CN101638435B - Blue-green algal virus protein N mutant, modified derivative and application thereof - Google Patents
Blue-green algal virus protein N mutant, modified derivative and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention belongs to the field of biological medicines, in particular to a blue-green algal virus protein N mutant, a PEG modified derivative and application thereof. The blue-green algal virus protein N mutant comprises two sequences, one is connecting peptide with hydrophilic flexibility, and the other is a blue-green algal virus protein N sequence optimized by trinucleotide. The derivative is obtained by PEG modification of the blue-green algal virus protein N mutant; the mutant and the derivative thereof show favorable anti-HIV activity. The invention is applied to preparing anti-HIV or other virus microorganisms.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of blue-green algal virus protein N (CVN) mutant, its PEG modified derivative and their application in pharmacy.
Background technology
(Cyanovirin-N is that people such as U.S. scientist BOYD are from the extractible a kind of albumen with HIV (human immunodeficiency virus)-resistant activity of blue-green algae CVN) to blue algae antiviral protein N.CVN can be specifically, thereby the capsid protein gp120 that is combined in human immunodeficiency virus HIV-1 goes up the performance antiviral activity high affinity, these characteristics make it can not be subjected to the interference of virus variation, simultaneously it also to have antiviral spectrum wide, characteristics such as stable in properties, this makes CVN albumen become a kind of of great value antiviral [BOYD M R, GUSTAFSON K R, MCMAHON J B, et al.Discovery of cyanovirin-N, a novel human immuno-deficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp 120:Potentialapplications to microbicide development[J] .Antimicrobial Agents and Chemotherapy, 1997,41 (7): 1521-30.].But because this molecular weight of albumen is less, and two disulfide linkage are arranged in the molecule, make this albumen, yield poorly in the expression in escherichia coli difficulty.Simultaneously as the small molecular protein class medicine in a kind of prokaryotic organism source, there is short, cytotoxicity of transformation period in it and causes shortcoming such as immunne response.
Polyoxyethylene glycol (PEG) is a kind of nontoxic, the water-soluble high-molecular substance of non-immunogenicity, can pass through covalent attachment mode modifying protein, polyethyleneglycol modified is with solving or alleviate the poor stability that albumen and polypeptide exist in medicinal process, effective way (the IANG Z Y of problems such as the transformation period is short, XU S W, WANG Y Q.Chemistry for pegylation ofprotein and peptide molecules[J] .Chinese Journal of Organic Chemistry, 2003,23 (12): 1340-7.) utilize the PEG-maleimide under acidic conditions, to modify the existing bibliographical information of CVN, people such as Zappe have selected 62 glutamine under the neutral PH condition it to be modified with mPEG-maleimide (mPEG-MAL) by cysteine alternate mutant (CVN (Q62C)), effectively improving its one-tenth property of medicine (ZAPPE H, SNELL M E, BOSSARD M J.PEGylation ofcyanovirin-N, an entry inhibitor of HIV[J] .Advanced Drug Delivery Reviews, 2008,60 (1): 79-87.).
The concrete modification strategy of people such as Zappe is: the mPEG-maleimide (mPEG-MAL) of selecting 62 and 14 glutamine to be respectively 20KD and 30KD by cysteine alternate mutant molecule amount is modified it under neutral PH and meta-alkalescence PH condition, 14 glutamine are by the unusual end of the modification efficient of cysteine alternate mutant (CVN (Q14C)) as a result, and 62 glutamine are that the modification rate is higher under 1: 3 the condition in neutral PH, CVN mutant and mPEG-MAL mol ratio by cysteine alternate mutant (CVN (Q62C)).But the CVN that the activity of the anti-HIV of CVN (Q62C) is not more suddenlyd change is low, and because the decorating site of selecting is the inside at CVN, so the activity of the anti-HIV of body variant that process mPEG-maleimide 30KDa (mPEG-MAL-30KDa) modifies is almost lost.
Summary of the invention
At the shortcoming and defect part that prior art exists, purpose of the present invention at first provides a kind of CVN mutant, and it is easier expresses in the host, is easier to purifying, and helps further modification.
Another object of the present invention provides the modified derivative of above-mentioned CVN mutant, to reduce the cytotoxicity and the immunogenicity of albumen itself, makes it be more suitable for using.
A further object of the present invention then is that said mutation body and modified derivative are used to prepare the medicine that prevents and/or treats acquired immune deficiency syndrome (AIDS).
For achieving the above object, the invention provides following technical scheme:
A kind of blue-green algal virus protein N mutant, its aminoacid sequence is made up of sequence A and sequence B, and sequence A is positioned at the N end of sequence B,
Sequence A such as one of following:
A. the SEQ ID NO:1 in the sequence table;
B. the amino acid residue sequence with the SEQ ID NO:1 in the sequence table passes through replacement, lacks or adds one or several amino acid, and it has the characteristics of hydrophilic flexibility;
Sequence B such as one of following:
C. the SEQ ID NO:2 in the sequence table;
D. the amino acid residue sequence with the SEQ ID NO:2 in the sequence table passes through replacement, lacks or adds one or several amino acid, but does not change first three residue of its N end, and it has anti HIV-1 virus activity specifically;
The present invention also provides the coding nucleotide sequence of blue-green algal virus protein N mutant.
Preferably, above-mentioned coding nucleotide sequence comprises one of following sequence:
E. the SEQ ID NO:3 in the sequence table;
F. the nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with SEQ ID NO:3 in the sequence table.
The high rigorous condition here is meant, less salt pyritous hybridization conditions, and as 0.1 * SSC, 0.1%SDS and 65 ℃ of temperature.
Above-mentioned nucleotide sequence mainly is used in expresses target protein among the host, contain the expression vector of above-mentioned nucleotide sequence, clone, and host bacterium etc. all can obtain by the technique means of routine.Protein purge process from the host also can adopt conventional method.
Obtaining on the basis of purifying protein, the present invention also provides CVN mutant modified derivative, is that N-terminal with above-mentioned CVN mutant carries out PEG and modifies, and decorating site generally is the alpha-amino group at the N-terminal glycine residue.
Preferably, the modifier that described PEG modifies uses mPEG-ALD (mono methoxy ether PEG-propionic aldehyde), and the molecular weight of described mPEG-ALD is preferably 10KD-20KD.
Said mutation body and modified derivative all are used in the application for preparing in the medicine that prevents and/or treats acquired immune deficiency syndrome (AIDS), based on identical antiviral-mechanism, mutant of the present invention and modified derivative can be applied to prepare the medicine that prevents and/or treats other viral micro-organisms associated diseases.
With respect to prior art, the present invention has following beneficial effect:
CVN mutant provided by the invention and modified derivative thereof are in order better to realize the antiviral function of vitro recombination CVN, improved L-CVN HIV (human immunodeficiency virus)-resistant activity enhancing (WST method and cytogamy method).Because the LCVN end of the present invention's preparation has been introduced the peptide sequence of hydrophilic flexibility, the fixed point that therefore can carry out the N end is amido modified.Our experimental result proves that LCVN not only can obtain activated modified outcome, and the antiviral activity of modified outcome strengthens further after N end fixed point is amido modified.
