CN1408875A - DNA type semliki forest cerebritis virus carrier for expressing IgG1 Fc chimeric protein as TNF receptor - Google Patents
DNA type semliki forest cerebritis virus carrier for expressing IgG1 Fc chimeric protein as TNF receptor Download PDFInfo
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- CN1408875A CN1408875A CN 01136032 CN01136032A CN1408875A CN 1408875 A CN1408875 A CN 1408875A CN 01136032 CN01136032 CN 01136032 CN 01136032 A CN01136032 A CN 01136032A CN 1408875 A CN1408875 A CN 1408875A
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Abstract
The present invention incldues soluble acceptor encoding human TNF; immune globulin G1 heavy strand constant region chimeric protein (husTNFR:IgG1 Fc) cDNA and its amino acid sequence; DNA type recombinant Semliki forest cerebritis virus expression carrier to express the recombinant protein in high efficiency and sTNFR produced in mammal cell with the recombinant expression carrier. The IgG1 Fc chimeric protem may have treating effect on adult's rheuamtic arthritis, children's multiple rheumatic arthritis, pyaemia, chronic heart failure, SLE and other autoimmune diseases.
Description
Huamn tumor necrosis factory alpha (hTNF α) is a kind of multifunctional cytokine that the monocytes/macrophages activation produces, has biologic activity widely, playing an important role aspect inflammatory reaction, immunomodulatory, antitumor, microorganism and the parasitic infection, in human cell factor immunomodulatory network, play the maincenter regulating effect.The biologic activity of TNF realizes that by the corresponding acceptor of cell surface TNF has two acceptor: P55 and P75 acceptor, is distributed widely in the surface of most cells in the body.TNF α, induces in a series of cells to change through complicated signal transmission with after film surface receptor (mTNFR) combines, and causes corresponding physiology and pathologic reaction.Oneself knows with TNF α diseases associated and comprises at present: autoimmune disease, cachexy, septic shock syndromes, rheumatoid arthritis, chronic heart failure, brain malaria, disseminated intravascular coagulation, diabetes, graft-rejection, Tiroidina hypertrophy etc.Therefore the biologic activity that suppresses TNF has great importance clinically.The biological function of TNF realizes that by the corresponding acceptor of cell surface (mTNFR) extracellular region of mTNFR can split away off under specific circumstances, forms soluble receptors (sTNFR).STNFR no longer mediates the acceptor transmission, but still can combine with TNF, thereby the biologic activity of TNF is sealed in combining of blocking-up TNF α and mTNFR.Utilizing soluble TNF acceptor to combine with receptor competition on the film, is the effective way that blocking-up TNF signal transmits.Studies show that in a large number TNF and TNFR are with tripolymer-trimerical form bonded, the polymerization of acceptor is the active basis of mediation TNF on the film.
STNFR and IgG Fc fragment are merged in the present invention, and " Y " shape chimeric molecule of formation guarantees the polymerization of sTNFR molecule.After studies show that sTNFR and IgG Fc fragment merge, the sTNFR transformation period in vivo prolongs, thereby has improved the neutralization activity of sTNFR, so the sTNFR polymer is obviously good than sTNFR monomer to the neutralization activity of TNF.In the present invention, we have snappiness flexibly at the connection peptides of design, Swiss Model software three-dimensional structural analysis shows, this connection peptides can guarantee that the functional subunit of two of chimeric protein keeps separately independently three-D space structure, thereby make the chimeric protein after the expression can form correct space conformation, keep the biologic activity of sTNFR.
