be separately added into different molar concentrations recombinant polypeptide or fusion protein (0.001~
2 μM), in 37 DEG C of 5%CO2It is incubated 4h;It addsAfter LDL (final concentration of 6 μ g/ml) is incubated 3h, discard
Clearly, cell is rinsed 3 times with PBS.200 μ l isopropanols, 37 DEG C of incubation 30min are added, fully extract cellular uptakeLDL is shifted in 150 μ l extract liquors to 96 black ELISA Plates, measures fluorescence intensity, excitation wavelength 488nm, inspection
Survey wavelength 520nm.
Separately take cell with 1 × 104Cells/ml, inoculating cell is in laser co-focusing Tissue Culture Dish (holes 1ml/).Set training
Plate is supported in 37 DEG C, 5%CO2In incubator, continue culture for 24 hours.Serum free medium is changed to continue to cultivate 16h;PCSK9 is added
(25 μ g/ml of final concentration), while it being separately added into EGF (AB) polypeptides or fusion protein (0.01~1 μM), it is placed in 37 DEG C of 5%CO2Training
It supports case to be incubated for 24 hours, be rinsed with PBS, harvest cell, the expression or degradation of LDL-R are observed using immunofluorescence technique, and carry out glimmering
Luminous intensity quantitative analysis.
Experimental result is shown, obviously inhibits PCSK9 with EGF provided by the present invention (AB) polypeptide mutant fusion protein
The LDLR of mediation degrades, and inhibiting effect is substantially better than EGF (AB) wild polypeptides and its fusion protein;At the same time, EGF
(AB) the LDL intakes that polypeptide mutant fusion protein also can be caused by antagonism PCSK9 are reduced, hence it is evident that are increased liver cell and taken the photograph to LDL
Taken amount, effect are also significantly better than EGF (AB) wild polypeptides and its fusion protein.Polypeptide and fusion protein half-inhibition concentration
(IC50) it is respectively:(EGF (AB) wt, 1196 white half-inhibition concentrations), (EGF (AB) wt-Fc, 707.0.0-Fc), (mEGF
(AB)55-Fc,498.5±19.28nM)、(2mEGF(AB)55-Fc,276.3±14.51nM)、(2mEGF(AB)56-Fc,
241.1±12.74nM)。
Embodiment 4 recombinates the half-life period of EGF (AB) mutant and its fusion protein in rat body
Healthy male SD rat is taken, EGF (AB) wt, EGF (AB) wt-Fc, mEGF (AB) 55-Fc, 2mEGF is randomly divided into
(AB) 55-Fc, 2mEGF (AB) 56-Fc groups, every group 5.Each group rat is subcutaneously injected according to weight and gives relative medicine respectively
(5mg/kg).Different time points orbital vein takes 250 μ l of blood before administration and after administration, prepares serum, and ELISA method detects blood
The content of each albumen in clear.
By table 4 the results show that rat skin lower injection with EGF (AB) polypeptides and its mutant fusion protein of dosage after, this
Invention EGF (AB) mutant fusion protein half-life period is obviously prolonged compared with the half-life period of EGF (AB) wild polypeptide, and 2mEGF
(AB) half-life period of 56-Fc is 71.6h.