Description of drawings
Fig. 1 is pET3c-6His-SUMO-LCVN recombinant plasmid figure.
Fig. 2 is reorganization pET3C-6His-SUMO-LCVN plasmid restriction enzyme and pcr analysis, wherein M:DL2000 DNA marker; Swimming lane 1:pET3C-6His-SUMO-LCVN/Nde I+BamH I; Swimming lane 2:PCR.
Fig. 3 is a SDS-PAGE electrophoretic analysis BL21/pET3c-6His-SUMO-LCVN protein expression characteristic, wherein: from left to right, each swimming lane be followed successively by do not add that IPTG induces, IPTG induces 20h, the broken centrifugal supernatant of abduction delivering, the broken centrifugation of abduction delivering.
Fig. 4 is purifying target protein LCVN, and wherein: from left to right, each swimming lane is followed successively by mixture, the LCVN albumen that Sumo-LCVN fusion rotein, Sumo-LCVN fusion rotein are cut through Sumo proteolytic enzyme enzyme.
(*) expression Sumo-LCVN fusion rotein
(* *) expression LCVN albumen.
Fig. 5 is the purity that RP-HPL analyzes reorganization LCVN.
Fig. 6 is the Tricine-SDS-PAGE electrophorogram that 10K mPEG-ALD modifies LCVN under different pH and the feed ratio condition, wherein: from left to right, swimming lane 1 is albumen Maker, swimming lane 2,3,4 is respectively PH3.5, and LCVN and 10KmPEG-ALD mol ratio are respectively 1: 1,1: 3,1: 5 o'clock modified outcome electrophoretic band; Swimming lane 5,6,7 is respectively PH4.0, and LCVN and 10KmPEG-ALD mol ratio are respectively 1: 1,1: 3,1: 5 o'clock modified outcome electrophoretic band; Swimming lane 8,9,10 is respectively PH5.0, and LCVN and 10KmPEG-ALD mol ratio are respectively 1: 1,1: 3,1: 5 o'clock modified outcome electrophoretic band; Swimming lane 11,12,13 is respectively PH6.0, and LCVN and 10KmPEG-ALD mol ratio are respectively 1: 1,1: 3,1: 5 o'clock modified outcome electrophoretic band; Swimming lane 14,15,16 is respectively PH7.0, and LCVN and 10K mPEG-ALD mol ratio are respectively 1: 1,1: 3,1: 5 o'clock modified outcome electrophoretic band.
Fig. 7 is the modification rate comparison diagram that 10K mPEG-ALD modifies LCVN under different PH and the feed ratio condition.
Fig. 8 is the Tricine-SDS-PAGE electrophorogram that 20K mPEG-ALD modifies LCVN under different PH and the feed ratio condition, wherein: from left to right, swimming lane 1 is albumen Maker, swimming lane 2,3,4 is respectively PH3.5, and LCVN and 20KmPEG-ALD mol ratio are respectively 1: 1,1: 3,1: 5 o'clock modified outcome electrophoretic band; Swimming lane 5,6,7 is respectively PH4.0, and LCVN and 20K mPEG-ALD mol ratio are respectively 1: 1,1: 3,1: 5 o'clock modified outcome electrophoretic band; Swimming lane 8,9,10 is respectively PH5.0, and LCVN and 20KmPEG-ALD mol ratio are respectively 1: 1,1: 3,1: 5 o'clock modified outcome electrophoretic band; Swimming lane 11,12,13 is respectively PH6.0, and LCVN and 20K mPEG-ALD mol ratio are respectively 1: 1,1: 3,1: 5 o'clock modified outcome electrophoretic band; Swimming lane 14,15,16 is respectively PH7.0, and LCVN and 20K mPEG-ALD mol ratio are respectively 1: 1,1: 3,1: 5 o'clock modified outcome electrophoretic band.
Fig. 9 is the modification rate comparison diagram that 20K mPEG-ALD modifies LCVN under different PH and the feed ratio condition.
Figure 10 is under the different time, 10K mPEG-ALD modifies the Tricine-SDS-PAGE electrophorogram of LCVN, wherein: swimming lane 1 is albumen Maker, and swimming lane 2-8 is respectively the sample electrophoresis result behind reaction 1h, 3h, 5h, 7h, 9h, 12h and the 24h, and swimming lane 9 is LCVN of unmodified.
Figure 11 is under the different time, 20K mPEG-ALD modifies the Tricine-SDS-PAGE electrophorogram of LCVN, wherein: swimming lane 1 is albumen Maker, and swimming lane 2-8 is respectively the sample electrophoresis result behind reaction 1h, 3h, 5h, 7h, 9h, 12h and the 24h, and swimming lane 9 is LCVN of unmodified.
Figure 12 is that 10K mPEG-ALD modifies the elution curve of LCVN through the SP-Sepharose separation and purification.
Figure 13 is that 10K mPEG-ALD modifies the Tricine-SDS-PAGE electrophorogram of LCVN through each component of SP-Sepharose separation and purification, wherein: swimming lane 1 is albumen Maker, swimming lane 2-5 be modification reaction mixture, go up the elution peak that sample penetrates the peak, contains the buffer A of 80mM NaCl; The elution peak that contains the buffer A of 400mM NaCl.
Figure 14 is that 20K mPEG-ALD modifies the elution curve of LCVN through the SP-Sepharose separation and purification.
Figure 15 is that 20K mPEG-ALD modifies the Tricine-SDS-PAGE electrophorogram of LCVN through each component of SP-Sepharose separation and purification, wherein: swimming lane 1 is albumen Maker, swimming lane 2-5 be modification reaction mixture, go up the elution peak that sample penetrates the peak, contains the buffer A of 70mM NaCl; The elution peak that contains the buffer A of 400mM NaCl.
Figure 16 is the cell survival rate (%) of MT-4 after different concns LCVN and modified outcome processing thereof.
Figure 17 is that CVN and LCVN are to HIV-1/IIIB inhibition of proliferation rate (%).
Figure 18 is the anti-human immunodeficiency virus activity of LCVN and PEG modified outcome thereof, wherein, co-cultured cell after cell after the common cultivation of the cell after cultivate altogether (1) the MOLT-4 cell under figure (a) phase microscope behind the observation cultivation 24h, (2) MOLT-4/IIIB cell, (3), (4) vacation processing, the CVN/LCVN/10kPEG-LCVN/20kPEG-LCVN processing that (5-8) concentration is 113nM is located the giant cell into merging shown in the black arrow.The fusion of figure (b) CNV, LCVN, 10kPEG-LCVN, 20kPEG-LCVN suppresses active, and all experiments are 3 statistical procedures results behind the independent experiment at least.
Figure 19 is the active CPE observations of LCVN and the anti-HSV-1 of PEG modified outcome thereof.Wherein:
A, normal control; B, virus control; C, positive drug ACV contrast (1 μ g/ml); D, L-CVN sample (1.562 μ g/ml); E, SUMO-L-CVN sample (3.125 μ g/ml); F, mPEG-ALD-10kDa-L-CVN (3.125 μ g/ml); G, mPEG-ALD-20kDa-L-CVN (3.125 μ g/ml).