Semliki fores encephalitis virus (SFV) expression vector is a kind of eukaryotic cell expression system of new development in recent years, and have following characteristics: (1) high infection rate Semliki SFV is to BHK, and the isocellular infection rate of CHO can reach 100%.(2) high replication rate, Semliki SFV be at present oneself know have one of virus of high replication rate, in the contamination back several hours, the RNA copy number of virus can reach 2 * 10
5, this RNA can directly participate in the synthetic of virus and foreign protein as mRNA, and individual cells can synthesize 10
8Individual viral genome encoded protein molecule.(3) duplicate in the tenuigenin, duplicate in tenuigenin behind the Semliki SFV cells infected, therefore add cap at gene, mRNA shears with the transhipment aspect has high-level efficiency.(4) cytopathy lags behind, and behind the SemlikiSFV cells infected 4-24 hour, structural protein were expressed fully, but obvious pathology does not appear in cell.(5) high security, Semliki SFV expression vector structural protein and regulatory gene and rdrp gene have been avoided viral reorganization not on same carrier.Structural protein are undergone mutation simultaneously, must just have capacity packing behind the protease cracking, the possibility of having avoided wild virus to produce.Compare with traditional SFV expression vector, DNA type Semliki SFV expression vector is added with CMV promotor/enhanser before the SFV26s promotor, avoided the RNA operation, has improved the transfection efficiency of plasmid, easy handling.The technical indicator that the present invention reaches: 1. (1) expresses sTNFRI:IgG
1The DNA type Semliki fores encephalitis virus expression vector total length 12837bp of Fc, nucleosides
Acid sequence is seen Fig. 3.(2) express sTNFRII:IgG
1The DNA type Semliki fores encephalitis virus expression vector total length 12855bp of Fc, nuclear
Nucleotide sequence is seen Fig. 4.2. (1) coding chimeric protein sTNFRI:IgG
1The full length gene of Fc is 1335bp, and nucleotide sequence is seen Fig. 5.(2) coding chimeric protein sTNFRII:IgG
1The full length gene of Fc is 1353bp, and nucleotide sequence is seen Fig. 6.3. (1) chimeric protein sTNFRI:IgG
1445 amino acid of Fc total length, aminoacid sequence is seen Fig. 5.(1) chimeric protein sTNFRIII:IgG
1453 amino acid of Fc total length, aminoacid sequence is seen Fig. 6.Advantage of the present invention: 1. express sTNFR:IgG
1The DNA type Semliki fores encephalitis virus expression vector of Fc has high expression level in mammalian cell, chimeric protein is with excretory form great expression behind the recombinant virus-infected cell, and every liter of nutrient solution can be secreted to produce and have the active chimeric protein 10-30mg of neutralization.2. utilize this recombinant expression vector in eukaryotic cell, to produce sTNFR:IgG
