Summary of the invention
The object of this invention is to provide a kind of fusion rotein, this fusion rotein is replaced/is suddenlyd change for the amino acid in EGF in LDLR (A) district and human IgG FC fusion rotein (representing with 3mEGF-FC), strengthen the keying action of EGF-FC and PCSK9, effectively suppress PCSK9 active.
Fusion rotein provided by the invention is realized by DNA recombinant technology or genetic engineering means, and expressed by the following formula:
X-connection peptides-Y-connection peptides-Z-connection peptides-IgG Fc fragment; Wherein, X, Y, Z are EGF (A) polypeptide mutant in people LDLR (low density lipoprotein receptor); Connection peptides is (GGGGS) n.
Fusion rotein provided by the invention, be connected to form fusion rotein by EGF (A) district's polypeptide mutant in people LDLR and human normal immunoglobulin Fc fragment, its concrete sequence is as follows:
The amino acid of EGF (A) polypeptide mutant is as described in the SEQ ID NO:1,3,5 in sequence table;
The amino acid of IgG1 Fc is as described in the SEQ ID NO:15 in sequence table;
The amino acid of IgG2 Fc is as described in the SEQ ID NO:17 in sequence table;
The amino acid of IgG3 Fc is as described in the SEQ ID NO:19 in sequence table;
The amino acid of IgG4 Fc is as described in the SEQ ID NO:21 in sequence table.
3mEGF-FC fusion rotein Nucleotide is as described in the SEQ ID NO:23 in sequence table.
Described above-mentioned fusion rotein nucleotide sequence of encoding is as follows:
The Nucleotide of EGF (A) is as described in the SEQ ID NO:2,4,6 in sequence table;
The Nucleotide of IgG1 Fc is as described in the SEQ ID NO:16 in sequence table;
The Nucleotide of IgG2 Fc is as described in the SEQ ID NO:18 in sequence table;
The Nucleotide of IgG3 Fc is as described in the SEQ ID NO:20 in sequence table;
The Nucleotide of IgG4 Fc is as described in the SEQ ID NO:22 in sequence table.
3mEGF-FC fusion rotein Nucleotide is as described in the SEQ ID NO:24 in sequence table.
Invention also provides the expression vector of above-mentioned nucleotide sequence, it can copy expression in transformed host cell.
Present invention also offers a kind of method preparing fusion rotein, above-mentioned expression vector is introduced suitable expression system, thus carry out the expression of fusion rotein.
Invention further provides a kind of pharmaceutical composition, it is made up of above-mentioned fusion rotein and pharmaceutically acceptable carrier or vehicle; The dosage form of described pharmaceutical composition is preferably injection, injection freeze-dried powder.
Present invention also offers above-mentioned fusion rotein and prepare the purposes in the disease medicaments such as prevention and therapy hypercholesterolemia, familial hypercholesterolemia, the metabolic disease that brought out by PCSK9.
The expression vector of fusion rotein of the present invention can be recombinant eukaryon expression vector, preferred mammal fibrocyte expression vector; Also can be recombinant virus expression vector, preferred adeno-associated virus or adenovirus carrier.
Containing expressing the host cell of above-mentioned fusion rotein, can be DG44, Chinese hamster ovary celI and subbreed thereof or 293 cells and subbreed thereof.
Embodiment
Following examples are provided to be further explained the present invention.Should be appreciated that these embodiments only have any restriction for illustration of the present invention to the present invention.Those skilled in the art under the enlightenment of this specification sheets to the invention process in any variation of doing all will drop in the scope of claims.
The preparation of embodiment 1 fusion rotein
1, plasmid construction
1.1 genes and primer synthesis
Expressed sequence by the gene of following synthesis and primer restructuring to expression vector.Gene and primer are synthesized by Beijing Jin Weizhi company of specialized company, and synthetic gene sequence is recombinated in plasmid vector pUC19, called after pUC19-EGFwt, pUC19-mEGF
14, pUC19-mEGF
16with pUC19-3M EGF.