Embodiment
Below enumerate some preferred embodiment of the invention, understand the present invention to help further, but embodiments of the present invention are not limited thereto.
The main raw that the embodiment of the invention relates to is as follows: host bacterium e. coli bl21 (DE3) (available from Novagen company), plasmid pET3c (available from Novagen company), pET3c-SUMO-CVN preserve (its construction process patent applied for " preparation method of recombined blue algae antiviral protein and application; application number: 200810198926.0 " by this chamber, the structure of plasmid also can adopt known gene engineering method, basic ideas are that at first the method by PCR obtains the SUMO-CVN fusion sequence, are connected into the pET3c carrier again and can obtain plasmid pET3c-SUMO-CVN); SUMO proteolytic enzyme (available from the biological company limited of sea base); Taq enzyme, T4DNA ligase enzyme, dna molecular amount standard, various restriction enzyme are available from the precious biotech firm in Dalian; Protein molecular weight standard product (available from the poly-bio tech ltd that grinds), primer are given birth to worker biotech firm available from Shanghai; Ni
2+Sepharose Fast Flow, SP Sepharose Fast Flow are available from GE Healthcare company; MTT, WST are available from U.S. SIGMA company; Herpes simplex virus type 1 (HSV-1) F strain is from Wuhan University institute of viruses (CGMCCNo.0396); Vero cell (CCL-81
TM), MOLT-4 cell (CRL-1582
TM), MT-4 cell (CRL-1942
TM), HIV-I/IIIB virus (CRL-1973
TM) wait DSMZ (ATCC) from U.S. typical case; MPEG-ALD (10K) and mPEG-ALD (20K) are available from the triumphant positive biotechnology in Beijing Development Co., Ltd.
NTA-0buffer (20mmol/L Tris-HCl, pH 8.0,0.15mol/L NaCl,), NTA-20buffer (20mmol/LTris-HCl, pH 8.0,0.15mol/LNaCl, the 20mmol/L imidazoles), NTA-250buffer (20mmol/L Tris-HCl, pH 8.0,0.15mol/LNaCl, 250mmol/L imidazoles), enzyme cutting buffering liquid (20mmol/L Tris-HCl, pH 8.0,0.15mol/LNaCl), bufferA (20mmol/LNaAc-Ac, pH 4.0).
Embodiment
1, construction recombination plasmid pET3c-6His-SUMO-LCVN
The structure of SUMO-L-CVN gene is synthetic to carry out in two steps, and at first by the synthetic L-CVN gene of twice PCR, PCR is a template with the pET3c-SUMO-CVN plasmid for the first time, is the upstream and downstream primer with F1-CVN, R-CVN.Reaction system is template 1ng, each 1 μ M of upstream and downstream primer, and 20 μ l Taq PCR MasterMix add water to 40 μ l, and 94 ℃ of sex change 1min of reaction mixture are annealed to 55 ℃, keep 1min, and 72 ℃ are extended 1min, carry out 29 circulations.Reaction product is carried out 1% agarose gel electrophoresis, and glue reclaims the purpose fragment, as the template of next round PCR.For the second time PCR is a template with last round of PCR product, F2-CVN, R-CVN be the upstream and downstream primer to (the flexible polypeptide that wherein contains 15 amino-acid residues of encode in the F2-CVN primer), synthesize the L-CVN full length sequence.The SUMO full length sequence is synthetic from pET3c-SUMO-CVN by PCR method.Utilize the terminal overlapping complementary sequence of 26bp with L-CVN sequence front end of SUMO sequence to carry out PCR: with SUMO sequence and L-CVN sequence serves as to extend template, F-SUMO, R-CVN is the upstream and downstream primer, under the PCR of routine condition, react, reaction product is carried out 1% agarose gel electrophoresis, and make glue and reclaim, obtain the SUMO-L-CVN full length sequence.
With plasmid pET3C and 6His-SUMO-LCVN full length sequence use respectively Nde I and with the BamHI double digestion, enzyme is cut product 1% agarose gel electrophoresis, reclaims enzyme and cuts product, T
4Dna ligase connects product transformed into escherichia coli JM109 competent cell, coats on the LB flat board that contains penbritin, and 37 ℃ of overnight incubation are extracted plasmid, and pcr amplification and Nde I and BamHI double digestion identify that positive plasmid is sent to the order-checking of Ying Jun company.
Table 1 is used for the primer of synthetic SUMO-LCVN full length sequence
2, the shake-flask culture of LCVN engineering bacteria, protein purification and purity testing
The plasmid transformation escherichia coli BL21 (DE3) that checks order correct gets engineering bacteria BL21[pET3c-6His-SUMO-LCVN], select mono-clonal and cultivate also abduction delivering, collect tropina and carry out the SDS-PAGE electrophoretic analysis.The result shows BL21[pET3c-6His-SUMO-LCVN] positive colony bacterial strain big or small fusion rotein that is about 28kDa of meeting expression after inducing, as seen do not induce control group not have naked eyes in the corresponding position expresses, behind the ultrasonication thalline, find that fusion rotein is arranged in supernatant, be solubility expression, through the gel densitometric scan, the solubility target protein accounts for 28.3 ± 3.4% (Fig. 3) of supernatant total protein.
Choose the high expression level inoculation and contain to 1L in the LB substratum of penbritin (100mg/L), 37 ℃, 180rpm is cultured to OD600=0.6～1.0 o'clock, is cooled to 20 ℃ and adds IPTG to final concentration 0.5mM abduction delivering 24h.4 ℃, the centrifugal collection thalline of 6000 * g, 10min, freeze thawing once is suspended in NTA-10buffer with bacterial sediment with 1: 10 ratio then again, ultrasonication (working hour 5s, intermittent time 5s, 99 times, repeat 3 times), 4 ℃, the centrifugal collection supernatant of 25000 * g, 30min.
The sample column volume is the Ni-NTA affinity column of 20ml on the supernatant liquor, flow velocity 0.6ml/min, and NTA-0buffer washes back baseline, and flow velocity is 1ml/min, and NTA-20buffer washes foreign protein, NTA-250buffer wash-out target protein.Purified target protein 6His-SUMO-LCVN carries out SUMO proteolytic enzyme enzyme and cuts after Sephadex G-25 molecular sieve takes off imidazoles, remove the SUMO fusion rotein.
6His-SUMO-LCVN adjusts concentration to 1mg/ml, adds 1U SUMO proteolytic enzyme/mg fusion rotein, and 30 ℃ of enzymes are cut 1h.Because of 6His-SUMO label, SUMO proteolytic enzyme all contain 6 * His label, sample after enzyme is cut is gone up the affine layer of sample Ni-NTA post once more and is carried out purifying, remove the SUMO of band 6His label, 6His-SUMO-LCVN and the SUMO proteolytic enzyme cut of enzyme not, obtain nonfused target protein LCVN, through G-25 molecular sieve column desalination postlyophilization, get the LCVN goods, modify for follow-up physical and chemical property determining, determination of activity or PEG and use.