1The Fc chimeric protein cycle is short, is easy to purifying.Behind the recombinant virus-infected cell 24 hours, chimeric protein just reached maximal secretory capacity, adopts serum-free culture simultaneously, and the chimeric protein of secretion in nutrient solution is easy to purifying.3. exist with dimeric form under the chimeric protein native state, compare with monomeric sTNFR, to the neutralising capacity raising of TNF, the transformation period prolongs in the body simultaneously.4. the connection small peptide that designs among the present invention has good snappiness, can make the sTNFR at two ends and IgG Fc structural domain keep separately independently space structure, thereby guarantee that sTNFR is to the neutralization activity of TNF and the dimerization of IgGFc molecule.Description of drawings: Fig. 1: express sTNFR:IgG
1DNA type SFV recombinant vectors structural representation Fig. 2 of Fc chimeric protein: sTNFR:IgG
1Fc chimeric protein structural representation Fig. 3: express sTNFRI:IgG
1DNA type SFV recombinant vectors nucleotide sequence Fig. 4 of Fc chimeric protein: express sTNFRII:IgG
1DNA type SFV recombinant vectors nucleotide sequence Fig. 5 of Fc chimeric protein: sTNFRI:IgG
1Fc gene and amino acids coding preface Fig. 6: sTNFRII:IgG thereof
1Fc gene and amino acids coding preface embodiment 1:sTNFR:IgG thereof
1The structure and the sequencing of Fc chimeric protein gene
Synthetic following six primers:
primer?sTNFR?N1：5’-ATG?AAT?TCC?AAT?ATG?GCG?CCC?GTC?GCC?GTC?TG-3’
primer?sTNFR?N2：5’-AAG?AAT?TCC?AAT?ATG?GGC?CTC?TCC?AC-3’
primer?sTNFR?C1：5’-AC?GAA?TCC?ACC?GCC?ACC?GGG?TAA?GTG?TAC?TG
primer?sTNFR?C2：5’-AA?GGA?TCC?ACC?GCC?ACC?TGT?GGT?GCC?TGA?GTC-3’
primer?IgG?Fc?N：5’-TA?GGA?TCC?GGA?GGT?GGA?GGT?AGC?GTT?GAG?CCC?AAA
TCT-3’
primer?IgG?Fc?C：5’-ATC?TCG?AGT?TAC?TAC?TGC?GTG?TAG?TGG?TTG-3’
Primer primer sTNFR N1 and primer sTNFR N2 introduce EcoR I restriction enzyme site, primer primer sTNFRC1, primer sTNFR C2 and primer IgG Fc N introduce BamH I restriction enzyme site, and primer primer IgG Fc C introduces Xhol I restriction enzyme site.
From fresh peripheral blood, separate mononuclear macrophage, utilize TRIZOL single stage method extracting RNA, six random primer reverse transcription cDNA, (1) is masterplate with reverse transcription cDNA then, primer sTNFR N1 and primer sTNFR C1 are that primer PCR amplifies the functional structure territory (comprising signal peptide) that four of TNFR I extracellular regions are rich in Cys, and introduce small peptide GlyGlyGlyGlySer at carboxyl terminal; (2) be masterplate with reverse transcription cDNA, primer sTNFR N2 and primer sTNFR C2 are that primer PCR amplifies the functional structure territory (comprising signal peptide) that four of TNFR II extracellular regions are rich in Cys, and to introduce small peptide GlyGlyGlyGlySer. (3) at carboxyl terminal be template with IgG Fc/TA, primer IgG Fc N and primer IgG Fc C are primer, and pcr amplification goes out IgG
1The Fc gene, and at aminoterminal introducing GlySerGlyGlyGlyGlySer, carboxyl terminal is introduced translation termination signal TAG TAA.
STNFR is inserted EcoR I and the BamHI restriction enzyme site of plasmid pBLUE SK, be built into recombinant cloning vector pBLUESK/sTNFR.Further IgG Fc gene is inserted BamH I and the EcoR I site of pBLUE SK/sTNFR, be built into recombinant cloning vector pBLUE SK/sTNFR:IgG Fc, thereby the gene of will encode sTNFR and IgG Fc links to each other, constitute coding chimeric protein huTNFR:IgG
1The Fc recombination, and the coding of introducing that is situated between connects small peptide ((the chimeric protein huTNFR:IgG of gene GlyGlyGlyGlySerGlyGlyGlyGlySer-)
1The Fc structural representation is seen Fig. 2).