The metabolizing parameters of 4 EGF of table (AB) and its mutant fusion protein in rat blood compare
SEQUENCE LISTING
<110>The Chengdu bio tech ltd Bei Aite
<120>The combination mutant of new EGF-A and B prepares and its application in bio-pharmaceutical
<130> 0
<160> 44
<170> PatentIn version 3.3
<210> 1
<211> 80
<212> PRT
<213>Artificial sequence
<400> 1
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Cys
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
<210> 2
<211> 240
<212> DNA
<213>Artificial sequence
<400> 2
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtctgcaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
<210> 3
<211> 80
<212> PRT
<213>Artificial sequence
<400> 3
Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
<210> 4
<211> 240
<212> DNA
<213>Artificial sequence
<400> 4
gggaccaacg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
<210> 5
<211> 80
<212> PRT
<213>Artificial sequence
<400> 5
Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Tyr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
<210> 6
<211> 240
<212> DNA
<213>Artificial sequence
<400> 6
gggaccaacg aatgcttgga caacaacggc ggctgttccc acgtctacaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
<210> 7
<211> 80
<212> PRT
<213>Artificial sequence
<400> 7
Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Cys
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
<210> 8
<211> 240
<212> DNA
<213>Artificial sequence
<400> 8
gggaccaacg aatgcttgga caacaacggc ggctgttccc acgtctgcaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
<210> 9
<211> 80
<212> PRT
<213>Artificial sequence
<400> 9
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
<210> 10
<211> 240
<212> DNA
<213>Artificial sequence
<400> 10
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
<210> 11
<211> 80
<212> PRT
<213>Artificial sequence
<400> 11
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Tyr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
<210> 12
<211> 240
<212> DNA
<213>Artificial sequence
<400> 12
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtctacaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
<210> 13
<211> 80
<212> PRT
<213>Artificial sequence
<400> 13
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Cys
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
<210> 14
<211> 240
<212> DNA
<213>Artificial sequence
<400> 14
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtctgcaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
<210> 15
<211> 80
<212> PRT
<213>Artificial sequence
<400> 15
Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
<210> 16
<211> 240
<212> DNA
<213>Artificial sequence
<400> 16
gggaccaacg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
<210> 17
<211> 80
<212> PRT
<213>Artificial sequence
<400> 17
Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Tyr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
<210> 18
<211> 240
<212> DNA
<213>Artificial sequence
<400> 18
gggaccaacg aatgcttgga caacaacggc ggctgttccc acgtctacaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
<210> 19
<211> 80
<212> PRT
<213>Artificial sequence
<400> 19
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
<210> 20
<211> 240
<212> DNA
<213>Artificial sequence
<400> 20
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
<210> 21
<211> 80
<212> PRT
<213>Artificial sequence
<400> 21
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Tyr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
<210> 22
<211> 240
<212> DNA
<213>Artificial sequence
<400> 22
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtctacaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
<210> 23
<211> 227
<212> PRT
<213>Artificial sequence
<400> 23
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 24
<211> 681
<212> DNA
<213>Artificial sequence
<400> 24
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540
gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 600
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660
ctctccctgt ctccgggtaa a 681
<210> 25
<211> 228
<212> PRT
<213>Artificial sequence
<400> 25
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
1 5 10 15
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45
His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
50 55 60
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
65 70 75 80
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
85 90 95
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro
100 105 110
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
130 135 140
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
145 150 155 160
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
180 185 190
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220
Ser Pro Gly Lys
225
<210> 26
<211> 684
<212> DNA
<213>Artificial sequence
<400> 26
gagcgcaaat gttgtgtcga gtgcccaccg tgcccagcac cacctgtggc aggaccgtca 60
gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120
acgtgcgtgg tggtggacgt gagccacgaa gaccccgagg tccagttcaa ctggtacgtg 180
gacggcgtgg aggtgcataa tgccaagaca aagccacggg aggagcagtt caacagcacg 240
ttccgtgtgg tcagcgtcct caccgttgtg caccaggact ggctgaacgg caaggagtac 300