Fig. 3 is engineering bacteria BL21[pET3c-6His-SUMO-LCVN] protein expression collection of illustrative plates after IPTG induces, as seen after inducing, thick protein band (swimming lane 3) appears obviously increasing at molecular weight 28kD place, conform to the theoretical molecular of 6His-SUMO-LCVN, after the ultrasonication, target protein is arranged in the supernatant behind the bacterial cell disruption, and content accounts for 40% (swimming lane 2) of thalline soluble proteins.Fig. 4 is that SDS-PAGE analyzes the LCVN purge process, wherein locates to 6His-SUMO-LCVN fusion rotein shown in the *, locates the albumen into LCVN shown in the * *.Fig. 5 is reverse high performance liquid chromatography (RP-HPLC) the purity check result of LCVN goods, plant type is the reverse post of C-18, the detector wavelength is 280nm, mobile phase A: the ultrapure water that contains 0.1% trifluoroacetic acid (TFA), Mobile phase B: second cyanogen, B through 40%-60% carries out gradient elution, and the elution peak of LCVN appears in retention time between 4-6min.
3, the pilot scale fermentation of LCVN engineering bacteria and protein Preparation
Engineering bacteria BL21[pET3c-6His-SUMO-LCVN], select that mono-clonal is cultivated and in test tube abduction delivering, choose the high expression level bacterial strain and carry out pilot scale fermentation.In Erlenmeyer flask, cultivate the firsts and seconds seed respectively, cultivate secondary seed, be inoculated into 10% inoculum size in the fermention medium of 15L to suitable concn.A.T.C is 37 ℃, adjusts rotating speed and air flow to keep suitable dissolved oxygen level in good time, and it is 7.0～7.2 that NaOH and HCl regulate PH.As cell density (OD
600) when being 12 left and right sides, temperature is reduced to 20 ℃, adds IPTG to final concentration 0.5mM abduction delivering 20h.100ml/min, 9000g, continuous flow centrifugation is collected thalline, and the fermention medium of 15L can be gathered in the crops the wet thallus of about 550g, and the thalline of collection is preserved in-20 ℃ of refrigerators.
Bacterial sediment is suspended in NTA-0buffer with 1: 10 ratio, ultrasonication (working hour 5s, intermittent time 5s 99 times, repeat 3 times), 4 ℃, the centrifugal collection supernatant of 25000 * g, 30min.Column volume is that the Ni-NTA filler applied sample amount of 20ml is the 250ml supernatant, flow velocity 1ml/min, and NTA-0buffer washes back baseline; Flow velocity is 1ml/min, and NTA-20buffer washes foreign protein, NTA-250buffer wash-out target protein.Purified target protein 6His-SUMO-LCVN carries out SUMO proteolytic enzyme enzyme and cuts after Sephadex G-25 molecular sieve takes off imidazoles, remove the SUMO fusion rotein.
6His-SUMO-LCVN adjusts concentration to 1mg/ml, adds 1U SUMO proteolytic enzyme/mg fusion rotein, and 30 ℃ of enzymes are cut 1h.Because of 6His-SUMO label, SUMO proteolytic enzyme all contain 6 * His label, sample after enzyme is cut is gone up the affine layer of sample Ni-NTA post once more and is carried out purifying, remove the SUMO of band 6His label, 6His-SUMO-LCVN and the SUMO proteolytic enzyme cut of enzyme not, obtain nonfused target protein LCVN, through G-25 molecular sieve column exchange buffering liquid, ultrafiltration pipe with interception 3KD concentrates LCVN albumen again, modifies for follow-up physical and chemical property determining, determination of activity or PEG and uses.
4, the proteic PEG of LCVN modifies
It is to improve the effective ways of its medicine for multiple patent medicine character such as characteristic, stability and immunogenicities that pharmaceutical protein is carried out the PEG modification.Protein can comprise that side chain amino, N end amino, side chain carboxyl group, C end carboxyl, side chain sulfydryl etc. are multiple for the site that PEG modifies.Existing carboxyl modified technology is easy to generate non-specific crosslinking reaction, and therefore amido modified more commonly used, technical development is also more ripe.The result of study of Zappe etc. shows, side chain amino or the amino modification of N end to wild-type CVN all can destroy this proteic activity, therefore, the author at first carries out rite-directed mutagenesis to CVN, obtain the Q62C mutant, Cys to 62 introducings carries out the side chain sulfydryl modification then, has obtained activated albumen.Owing to form 2 pairs of disulfide linkage among the natural CVN between existing 4 Cys residues, and in solution, proteinic disulfide linkage is in dynamic heterogeneous and equilibrium state, therefore after introducing the Q62C sudden change, be difficult to avoid the Cys that introduces not disturb the correct collocation of disulfide linkage, perhaps non-62 Cys modifies, and the last experimental result of author proves that also the activity of CVN Q62C mutant and modified outcome thereof all is lower than wild-type CVN.
Because the LCVN end of the present invention preparation has been introduced 15 peptide sequences of hydrophilic flexibility, the fixed point that therefore can attempt carrying out the N end is amido modified.Our experimental result proves that LCVN not only can obtain activated modified outcome, and the antiviral activity of modified outcome strengthens further after N end fixed point is amido modified.
PEG modifying method to amino is a lot, alternative modifier is also a lot, the present invention selects mPEG-ALD (10K) and mPEG-ALD (20K) as modifier, and from substrate: the modifier ratio, modify 3 aspects such as pH, modification reaction time, the best PEG that has screened LCVN modifies condition.
Selecting pH is 3.5,4.0,5.0,60,7.0, and ionic strength is the Na-Ac damping fluid of 20mM, and the concentration of LCVN is 5mg/ml in the reaction system, and the mol ratio of LCVN and mPEG-ALD (10K) is chosen as 1: 1 respectively, 1: 3, and 1: 5; NaCNBH
3Final concentration be 5mg/ml.Under the room temperature, shake reaction on the vibrator, take a sample behind reaction 3h, electrophoresis is identified the effect of modification reaction.MPEG-ALD (10K) modifies the about 30KD of the apparent molecular weight of mono-modified product on SDS-PAGE of LCVN, and the SDS-PAGE analytical results shows, at pH4.0, the mol ratio of LCVN and mPEG-ALD (10K) is under 1: 3 the condition, mono-modified rate the highest (Fig. 6).
Similar approach, research mPEG-ALD (20K) modifies the best of LCVN and modifies pH and feed ratio.After mPEG-ALD (20K) modifies LCVN, the about 50KD of the apparent molecular weight of mono-modified product on SDS-PAGE, experimental result shows: at pH5.0, the mol ratio of LCVN and mPEG-ALD (20K) is under 1: 1 the condition, mono-modified rate the highest (Figure 18).