Synthesis fusion protein gene fraction:
EGFwt:
ATCACCGGTATGGGGCCCTGGGGCTGGAAATTGCGCTGGACCGTCGCCTTGCTCCTCGCCGCGGCGGGGACTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTGCAATGACCTTAAGATCGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGGCGGCGGCGGCTCCGGAGGAGGAGGATCCGGAGGAGGAGGATCCGACAAAACTCACACATGCCCAC
mEGF
14:
ATCACCGGTATGGGGCCCTGGGGCTGGAAATTGCGCTGGACCGTCGCCTTGCTCCTCGCCGCGGCGGGGACTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCTACGTCTGCAATGACCTTAAGATCGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGGCGGCGGCGGCTCCGGAGGAGGAGGATCCGGAGGAGGAGGATCCGACAAAACTCACACATGCCCAC
mEGF
16:
ATCACCGGTATGGGGCCCTGGGGCTGGAAATTGCGCTGGACCGTCGCCTTGCTCCTCGCCGCGGCGGGGACTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTACAATGACCTTAAGATCGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGGCGGCGGCGGCTCCGGAGGAGGAGGATCCGGAGGAGGAGGATCCGACAAAACTCACACATGCCCAC
3mEGF:
CCTAGGGCCACCATGGGGCCCTGGGGCTGGAAATTGCGCTGGACCGTCGCCTTGCTCCTCGCCGCGGCGGGGACTGGGACCAGCGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTGCAATGACCTTAAGATCGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGGCGGCGGCGGCTCCGGAGGAGGAGGATCCGGAGGAGGAGGATCCGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTACAATGACCTTAAGATCGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGGCGGCGGCGGCTCCGGAGGAGGAGGATCCGGAGGAGGAGGATCCGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTGCAATGACCTTAAGATCGGCTACGAGTGCCCGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGGCGGCGGCGGCTCCGGAGGAGGAGGATCCGGAGGAGGAGGATCCGACAAAACTCACACATGCCCAC
Synthetic primer:
E1:5’-ATCCCTAGGGCCACCATGGGGCCCTGGGGCTGGAAATTGCGCTGGACCGTC-3’
E2:5’-GTGGGCATGTGTGAGTTTTGTCGGATCCT-3’
E3:5’-GACAAAACTCACACATGCCCACCGTGC-3’
E4:5’-GTCGTATACTCATTTACCCGGAGACAGGGA-3’
The amplification of 1.2 gene fragments
Special primer E1 and E2 is utilized to obtain partial gene fragments by PCR method amplification.PCR reaction system 1 (cumulative volume 50 μ l): 5 × Buffer 10 μ l, dNTP 2 μ l, primer 1 μ l (E1 and E2), template are respectively synthesis fragment pUC19-EGFwt, pUC19-mEGF
14, pUC19-mEGF
16with pUC19-3mEGF plasmid 1 μ l, Phusion enzyme 0.5 μ l, finally mend to 50 μ l with distilled water; Reaction conditions: 98 DEG C of denaturation 30s, 98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations, last 72 DEG C of 5min.Gene product detects with agarose gel electrophoresis, obtains gene fragment called after EGFwt, mEGF
14, mEGF
16and 3mEGF.
Special primer E3 and E4 is utilized to obtain partial gene fragments by PCR method amplification.PCR reaction system 2 (cumulative volume 50 μ l): 5 × Buffer 10 μ l, dNTP 2 μ l, primer 1 μ l (E3 and E4), template are pFUSE-hIgG1-Fc plasmid 1 μ l, Phusion enzyme 0.5 μ l, finally mends to 50 μ l with distilled water; Reaction conditions: 98 DEG C of denaturation 30s, 98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations, last 72 DEG C of 5min.Gene product detects with agarose gel electrophoresis, obtains gene fragment called after Fc.
Special primer E1 and E4 is utilized to obtain goal gene fragment by the amplification of bridge joint PCR method.PCR reaction system 3 (cumulative volume 50 μ l): 5 × Buffer 10 μ l, dNTP 2 μ l, primer 1 μ l (P1 and P2), template are above-mentioned amplification gene fragment EGFwt, mEGF
14, mEGF
16, 3mEGF respectively with Fc mix each 1 μ l, Phusion enzyme 0.5 μ l, finally with distilled water mend to 50 μ l; Reaction conditions: 98 DEG C of denaturation 30s, 98 DEG C of 10s, 68 DEG C of 60s, 3 circulations, 98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 50s, 30 circulations, last 72 DEG C of 5min.Gene product detects with agarose gel electrophoresis, obtains gene fragment called after EGFwt-Fc, mEGF
14-Fc, mEGF
16-Fc and 3mEGF-Fc.