With pBLUE SK/sTNFR:IgG Fc is template, the order-checking of ABI377 automatic sequencer, and sequencing result shows that the gene of sTNFRII and IgGFc is entirely true, the insertion site is connected small peptide with the centre and also conforms to design.Embodiment 2:pSFV/sTNFR:IgG
1The structure of Fc recombinant expression vector
Synthetic following primer:
pSFV?N1：5’-A?TTG?ATC?AAT?ATG?GCG?CCC?GTC-3’
pSFV?N2：5’-A?TTG?ATC?AAT?ATG?GGC?CTC?TCC?A-3’
pSFV?C：5’-GGC?TGA?TCA?TTA?CTA?CTG?CGT?GTA-3’
Bcl I restriction enzyme site (BamH I isocaudarner) is introduced at the primer two ends, and the N section is introduced the Kozak sequence simultaneously.With pBLUESK/sTNFRI:IgG Fc is template, and pSFV N1 and pSFV C are primer, and pfu amplifies sTNFRI:IgG
1The Fc gene after Bcl I enzyme is cut digestion, inserts the BamH I site of pSFV, is built into pSFV/sTNFRI:IgG
1The Fc recombinant expression vector; With pBLUE SK/sTNFRII:IgG
1Fc is a template, and pSFVN1 and pSFVC are primer, and pfu amplifies sTNFRII:IgG
1The Fc gene after Bel I enzyme is cut digestion, inserts the BamH I site of pSFV, is built into pSFV/sTNFRII:IgG
1The Fc recombinant expression vector.Enzyme is cut and PCR identifies that the gene of recombinant vectors and insertion is correct.(pSFV/sTNFR:IgG
1The structure of Fc recombinant expression vector is seen Fig. 1) embodiment 3:pSFV/sTNFR:IgG
1The Fc recombinant expression vector is expressed in bhk cell and is identified
Utilize Qiagen Large Scale Plasmid Purification Kit purifying pSFV/sTNFR:IgG
1Fc recombinant vectors and pSFV-h assistant carrier, the LF plus transfection reagent of Gibco company is with pSFV/sTNFR:IgG
1Fc and pSFV-h change bhk cell in 1: 1 ratio of mole number, gather in the crops culture supernatant after 24 hours, obtain to contain sTNFR:IgG
1The recombinant virus of Fc structure gene.Recombinant virus activates virus with Quimotrase A4 (Chymotrypsin A4,2mg/ml inPBS) the room temperature cracking of 1/10 volume 4 hours; (Aprotintin, 5-10TIU/ml) the room temperature inactivated proteases is 5 minutes to use the Trypsin inhibitor,Trasylol of 1/4 volume then.With this virus infection bhk cell, change serum free medium into after 4 hours, gather in the crops supernatant after 24 hours.
After expressing supernatant SDS-PAGE, 80V2hr changes film, and the skimmed milk sealing is spent the night, respectively with anti-sTNFR antibody and anti-human IgG
1Fc antibody is one anti-, and the sheep anti-mouse igg of horseradish peroxidase-labeled is two anti-ly to carry out Western-Blot, and the result confirms to exist in the supernatant a large amount of chimeric proteins.
Express supernatant and can suppress TNF, have among the tangible TNF and active the killing and wounding of L929 cell.Embodiment 4:sTNFR:IgG
1The purifying of Fc chimeric protein and evaluation
The culture supernatant of virus infection bhk cell after 24 hours after the processing of saltouing, directly goes up affinity column, and the target protein under the wash-out is the sTNFR:IgG of purifying
1The Fc chimeric protein.
Purifying protein is behind reduction and non-reduced SDS-PAGE, and 80V2hr changes film, and the skimmed milk sealing is spent the night, respectively with anti-sTNFR antibody and anti-human IgG
1Fc antibody is one anti-, and the sheep anti-mouse igg of horseradish peroxidase-labeled is two anti-ly to carry out Western-Blot, and the result shows that the albumen that is purified into is our target protein, show simultaneously chimeric protein under native state with single aggressiveness and the coexistence of dimeric form.
Bag is antigen by the pure product albumen of TNF with the chimeric protein, is one anti-with anti-sTNFR antibody, and the sheep anti-mouse igg of horseradish peroxidase-labeled is two anti-, carries out " double antibodies sandwich method " and detects, and the result confirms that the chimeric protein of purifying can discern TNF albumen and combination with it.
Bag is by hTNF α albumen, by sample sTNFR:IgG on the automatic sample adding system on the BIACORE protein chip
1The Fc chimeric protein utilizes the BIACORE system to detect interaction between hTNF α and chimeric protein, and the result confirms under the native state that chimeric protein can be discerned and in conjunction with TNF albumen.