Determining under best the modification pH and feed ratio that mPEG-ALD (10K) and mPEG-ALD (20K) modify LCVN respectively in reaction 1h, 3h, 5h, 7h, 9h, 12h and 24h sampling, the Tricine-SDS-PAGE electrophoresis is identified the best reaction times.The SDS-PAGE analytical results shows: the reaction of reaction times to mPEG-ALD (10K) and mPEG-ALD (20K) modification LCVN has no significant effect, take all factors into consideration time cost, the preferred best modification reaction time is room temperature oscillatory reaction 2h (Figure 10 and Figure 11).
5, the separation and purification of the PEG modified outcome of LCVN
LCVN mainly contains the LCVN of unmodified in the reaction mixture after PEG-ALD modifies, remaining PEG, the PEG LCVN that modify, the LCVN that PEG is mono-modified more.Adopt AKTA prime plus separation and purification system, the mono-modified mPEG-ALD-LCVN of SP sepharose chromatography column separation and purification, moving phase is the Na-Ac damping fluid (buffer A) of 20mM, first bufferA balance pillar before the last sample with 5 times of column volumes, collect behind the last sample and penetrate the peak, carry out wash-out with the bufferA that contains different concns NaCl then, collect each elution peak.Flow velocity is 1ml/min, and detecting wavelength is 280nm.The sample of collecting carries out the SDS-PAGE detected through gel electrophoresis.
MPEG-ALD (10K) modifies the mixture of LCVN, after SP sepharose positively charged ion chromatography purifying, modifier mPEG-ALD and many modified outcomes do not combine and are penetrated with SP sepharose, and the bufferA eluted product that contains 80mM NaCl is mono-modified mPEG-ALD
(10K)-LCVN; The buffer A eluted product that contains 400mM NaCl is the LCVN of unmodified.Figure 12 is mPEG-ALD
(10K)The elution curve of-LCVN separation and purification, peak 1 are modifier and many modified outcomes, and peak 2 is mono-modified product, and peak 3 is the unmodified substrate.Figure 13 analyzes each wash-out component for SDS-PAGE, and swimming lane M is a protein molecular weight standard; Swimming lane A is last all product, visible unmodified substrate, mono-modified product and many modified outcomes; Swimming lane 1 is to penetrate the peak; Swimming lane 2 is mono-modified products, just target protein mPEG-ALD
(10K)-LCVN; Swimming lane 3 is the unmodified product.
MPEG-ALD (20K) modifies the mixture of LCVN, after SP sepharose positively charged ion chromatography purifying, modifier mPEG-ALD and many modified outcomes also are present in sample and penetrate in the peak, and the buffer A eluted product that contains 70mM NaCl is mono-modified mPEG-ALD
(20K)-LCVN; The buffer A eluted product that contains 400mM NaCl is the LCVN of unmodified.
Figure 14 is mPEG-ALD
(20K)The elution curve of-LCVN separation and purification, peak 1 are modifier and many modified outcomes, and peak 2 is mono-modified product, and peak 3 is the unmodified substrate.Figure 15 analyzes each wash-out component for SDS-PAGE, and swimming lane M is a protein molecular weight standard; Swimming lane A is last all product, visible unmodified substrate, mono-modified product and many modified outcomes; Swimming lane 1 is to penetrate the peak; Swimming lane 2 is mono-modified products, just target protein mPEG-ALD
(10K)-LCVN; Swimming lane 3 is a 400mMNaCl wash-out component, visible unmodified product, but also contain
The mono-modified product of part is arranged.
Application Example 1WST method is measured the cytotoxicity of LCVN to the T cell
CVN, LCVN and PEG modified outcome are made 5 times of serial dilutions with the RPMI-1640 nutrient solution, add in the 96 porocyte culture plates, every hole 50 μ l, CVN and LCVN dilution range are 10 μ g/ml to 0.04 μ g/ml, the dilution range that PEG modifies LCVN is 50 μ g/ml to 0.19 μ g/ml.The MT-4 cell is adjusted concentration to 1 * 10
5/ ml, every hole 100 μ l, mixing is put 37 ℃, 5%CO
2Cultivate in the cell culture incubator, (the 63nM Zidovodine, AZT), holes are answered in 3 of the equal replicate(determination)s of each sample to establish cell contrast and positive drug contrast simultaneously.After 4 days in the culture plate every hole add 10 μ l WST-1 (water-soluble tetrazoles, 5mmol/L) solution, continue to cultivate 4 hours, put and read to read on the plate instrument light absorption value (A), wavelength 450/650nm calculates cell survival rate (Relative percentage, RP, %), and according to RP calculate 50% toxic concentration (CC
50).
Cell survival rate (Relative percentage, %)=drug treating group A value/cell control group A value * 100%.
Figure 16 is the cell survival rate after 4 kinds of albumen such as different concns CVN, LCVN and modified outcome thereof are handled MT-4, and N.C. refers to untreated cell contrast among the figure, and the density that contrasts with cell is 100%.The positive control drug of AZT, the 63nM Zidovodine.50% toxic concentration as calculated is as shown in table 2.
50% toxic concentration of table 2LCVN and PEG modified outcome thereof
Application Example 2WST method is measured the anti-human immunodeficiency virus activity of LCVN
CVN, LCVN and PEG modified outcome are made 5 times of serial dilutions with the RPMI-1640 nutrient solution, add in the 96 porocyte culture plates every hole 50 μ l.The MT-4 cell is adjusted concentration to 1 * 10
5/ ml, every hole 100 μ l, mixing adds HIV-1/IIIB viral suspension 50 μ l then, and titre is 100TCID50.Put 37 ℃, 5%CO
2Cultivate in the cell culture incubator, (Zidovodine, AZT), holes are answered in 3 of the equal replicate(determination)s of each sample to establish cell contrast, virus control and positive drug contrast simultaneously.After 4 days in the culture plate every hole add 10 μ l WST-1 (water-soluble tetrazole, 5mmol/L) solution continue to be cultivated 4 hours, put and read to read on the plate instrument light absorption value (A), wavelength 450/650nm.
Cell survival rate (Relative percentage, %)=drug treating group A value/cell control group A value * 100%.
Virus inhibiting rate (%)=(drug treating group A
450/650-virus control group A
450/650)/(cell control group A
450/650-virus control group A
450/650) * 100%
The Reed-Muench method is calculated 50% inhibition concentration (IC50), and according to embodiment * 50% toxic concentration (CC50), and calculate selectivity index (TI), TI=CC50/IC50.
As can be seen from Table 3, for positive drug AZT, CVN, LCVN are stronger to HIV-1/IIIB inhibition of proliferation activity.
Table 3CVN and LCVN are to HIV-1/IIIB inhibition of proliferation activity
Application Example 3 merges the anti-human immunodeficiency virus activity that the inhibition method is measured LCVN and PEG modified outcome thereof
Existing studies show that the propagation of HIV between the T cell mainly is the mediation by the gp120 of cells infected surface expression, and with the cell that does not infect on receptors bind, form fused cell and take place.CVN can specificity suppresses fusion between cells infected and the normal cell and the propagation of blocking virus in conjunction with gp120, therefore, our application cell merges antiviral activity [the Tochikura TS that suppresses model determination LCVN and derivative thereof, Nakashima H, Tanabe A, YamamotoN.Human immunodeficiency virus (HIV)-induced cell fusion:quantification and its applicationfor the simple and rapid screening of anti-HIV substances in vitro.Virology, 1988,164 (2): 542-546].