The enzyme of 1.3 carriers and gene fragment cuts process
To pCHO1.0 plasmid and EGFwt-Fc, mEGF
14-Fc, mEGF
16-Fc and 3mEGF-Fc gene fragment carry out double digestion process respectively, and enzyme cuts the foundation of system: in 1.5ml EP pipe, add following composition: enzyme cuts the foundation of system 1: in 1.5mlEP pipe, add following composition: pCHO1.0 plasmid or EGFwt-Fc, mEGF
14-Fc, mEGF
16-Fc and 3mEGF-Fc gene fragment 40 μ l, each 5 μ l of Buffer410 μ l, Avr II and BstZ17I, aqua sterilisa 45 μ l, reacts 5h after mixing at 37 DEG C, utilizes QIAGEN Product Purification Kit to reclaim.
The connection of 1.4 recombinant plasmids transforms
Under the effect of T4 ligase enzyme, by with same enzyme cut rear reclaim obtain gene fragment pCHO1.0 (Avr II and BstZ17I) large fragment cut through with enzyme respectively after EGFwt-Fc (Avr II and BstZ17I), mEGF
14-Fc (Avr II and BstZ17I), mEGF
16-Fc (Avr II and BstZ17I) is connected with 3mEGF-Fc (Avr II and BstZ17I) gene fragment.Ligation system is as follows: in 1.5ml EP pipe, add following composition pCHO1.0 (Avr II and BstZ17I) 2 μ l, EGFwt-Fc (Avr II and BstZ17I) or mEGF
14-Fc (Avr II and BstZ17I) or mEGF
16-Fc (Avr II and BstZ17I) or 3mEGF-Fc (Avr II and BstZ17I) 6 μ l, 10 × T4 Buffer 1 μ l, T4 DNA ligase 1 μ l, under room temperature (about 20 DEG C), more than 4h is reacted after mixing, connect product conversion in Top10 competent escherichia coli cell, coat 37 DEG C of left undisturbed overnight on 2YT (KANA) plate culture medium, dull and stereotyped numbering pCHO-EGFwt-Fc, pCHO-mEGF
14-Fc, pCHO-mEGF
16-Fc and pCHO-3mEGF-Fc.
The bacterium colony PCR of 1.5 recombinant plasmids screens
From pCHO-EGFwt-Fc, pCHO-mEGF
14-Fc, pCHO-mEGF
16in-Fc and pCHO-3mEGF-Fc flat board, the single bacterium colony of the several restructuring of each picking is as pcr template after cultivating, and carries out PCR Screening and Identification respectively.Bacterium liquid pcr amplification reaction system (cumulative volume 20 μ L): 2 × Taq HS 10 μ L, bacterium liquid template 2 μ L, each 1 μ L of upstream and downstream primer (E1 and E4) (final concentration 0.3 μm of ol/L), finally mends to 20 μ L with distilled water; Reaction conditions: 95 DEG C of 3min, 94 DEG C of 60s, 53 DEG C of 60s, 72 DEG C of 90s, 30 circulations; Last 72 DEG C of 5min.Agarose gel electrophoresis analytical results.
The enzyme of 1.6 recombinant plasmids cuts qualification
Extract after identifying correct colony inoculation by bacterium colony PCR and carry out enzyme again and cut qualification.First carry out the plasmid extraction of recombinant bacterium, then carry out restriction analysis, enzyme cuts system: in 1.5ml EP pipe, add following composition: plasmid 2 μ l, the each 1 μ l of Buffer11 μ l, BSA0.1 μ l, Avr II and BstZ17I, mend aqua sterilisa to 10 μ l, after mixing, at 37 DEG C, react 4h.Agarose gel electrophoresis analytical results.
The order-checking qualification of 1.7 recombinant plasmids
Identify that correct bacterium colony is inoculated several bacterium colony at random and carried out order-checking qualification again by being cut by bacterium colony PCR and enzyme.There is provided bacterium liquid sample recombinant plasmid to deliver to company and carry out order-checking qualification.Expression plasmid saves backup.
2, plasmid transfection and cell screening
Plasmid extraction adopts QIAGEN Plasmid Midi Kit, adopts host cell CHO-S or DG44, according to Freedom
tMcHO-S
tMkit test kit specification sheets carries out plasmid transfection respectively.The cell proceeding to plasmid is placed in shake-flask culture (37 DEG C, 8%CO2,110rpm/min) respectively to 48h, and cell counter detects Cell viability and cell count.
Two step pressurization screenings are carried out: 10P/100M, 20P/200M (P=10 μ g/mL Puromycin, M=nM MTX) after transfection 48h; 30P/500M, 50P/1000M, obtain just sieve cell.Carry out monoclonal cell screening with limiting dilution assay, therefrom preferably clone enlarged culturing step by step by detection expressing quantity and purity.