SDS-PAGE shows, chimeric protein sTNFRI:IgG
1Fc and sTNFRII:IgG
1The molecular weight of Fc all is about 50kD; Uv scan shows that absorbing wavelength is 280nm outside the maximum; Isoelectric analysis shows, chimeric protein sTNFRI:IgG
1The iso-electric point of Fc is about 6.5, chimeric protein sTNFRII:IgG
1The iso-electric point of Fc is about 7.0.Embodiment 5:sTNFR:IgG
1The biologic activity analysis of Fc chimeric protein
With mouse L929 cell detection hTNF biologic activity, the chimeric protein behind the purifying is with 1640 dilutions, with the hTNF protein 37 of 0.2ng ℃ insulation 2h, and then with L929 cell cultures 24 hours, supernatant discarded, the MTT colour developing, 570nm surveys the OD value.The result shows, sTNFR:IgG
1The Fc chimeric protein has obvious inhibiting activity to TNF albumen, can effectively suppress TNF albumen killing and wounding the L929 cell.
Claims (4)
1. the present invention relates to a kind ofly can efficiently express the human tumor necrosis factor soluble receptors: immunoglobulin G 1 CH (containing hinge region) chimeric protein (sTNFR:IgG
1Fc) DNA type Semliki fores encephalitis virus recombinant expression vector (pSFV/sTNFR:IgG
1Fc).Its essential characteristic is: (1) pSFV/sTNFRI:IgG
1Fc expression vector full length gene is 12837bp, contains coding sTNFRI:IgG
1The structure gene of Fc chimeric protein (7407-8741 bit base, mrna length is 1335bp), contain CMV promotor/enhanser on the carrier, the helper virus plasmid pSFV-h of nonstructural protein gene nsp1, nsp2, nsp3 and the nsp4 of SFV26s promotor and SFV and coding SFV structural protein.(2) pSFV/sTNFRII:IgG
1Fc expression vector full length gene is 12855bp, contains coding sTNFRII:IgG
1The structure gene of Fc chimeric protein (7407-8759 bit base, mrna length is 1353bp), contain CMV promotor/enhanser on the carrier, the helper virus plasmid pSFV-h of the nonstructural protein gene nsp1 of SFV26s promotor and SFV, nsp2 nsp3 and nsp4 and coding SFV structural protein.
2. according to claim 1, coding chimeric protein sTNFR:IgG
1The gene of Fc, its essential characteristic is: (1) sTNFRI:IgG
1The Fc full length gene is 1335bp, 445 amino acid of encoding, molecular weight is about 50kD, have soluble tumor necrosis factor receptor I (sTNFRI) encoding gene (1-633 bit base), immunoglobulin G while 1 CH (containing hinge region) (IgGFc) encoding gene (664-1335 bit base) be connected small peptide encoding gene three parts and form (634-663 bit base, nucleotides sequence are classified 5 '-ggt ggc ggt gga tcc gga ggt gga ggt agc-3 ' as).(2) sTNFRII:IgG
1The Fc full length gene is 1353bp, 453 amino acid of encoding, molecular weight is about 50kD, soluble tumor necrosis factor receptor II (sTNFRI) encoding gene (1-651 bit base), immunoglobulin G while 1 CH (containing hinge region) (IgG Fc) encoding gene (681-1353 bit base) are arranged and be connected small peptide encoding gene (652-681 bit base, nucleotides sequence are classified 5 '-ggt ggc ggt gga tcc gga ggt gga ggt agc-3 ' as) three parts and form.