The MOLT-4 cell and the MOLT-4/IIIB cell that grow to logarithmic phase are adjusted density to 1 * 10
6/ ml respectively gets 250 μ l cell suspensions, and equal-volume mixes, and adds to that (total cellular score is 5 * 10 in the 24 porocyte culture plates
5/ 500 μ l/ holes); CVN, LCVN and PEG modified outcome thereof are made 4 times of serial dilutions to contain foetal calf serum and antibiotic RPMI-1640 substratum, every kind is subjected to the reagent thing to select 3 concentration (452nM, 113nM, 28nM), equal-volume adds in the cell suspension, various control groups are set simultaneously, parallel 2 the multiple holes of all samples, 37 ℃, 5%CO
2Cultivated 24 hours.Obtained cell suspension behind the 24h charges into the cell counting pond after the trypan blue dyeing, the MOLT-4 cell number of microscopically counting survival.Because the MOLT-4/IIIB cell can constantly produce the HIV-I/IIIB virion, and by with the fusion of normal MOLT-4 cell, form big syncyte (synplasm) and infect the normal host cell, when cell counting, fused cell can't enter counting cell, therefore, normal size MOLT-4 or MOLT-4/IIIB cell count in the counting cells counting cell, can calculate and form plasmodial cell number, the cell count of administration cultivation group altogether and do not carry out the MOLT-4 cell count of common cultivation group relatively, the calculating fusion index (FI, fusionindex):
FI=1-(the cell count ÷ in co-cultured cell hole only contains the cell count in the control wells of MOLT-4 cell)
Relatively administration co-cultured cell and the not fusion index of administration co-cultured cell, calculate and merge inhibiting rate (FIR, fusioninhibition rate):
FIR (%)=[1-(FI
T/ FI
C)] * 100, FI wherein
TBe the fusion index of administration sample, FI
CIt is the fusion index of administration co-cultured cell not.
The fusion inhibiting rate (%) of table 4LCVN and PEG modified outcome pair cell thereof
Table 4 as can be seen, no matter LCVN suppresses active in what dosage group, all apparently higher than CVN to the fusion of HIV-1/IIIB; In addition, when middle dosage and high dosage, the activity of the PEG modified outcome of LCVN will be higher than the LCVN of unmodified.
Figure 18 (a) is the co-cultured cell after the cell after the false common cultivation of handling in cell, (4) of observe cultivating under the phase microscope after cultivate altogether (1) MOLT-4 cell, (2) MOLT-4/IIIB cell, (3) behind the 24h, CVN/LCVN/10kPEG-LCVN/20kPEG-LCVN that (5-8) concentration is 113nM handle, locate giant cell shown in the black arrow into merging, as seen be not total to amixis cell in the cultivation group, typical fused cell occurs in the cultivation group altogether, and the administration group be can't see fused cell substantially.
Figure 18 (b) is that the fusion of CNV, LCVN, 10kPEG-LCVN, 20kPEG-LCVN suppresses active, and all experiments are 3 statistical procedures results behind the independent experiment at least.No matter as seen LCVN suppresses active in what dosage group, all apparently higher than CVN to the fusion of HIV-1/IIIB; In addition, when middle dosage and high dosage, the PEG modified outcome of LCVN is along with the increase of molecular weight, and activity progressively strengthens, but in low dose group, the activity of PEG modified outcome increases and progressively reduces with molecular weight.
Application Example 4MTT method is measured anti-herpes simplex virus (HSV-1) activity of LCVN
Herpes simplex virus I-type (HSV-I) is a kind of dna virus that causes extensive infection in the crowd, and the mankind are its unique hosts, has 90% to infect HSV-1 in the health adult approximately.HSV-I can cause multiple diseases such as herpes labialis, herpetic keratoconjunctivitis, encephalitis neonatorum and since HSV-I can be in neuroganglion latent infection, so symptom easily recurs.This experiment is measured reorganization LCVN and modified outcome thereof, the wild-type CVN toxicity to the Vero cell with mtt assay; The CPE method is observed the activity of medicine pair cell.
In the individual layer Vero cell, add different dilution each 50 μ l of HSV-1 virus liquid that are subjected to reagent thing and 100TCID50, establish normal cell contrast and virus control group simultaneously.5%CO
2Cultivate 48h, every hole adds 5mg/ml MTT 10 μ l, 5%CO
2Continue to cultivate 4h, abandon supernatant liquor, every hole adds 200 μ l DMSO, the room temperature lucifuge is placed 30min, about jolting culture plate 10min, elisa reading instrument colorimetric (wavelength 570nm, reference wavelength 630nm), measure 50% toxic concentration (50%cytotoxic concentration, the CC of absorbancy and calculation sample
50).