aagtgcaagg tctccaacaa aggcctccca gcccccatcg agaaaaccat ctccaaaacc 360
aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 420
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 480
gagtgggaga gcaatgggca gccggagaac aactacaaga ccacacctcc catgctggac 540
tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660
agcctctccc tgtctccggg taaa 684
<210> 27
<211> 264
<212> PRT
<213>Artificial sequence
<400> 27
Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys
1 5 10 15
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
20 25 30
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala
35 40 45
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
50 55 60
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
65 70 75 80
Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val
85 90 95
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
100 105 110
Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln
115 120 125
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
130 135 140
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
145 150 155 160
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
165 170 175
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
180 185 190
Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr
195 200 205
Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
210 215 220
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe
225 230 235 240
Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys
245 250 255
Ser Leu Ser Leu Ser Pro Gly Lys
260
<210> 28
<211> 792
<212> DNA
<213>Artificial sequence
<400> 28
gagctcaaaa ccccacttgg tgacacaact cacacatgcc cacggtgccc agagcccaaa 60
tcttgtgaca cacctccccc gtgcccacgg tgcccagagc ccaaatcttg tgacacacct 120
cccccatgcc cacggtgccc agcacctgaa ctcctgggag gaccgtcagt cttcctcttc 180
cccccaaaac ccaaggatac ccttatgatt tcccggaccc ctgaggtcac gtgcgtggtg 240
gtggacgtga gccacgaaga ccccgaggtc cagttcaagt ggtacgtgga cggcgtggag 300
gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgtt ccgtgtggtc 360
agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 420
tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaaaccaa aggacagccc 480
cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 540
agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 600
agcgggcagc cggagaacaa ctacaacacc acgcctccca tgctggactc cgacggctcc 660
ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacatcttc 720
tcatgctccg tgatgcatga ggctctgcac aaccgcttca cgcagaagag cctctccctg 780
tctccgggta aa 792
<210> 29
<211> 229
<212> PRT
<213>Artificial sequence
<400> 29
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
1 5 10 15
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
20 25 30
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
35 40 45
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
50 55 60
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
65 70 75 80
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
85 90 95
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
100 105 110
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
115 120 125
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
130 135 140
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
145 150 155 160
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
165 170 175
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
180 185 190
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
195 200 205
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220
Ser Leu Ser Leu Gly
225
<210> 30
<211> 687
<212> DNA
<213>Artificial sequence
<400> 30
gctgagtcca agtatggccc tccctgccct ccttgccctg ctcctgaggc tgctggaggc 60
cctagcgtgt tcctgttccc ccctaagcct aaggacaccc tgatgatttc ccggaccccc 120
gaggtgacct gtgtggtggt ggatgtgtcc caggaggacc ctgaagtgca gttcaactgg 180
tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggaaga gcagttcaac 240
agcacctaca gggtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 300
gagtacaagt gcaaggtgag caataagggc ctgccctcct ccatcgagaa gaccatttcc 360
aaggccaagg gccagcccag ggaaccccag gtgtacaccc tccctcccag ccaggaggag 420
atgaccaaga accaggtgtc cctgacctgc ctggtgaaag gcttctaccc ctccgacatt 480
gccgtcgagt gggaaagcaa cggccagccc gagaacaatt acaagaccac accccccgtg 540
ctggacagcg atggcagctt tttcctgtac tccaggctga ccgtcgacaa gtccaggtgg 600
caggagggca acgtcttctc ctgctccgtg atgcatgagg ccctgcacaa ccactacacc 660
cagaagtccc tgtccctgag cctgggc 687
<210> 31
<211> 324
<212> PRT
<213>Artificial sequence
<400> 31
Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly
<210> 32
<211> 975
<212> DNA
<213>Artificial sequence
<400> 32
gggaccaacg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gctga 975
<210> 33
<211> 324
<212> PRT
<213>Artificial sequence
<400> 33
Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Tyr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly
<210> 34
<211> 975
<212> DNA
<213>Artificial sequence
<400> 34
gggaccaacg aatgcttgga caacaacggc ggctgttccc acgtctacaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gctga 975
<210> 35
<211> 324
<212> PRT
<213>Artificial sequence
<400> 35
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly
<210> 36
<211> 975
<212> DNA