3. according to claim 1, chimeric protein sTNFR:IgG
1The aminoacid sequence of Fc, its essential characteristic is: (1) chimeric protein sTNFRI:IgG
1445 amino acid of Fc total length, molecular weight is about 50kD, form by three parts: soluble tumor necrosis factor receptor I (sTNFRI) (1-211 amino acids residue), connect small peptide (212-221 amino acids residue, aminoacid sequence is-GlyGlyGlyGlySerGlyGlyGlyGlySer-), and immunoglobulin G while 1 CH (containing hinge region) (IgG Fc) (222-445 amino acids residue).(2) chimeric protein huTNFRII:IgG
1453 amino acid of Fc total length, molecular weight is about 50kD, form by three parts: soluble tumor necrosis factor receptor II (sTNFRII) (1-217 amino acids residue), connect small peptide (218-226 amino acids residue, aminoacid sequence is-GlyGlyGlyGlySerGlyGlyGlyGly Ser-), immunoglobulin G while 1 CH (containing hinge region) is (218-451 amino acids residue) (IgGFc).
4. according to claim 1, the pharmaceutical salts of utilizing this recombinant expression vector to produce is characterized by: the treatment of can be used for being grown up rheumatoid arthritis (RA), the multiple rheumatic arthritis of children (JRA), Sepsis, chronic heart failure and systemic lupus erythematous autoimmune disorders such as (SLE).
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| Application Number | Priority Date | Filing Date | Title |
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| CN 01136032 CN1408875A (en) | 2001-09-29 | 2001-09-29 | DNA type semliki forest cerebritis virus carrier for expressing IgG1 Fc chimeric protein as TNF receptor |
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| CN 01136032 CN1408875A (en) | 2001-09-29 | 2001-09-29 | DNA type semliki forest cerebritis virus carrier for expressing IgG1 Fc chimeric protein as TNF receptor |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007041964A1 (en) * | 2005-10-14 | 2007-04-19 | Hai Li | The use of long half-life human recombinant soluble tumor necrosis factor alpha receptor in preparing the medicine which can prevent hepatic failure |
| CN100382845C (en) * | 2005-10-14 | 2008-04-23 | 李海 | Use of long-acting human recombined soluble TNF alpha receptor in preparation of medicament for treating liver failure |
| CN102574907A (en) * | 2009-10-19 | 2012-07-11 | 韩诺生物制药株式会社 | Modified human tumor necrosis factor receptor-1 polypeptide or fragment thereof, and method for preparing same |
| CN109705222A (en) * | 2018-12-27 | 2019-05-03 | 中国科学院武汉病毒研究所 | The fusion protein and the preparation method and application thereof of flaviviridae envelope protein |
-
2001
- 2001-09-29 CN CN 01136032 patent/CN1408875A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007041964A1 (en) * | 2005-10-14 | 2007-04-19 | Hai Li | The use of long half-life human recombinant soluble tumor necrosis factor alpha receptor in preparing the medicine which can prevent hepatic failure |
| CN100382845C (en) * | 2005-10-14 | 2008-04-23 | 李海 | Use of long-acting human recombined soluble TNF alpha receptor in preparation of medicament for treating liver failure |
| US8227404B2 (en) | 2005-10-14 | 2012-07-24 | Hai Li | Method of preventing acute or sub-acute hepatic failure in a subject by administering a soluble human tumor necrosis factor alpha fusion protein |
| US8575088B2 (en) | 2005-10-14 | 2013-11-05 | Zhenyi Li | Method of preventing acute or sub-acute hepatic failure by administering a long-acting recombinant human soluble tumor necrosis factor alpha receptor II fusion protein |
| CN102574907A (en) * | 2009-10-19 | 2012-07-11 | 韩诺生物制药株式会社 | Modified human tumor necrosis factor receptor-1 polypeptide or fragment thereof, and method for preparing same |
| CN102574907B (en) * | 2009-10-19 | 2015-10-21 | 韩诺生物制药株式会社 | The human tumor necrosis factor receptor I polypeptide of modifying or its fragment and prepare their method |
| CN109705222A (en) * | 2018-12-27 | 2019-05-03 | 中国科学院武汉病毒研究所 | The fusion protein and the preparation method and application thereof of flaviviridae envelope protein |
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