Figure 19 is the form of cell under phase microscope behind model experiment, the cytopathic effect due to the visible viral pair cell, and the protective effect of medicine pair cell.The result shows that LCVN and modified outcome thereof all have good anti-HSV-1 activity, and under the condition approaching in mass concentration, that volumetric molar concentration is lower, LCVN and PEG modified outcome thereof show the approaching substantially antiviral activity with positive control medicine ACV.Toxicity test result shows that the LCVN after PEG modifies also obviously reduces the toxicity of Vero cell.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
SEQUENCE?LISTING
＜110〉Ji'nan University
＜120〉a kind of blue-green algal virus protein N mutant, its modified derivative and application
<130>090831
<160>11
<170>PatentIn?version?3.3
<210>1
<211>15
<212>PRT
＜213〉artificial sequence
<220>
<221>PEPTIDE
<222>(1)..(15)
＜223〉flexible peptide linker (linker)
<400>1
Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
1 5 10 15
<210>2
<211>101
<212>PRT
＜213〉artificial sequence
<220>
<221>PEPTIDE
<222>(1)..(101)
＜223〉aminoacid sequence of CVN
<400>2
Leu?Gly?Lys?Phe?Ser?Gln?Thr?Cys?Tyr?Asn?Ser?Ala?Ile?Gln?Gly?Ser
1 5 10 15
Val?Leu?Thr?Ser?Thr?Cys?Glu?Arg?Thr?Asn?Gly?Gly?Tyr?Asn?Thr?Ser
20 25 30
Ser?Ile?Asp?Leu?Asn?Ser?Val?Ile?Glu?Asn?Val?Asp?Gly?Ser?Leu?Lys
35 40 45
Trp?Gln?Pro?Ser?Asn?Phe?Ile?Glu?Thr?Cys?Arg?Asn?Thr?Gln?Leu?Ala
50 55 60
Gly?Ser?Ser?Glu?Leu?Ala?Ala?Glu?Cys?Lys?Thr?Arg?Ala?Gln?Gln?Phe
65 70 75 80
Val?Ser?Thr?Lys?Ile?Asn?Leu?Asp?Asp?His?Ile?Ala?Asn?Ile?Asp?Gly
85 90 95
Thr?Leu?Lys?Tyr?Glu
100
<210>3
<211>348
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)..(348)
＜223〉coding nucleotide sequence of LCVN
<400>3
ggt?ggc?gga?ggg?agc?ggt?gga?ggg?ggc?agt?ggc?gga?gga?ggt?agc?ctt 48
Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Leu
1 5 10 15
ggt?aaa?ttc?tcc?cag?acc?tgc?tac?aac?tcc?gct?atc?cag?ggt?tct?gtt 96
Gly?Lys?Phe?Ser?Gln?Thr?Cys?Tyr?Asn?Ser?Ala?Ile?Gln?Gly?Ser?Val
20 25 30
ctg?acc?tct?acc?tgc?gaa?cgt?acc?aac?ggt?ggt?tac?aac?acc?tcc?tct 144
Leu?Thr?Ser?Thr?Cys?Glu?Arg?Thr?Asn?Gly?Gly?Tyr?Asn?Thr?Ser?Ser
35 40 45
atc?gac?ctg?aac?tcc?gtt?atc?gaa?aac?gtt?gac?ggt?tct?ctg?aaa?tgg 192
Ile?Asp?Leu?Asn?Ser?Val?Ile?Glu?Asn?Val?Asp?Gly?Ser?Leu?Lys?Trp
50 55 60
cag?ccg?tct?aac?ttc?atc?gaa?acc?tgc?cgt?aac?acc?cag?ctg?gct?ggt 240
Gln?Pro?Ser?Asn?Phe?Ile?Glu?Thr?Cys?Arg?Asn?Thr?Gln?Leu?Ala?Gly
65 70 75 80
tcc?tct?gaa?ctg?gct?gct?gaa?tgc?aaa?acc?cgt?gct?cag?cag?ttc?gtt 288
Ser?Ser?Glu?Leu?Ala?Ala?Glu?Cys?Lys?Thr?Arg?Ala?Gln?Gln?Phe?Val
85 90 95
tct?acc?aaa?atc?aac?ctg?gac?gac?cac?atc?gct?aac?atc?gac?ggt?acc 336
Ser?Thr?Lys?Ile?Asn?Leu?Asp?Asp?His?Ile?Ala?Asn?Ile?Asp?Gly?Thr
100 105 110
ctg?aaa?tac?gaa 348
Leu?Lys?Tyr?Glu
115
<210>4
<211>116
<212>PRT
＜213〉artificial sequence
<400>4
Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Leu
1 5 10 15
Gly?Lys?Phe?Ser?Gln?Thr?Cys?Tyr?Asn?Ser?Ala?Ile?Gln?Gly?Ser?Val
20 25 30
Leu?Thr?Ser?Thr?Cys?Glu?Arg?Thr?Asn?Gly?Gly?Tyr?Asn?Thr?Ser?Ser
35 40 45
Ile?Asp?Leu?Asn?Ser?Val?Ile?Glu?Asn?Val?Asp?Gly?Ser?Leu?Lys?Trp
50 55 60
Gln?Pro?Ser?Asn?Phe?Ile?Glu?Thr?Cys?Arg?Asn?Thr?Gln?Leu?Ala?Gly
65 70 75 80
Ser?Ser?Glu?Leu?Ala?Ala?Glu?Cys?Lys?Thr?Arg?Ala?Gln?Gln?Phe?Val
85 90 95
Ser?Thr?Lys?Ile?Asn?Leu?Asp?Asp?His?Ile?Ala?Asn?Ile?Asp?Gly?Thr
100 105 110
Leu?Lys?Tyr?Glu
115
<210>5
<211>669
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)..(669)
＜223〉nucleotide sequence of coding His-SUMO-LCVN in the pET3c-6His-SUMO-LCVN carrier
<400>5
atg?cat?cat?cat?cat?cat?cac?ggc?atg?tcg?gac?tca?gaa?gtc?aat?caa 48
Met?His?His?His?His?His?His?Gly?Met?Ser?Asp?Ser?Glu?Val?Asn?Gln
1 5 10 15
gaa?gct?aag?cca?gag?gtc?aag?cca?gaa?gtc?aag?cct?gag?act?cac?atc 96
Glu?Ala?Lys?Pro?Glu?Val?Lys?Pro?Glu?Val?Lys?Pro?Glu?Thr?His?Ile
20 25 30
aat?tta?aag?gtg?tcc?gat?gga?tct?tca?gag?atc?ttc?ttc?aag?atc?aaa 144
Asn?Leu?Lys?Val?Ser?Asp?Gly?Ser?Ser?Glu?Ile?Phe?Phe?Lys?Ile?Lys
35 40 45
aag?acc?act?cct?tta?aga?agg?ctg?atg?gaa?gcg?ttc?gct?aaa?aga?cag 192
Lys?Thr?Thr?Pro?Leu?Arg?Arg?Leu?Met?Glu?Ala?Phe?Ala?Lys?Arg?Gln
50 55 60
ggt?aag?gaa?atg?gac?tcc?tta?aga?ttc?ttg?tac?gac?ggt?att?aga?att 240
Gly?Lys?Glu?Met?Asp?Ser?Leu?Arg?Phe?Leu?Tyr?Asp?Gly?Ile?Arg?Ile
65 70 75 80
caa?gct?gat?cag?acc?cct?gaa?gat?ttg?gac?atg?gag?gat?aac?gat?atc 288
Gln?Ala?Asp?Gln?Thr?Pro?Glu?Asp?Leu?Asp?Met?Glu?Asp?Asn?Asp?Ile
85 90 95
att?gag?gct?cac?aga?gaa?cag?att?ggt?ggt?ggt?ggc?gga?ggg?agc?ggt 336
Ile?Glu?Ala?His?Arg?Glu?Gln?Ile?Gly?Gly?Gly?Gly?Gly?Gly?Ser?Gly
100 105 110
gga?ggg?ggc?agt?ggc?gga?gga?ggt?agc?ctt?ggt?aaa?ttc?tcc?cag?acc 384
Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Leu?Gly?Lys?Phe?Ser?Gln?Thr
115 120 125
tgc?tac?aac?tcc?gct?atc?cag?ggt?tct?gtt?ctg?acc?tct?acc?tgc?gaa 432
Cys?Tyr?Asn?Ser?Ala?Ile?Gln?Gly?Ser?Val?Leu?Thr?Ser?Thr?Cys?Glu
130 135 140
cgt?acc?aac?ggt?ggt?tac?aac?acc?tcc?tct?atc?gac?ctg?aac?tcc?gtt 480
Arg?Thr?Asn?Gly?Gly?Tyr?Asn?Thr?Ser?Ser?Ile?Asp?Leu?Asn?Ser?Val
145 150 155 160
atc?gaa?aac?gtt?gac?ggt?tct?ctg?aaa?tgg?cag?ccg?tct?aac?ttc?atc 528
Ile?Glu?Asn?Val?Asp?Gly?Ser?Leu?Lys?Trp?Gln?Pro?Ser?Asn?Phe?Ile
165 170 175
gaa?acc?tgc?cgt?aac?acc?cag?ctg?gct?ggt?tcc?tct?gaa?ctg?gct?gct 576