<213>Artificial sequence
<400> 36
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gctga 975
<210> 37
<211> 324
<212> PRT
<213>Artificial sequence
<400> 37
Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly
<210> 38
<211> 975
<212> DNA
<213>Artificial sequence
<400> 38
gggaccaacg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gctga 975
<210> 39
<211> 324
<212> PRT
<213>Artificial sequence
<400> 39
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly
<210> 40
<211> 975
<212> DNA
<213>Artificial sequence
<400> 40
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gctga 975
<210> 41
<211> 419
<212> PRT
<213>Artificial sequence
<400> 41
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr Asn
100 105 110
Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln Leu
115 120 125
Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro Asp
130 135 140
Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys Gln
145 150 155 160
Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys Gly
165 170 175
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
180 185 190
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
195 200 205
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
210 215 220
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
225 230 235 240
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
245 250 255
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
260 265 270
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
275 280 285
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
290 295 300
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
305 310 315 320
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
325 330 335
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
340 345 350
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
355 360 365
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
370 375 380
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
385 390 395 400
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
405 410 415
Ser Leu Gly
<210> 42
<211> 1260
<212> DNA
<213>Artificial sequence
<400> 42
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggcggtggcg gctccggagg aggcggatcc ggtggaggcg gatccgggac cagcgaatgc 300
ttggacaaca acggcggctg ttcccacgtc accaatgacc ttaagatcgg ctacgagtgc 360
cggtgccccg acggcttcca gctggtggcc cagcgaagat gcgaagatgt cgatgagtgt 420
caggatcccg acacctgcag ccagcactgc gtgaacctgg agggtggcta caagtgccag 480
tgtgaggaag gcttccagct ggacccccac acgaaggcct gcaagggtgg cggagggtcc 540
ggaggtggcg gtagcggcgg tgggggatcc gctgagtcca agtatggccc tccctgccct 600
ccttgccctg ctcctgaggc tgctggaggc cctagcgtgt tcctgttccc ccctaagcct 660
aaggacaccc tgatgatttc ccggaccccc gaggtgacct gtgtggtggt ggatgtgtcc 720
caggaggacc ctgaagtgca gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc 780
aagaccaagc cccgggaaga gcagttcaac agcacctaca gggtggtgag cgtgctgacc 840
gtgctgcacc aggactggct gaacggcaaa gagtacaagt gcaaggtgag caataagggc 900
ctgccctcct ccatcgagaa gaccatttcc aaggccaagg gccagcccag ggaaccccag 960
gtgtacaccc tccctcccag ccaggaggag atgaccaaga accaggtgtc cctgacctgc 1020
ctggtgaaag gcttctaccc ctccgacatt gccgtcgagt gggaaagcaa cggccagccc 1080
gagaacaatt acaagaccac accccccgtg ctggacagcg atggcagctt tttcctgtac 1140
tccaggctga ccgtcgacaa gtccaggtgg caggagggca acgtcttctc ctgctccgtg 1200
atgcatgagg ccctgcacaa ccactacacc cagaagtccc tgtccctgag cctgggctga 1260
<210> 43
<211> 419
<212> PRT
<213>Artificial sequence
<400> 43
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
325 330 335
Gly Gly Ser Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser
340 345 350
His Val Thr Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp
355 360 365
Gly Phe Gln Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys
370 375 380
Gln Asp Pro Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly
385 390 395 400
Tyr Lys Cys Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys
405 410 415
Ala Cys Lys
<210> 44
<211> 1260
<212> DNA
<213>Artificial sequence
<400> 44
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gcggcggtgg cggctccgga ggaggcggat ccggtggagg cggatccggg 1020
accagcgaat gcttggacaa caacggcggc tgttcccacg tcaccaatga ccttaagatc 1080
ggctacgagt gccggtgccc cgacggcttc cagctggtgg cccagcgaag atgcgaagat 1140
gtcgatgagt gtcaggatcc cgacacctgc agccagcact gcgtgaacct ggagggtggc 1200
tacaagtgcc agtgtgagga aggcttccag ctggaccccc acacgaaggc ctgcaagtga 1260
Claims (9)
1. EGF (AB) mutant of new combination PCSK9, by appointing in 294-319, areas ldl receptor extracellular domain EGF-A
Arbitrary amino acid substitution (or displacement) in meaning amino acid and 333-347, the areas EGF-B obtains.
2. EGF (AB) mutant described in claim 1, which is characterized in that the asparagus fern in the 295th, the areas LDLR extracellular domain EGF-A
Amide (Asn) replaces (N295S) by serine (Ser), and the 308th cysteine acid (Cys) is by threonine (Thr) or tyrosine
(Tyr) replace (C308T or C308Y), the 318th leucine (Leu) replaces (L318R) by arginine (Arg);Extracellular domain EGF-
The 334th, the areas B isoleucine (Ile) replaces (I334V) by valine (Val), and the 346th leucine (Leu) is by histidine
(His) replace (L346H).