3, Protein expression and purification
Screening High producing clones cell expands to 24 orifice plate growths from 96 orifice plates, then expands to 6 orifice plate growths, expands afterwards to 50mL Shake flask grown.
Collect cultivation 7 days cell culture supernatants, centrifugal removing cell debris, supernatant liquor is with 0.45 μm of membrane filtration, regulate pH to 7.4, with HiTrap Protein-A Sepharose affinity column purified fusion protein, the deionized water rinsing pillar of 5 times of column volumes, use PBS damping fluid (the 20mM phosphoric acid salt of 5 times of column volumes again, pH 7.4) balance pillar, loading, collection effluent liquid detects, with 10 times of volume PBS damping fluid (0.02mol/L phosphoric acid salt, pH 7.4) wash except foreigh protein removing, then 0.1M glycine buffer (pH3) is used by target protein from wash-out post, purity of protein is detected more than 90% with SDS-PAGE.
Embodiment 2 fusion rotein biological activity determination
The binding affinity of EGF-Fc fusion rotein and PCSK9 is determined at Otect QK (Fortebio) by microbial film interferometric method and above measures.SA sensor sensor (Fortebio, article No. 18-5063) be cured in containing the PCSK9 in TrisHCl pH 7.4 damping fluid of 0.5%BSA and 1mM CaCl2, wash in same buffer, and to be transferred to containing concentration in same buffer be in the hole of EGF-FC fusion rotein of 0-500nM.Signal for the reference hole only containing damping fluid is deducted from all in conjunction with data.Avidity KD uses Octet software to obtain by fitting to steady-state algorithm.Summarize the KD value display of determination in tablei: compared with EGFwt-Fc, the avidity of mEGF-Fc mutant and 3mEGF-FC series connection mutein fusion protein increases by 15 to 50 times.
Table I, the binding affinity of EGFwt-Fc, mEGF-Fc mutant and 3mEGF-FC series connection sudden change and PCSK9.KD value is by being that steady-state equation is determined by data fitting.Data represent with mean ± SD.
Recombination fusion protein |
K
DValue (nM)
|
EGFwt-Fc |
798±90 |
mEGF
14-Fc
|
121±14 |
mEGF
16-Fc
|
58±9 |
3mEGF-Fc |
17±4 |
Embodiment 3 fusion rotein biological activity determination
Human hepatoma HepG2 cell is cultured to logarithmic phase, washes cell once with PBS, and making cell suspension through trysinization and adjust cell density is 1 × 10
6cells/ml.Inoculating cell is in 96 well culture plates (100 μ l/ hole), and marginal pore adds 200 μ l substratum.Put culture plate in 37 DEG C, 5%CO
2in incubator, continue cultivation 24 hours.Remove cells and supernatant, every hole adds 100 μ l serum-free cell culture mediums, and CO2 incubator continues cultivation 16 hours; Abandon supernatant, again change serum-free cell culture medium into, add PCSK9 (final concentration 25 μ g/ml), add EGFwt-FC (10 ~ 50 μMs) respectively or 3mEGF-Fc (03 ~ 3 μM) puts 37 DEG C simultaneously, 5%CO2 incubator hatches 4 hours; Add again
(final concentration is 6 μ g/ml) hatches 3h, removes supernatant, and rinse cell 3 times with PBS, observe under being placed in inverted fluorescence microscope and obtain image, the result that obtains carries out fluorescent quantitation again; Get cell in addition according to preceding method process cell, add PCSK9 (final concentration 25 μ g/ml) and EGFwt-FC (10 ~ 50 μMs) or 3mEGF-Fc (03 ~ 3 μM) effect respectively after 4 hours, harvested cell, adopt immunofluorescence technique, observe expression or the degraded of LDL-R, and carry out fluorescent quantitation.
Intervene PCSK9 process HepG2 cell with EGFwt-FC and 3mEGF-Fc fusion rotein provided by the invention, result shows, EGFwt-FC and 3mEGF-Fc fusion rotein of the present invention all can obviously suppress PCSK9 to the degraded (Fig. 1) of LDL-R; Consistent therewith, EGFwt-FC and 3mEGF-Fc of the present invention also obviously increases liver cell to the picked-up (Fig. 2) of LDL-C, the effect that 3mEGF-Fc suppresses PCKS9 activity and increase liver cell to absorb LDL-C is the most obvious, and present significant concn dependence, illustrate and obviously strengthened by the 3mEGF-FC biological activity of EGF amino acid mutation.