3. EGF (AB) mutant described in claim 2, which is characterized in that by forming selected from following any form:
N295S-I334V-L346H;
C308T-I334V-L346H or C308Y-I334V-L346H;
L318R-I334V-L346H;
N295S-C308T-I334V-L346H or N295S-C308Y-I334V-L346H;
N295S-L318R-I334V-L346H;
C308T-L318R-I334V-L346H or C308Y-L318R-I334V-L346H;
N295S-C308T-L318R-I334V-L346H or N295S-C308Y-L318R-I334V-L346H.
4. EGF (AB) mutant described in claim 1, by selected from any one of following amino acid sequence:
a)SEQ ID NO:1
GTSECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLD
PHTKACK
b)SEQ ID NO:3
GTNECLDNNGGCSHVTNDLKIGYECLCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLD
PHTKACK
c)SEQ ID NO:5
GTNECLDNNGGCSHVYNDLKIGYECLCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLD
PHTKACK
d)SEQ ID NO:7
GTNECLDNNGGCSHVCNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLD
PHTKACK
e)SEQ ID NO:9
GTSECLDNNGGCSHVTNDLKIGYECLCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLD
PHTKACK
f)SEQ ID NO:11
GTSECLDNNGGCSHVYNDLKIGYECLCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLD
PHTKACK
g)SEQ ID NO:13
GTSECLDNNGGCSHVCNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLD
PHTKACK
h)SEQ ID NO:15
GTNECLDNNGGCSHVTNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLD
PHTKACK
i)SEQ ID NO:17
GTNECLDNNGGCSHVYNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLD
PHTKACK
j)SEQ ID NO:19
GTSECLDNNGGCSHVTNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLD
PHTKACK
k)SEQ ID NO:21
GTSECLDNNGGCSHVYNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLD
PHTKACK。
5. the nucleotide sequence of EGF (AB) mutant described in claim 4 (a-k) is encoded, such as SEQ ID NO:2、4、6、8、
10、12、14、16、、18、20、22。
6. the fusion protein of any one EGF (AB) mutant and human immunoglobulin(HIg) Fc segments described in claim 4 (a-k),
It is characterized in that EGF (AB) mutant is connect with Fc segments by connection peptide appropriate or is directly connect with Fc segments, choosing
From the connection type being made up of:
EGF (AB)-connections peptide-Fc
Or:EGF(AB)—Fc
EGF (AB)-connection peptide-Fc-connection peptides-EGF (AB)
Or:EGF(AB)—Fc—EGF(AB)
[EGF (AB)] n-connection peptide-Fc-connection peptides-[EGF (AB)] n
Or:EGF(AB)n—Fc—EGF(AB)n
Wherein, n takes 1~3 integer, connection peptide to be made of 3-16 amino acid.
7. the fusion protein of EGF (AB) mutant and human immunoglobulin(HIg) Fc segments described in claim 4, it is characterized in that described
Immunoglobulin be by the Fc segments selected from human IgG1-4.
8. the method for preparing genetic engineering EGF (AB) mutant fusion protein, it is characterised in that the method includes such as lower part
Point:
A) using human liver cell as template, EGF (AB) gene of human ldl receptor is obtained;
B) method for synthesizing gene (amino acid substitution or method of replacing) is used
C) eukaryotic expression vector is built, it includes any one of described EGF (AB) mutant genes or at least one connection Fc
Gene order;
D) expression plasmid is site-specific integrated into CHO cell line genome or 293 cell line genomes, expresses the EGF (AB) mutation
Body or the fusion protein for connecting Fc;
E) collect expression supernatant, through affinity capture or (and) ion-exchange chromatography obtains EGF (AB) mutant or it melts
Hop protein.
9. EGF (AB) mutant or its fusion protein described in claim 1-4 are being prepared for treating hypercholesterolemia, moving
The angiocardiopathies such as pulse atherosclerosis and the purposes by reducing the other diseases be benefited by blood plasma PCSK9 is horizontal.
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