Glu?Thr?Cys?Arg?Asn?Thr?Gln?Leu?Ala?Gly?Ser?Ser?Glu?Leu?Ala?Ala
180 185 190
gaa?tgc?aaa?acc?cgt?gct?cag?cag?ttc?gtt?tct?acc?aaa?atc?aac?ctg 624
Glu?Cys?Lys?Thr?Arg?Ala?Gln?Gln?Phe?Val?Ser?Thr?Lys?Ile?Asn?Leu
195 200 205
gac?gac?cac?atc?gct?aac?atc?gac?ggt?acc?ctg?aaa?tac?gaa?tga 669
Asp?Asp?His?Ile?Ala?Asn?Ile?Asp?Gly?Thr?Leu?Lys?Tyr?Glu
210 215 220
<210>6
<211>222
<212>PRT
＜213〉artificial sequence
<400>6
Met?His?His?His?His?His?His?Gly?Met?Ser?Asp?Ser?Glu?Val?Asn?Gln
1 5 10 15
Glu?Ala?Lys?Pro?Glu?Val?Lys?Pro?Glu?Val?Lys?Pro?Glu?Thr?His?Ile
20 25 30
Asn?Leu?Lys?Val?Ser?Asp?Gly?Ser?Ser?Glu?Ile?Phe?Phe?Lys?Ile?Lys
35 40 45
Lys?Thr?Thr?Pro?Leu?Arg?Arg?Leu?Met?Glu?Ala?Phe?Ala?Lys?Arg?Gln
50 55 60
Gly?Lys?Glu?Met?Asp?Ser?Leu?Arg?Phe?Leu?Tyr?Asp?Gly?Ile?Arg?Ile
65 70 75 80
Gln?Ala?Asp?Gln?Thr?Pro?Glu?Asp?Leu?Asp?Met?Glu?Asp?Asn?Asp?Ile
85 90 95
Ile?Glu?Ala?His?Arg?Glu?Gln?Ile?Gly?Gly?Gly?Gly?Gly?Gly?Ser?Gly
100 105 110
Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Leu?Gly?Lys?Phe?Ser?Gln?Thr
115 120 125
Cys?Tyr?Asn?Ser?Ala?Ile?Gln?Gly?Ser?Val?Leu?Thr?Ser?Thr?Cys?Glu
130 135 140
Arg?Thr?Asn?Gly?Gly?Tyr?Asn?Thr?Ser?Ser?Ile?Asp?Leu?Asn?Ser?Val
145 150 155 160
Ile?Glu?Asn?Val?Asp?Gly?Ser?Leu?Lys?Trp?Gln?Pro?Ser?Asn?Phe?Ile
165 170 175
Glu?Thr?Cys?Arg?Asn?Thr?Gln?Leu?Ala?Gly?Ser?Ser?Glu?Leu?Ala?Ala
180 185 190
Glu?Cys?Lys?Thr?Arg?Ala?Gln?Gln?Phe?Val?Ser?Thr?Lys?Ile?Asn?Leu
195 200 205
Asp?Asp?His?Ile?Ala?Asn?Ile?Asp?6ly?Thr?Leu?Lys?Tyr?Glu
210 215 220
<210>7
<211>45
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(45)
＜223〉primers F 1-CVN
<400>7
ggagggggca?gtggcggagg?aggtagcctt?ggtaaattct?cccag 45
<210>8
<211>49
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(69)
＜223〉primers F 2-CVN
<220>
<221>misc_feature
<222>(1)..(49)
＜223〉primers F-CVN
<400>8
cagattggtg?gtggtggcgg?agggagcggt?ggagggggca?gtggcggag 49
<210>9
<211>32
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(32)
＜223〉primer R-CVN
<400>9
agaggatcct?catcattcgt?atttcagggt?ac 32
<210>10
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(22)
＜223〉primers F-SUM0
<400>10
cagcatatgc?atcatcatca?tc 22
<210>11
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(33)
＜223〉primer R-SUMO
<400>11
ctccctccgc?caccaccacc?aatctgttct?ctg 33
Claims (10)
1. blue-green algal virus protein N mutant, it is characterized in that: its aminoacid sequence is made up of sequence A and sequence B, and sequence A is positioned at the N end of sequence B,
Sequence A is shown in the SEQ ID NO:1 in the sequence table;
Sequence B is shown in the SEQ ID NO:2 in the sequence table.
2. the coding nucleotide sequence of the described blue-green algal virus protein N mutant of claim 1.
3. according to the coding nucleotide sequence of the described blue-green algal virus protein N mutant of claim 2, it is characterized in that: shown in the SEQ ID NO:3 in the sequence table.
4. an expression vector is characterized in that: contain claim 2 or 3 described nucleotide sequences.
5. a host bacterium is characterized in that: contain claim 2 or 3 described nucleotide sequences.
6. blue-green algal virus protein N mutant modified derivative is characterized in that: be that N-terminal with the described blue-green algal virus protein N mutant of claim 1 carries out PEG and modifies.
7. according to the described blue-green algal virus protein N mutant modified derivative of claim 6, it is characterized in that: the modifier that described PEG modifies uses mono methoxy ether PEG-propionic aldehyde.
8. according to the described blue-green algal virus protein N mutant modified derivative of claim 7, it is characterized in that: the molecular weight of described mono methoxy ether PEG-propionic aldehyde is 10KD-20KD.
9. the application of the described blue-green algal virus protein N mutant of claim 1 in the medicine of preparation HIV-I resisting.
10. the application of each described blue-green algal virus protein N mutant modified derivative in the medicine of preparation HIV-I resisting among the claim 6-8.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101920630A CN101638435B (en) | 2009-09-04 | 2009-09-04 | Blue-green algal virus protein N mutant, modified derivative and application thereof |
| IN2837DEN2012 IN2012DN02837A (en) | 2009-09-04 | 2010-05-24 | |
| PCT/CN2010/073124 WO2011026351A1 (en) | 2009-09-04 | 2010-05-24 | A cyanovirin n mutant, modified derivative and uses thereof |
| JP2012527184A JP5592490B2 (en) | 2009-09-04 | 2010-05-24 | Cyanovirin-N mutant, modified derivative thereof and application thereof |
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| CN2009101920630A CN101638435B (en) | 2009-09-04 | 2009-09-04 | Blue-green algal virus protein N mutant, modified derivative and application thereof |
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| CN101638435A CN101638435A (en) | 2010-02-03 |
| CN101638435B true CN101638435B (en) | 2011-11-16 |
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| JP (1) | JP5592490B2 (en) |
| CN (1) | CN101638435B (en) |
| IN (1) | IN2012DN02837A (en) |
| WO (1) | WO2011026351A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101638435B (en) * | 2009-09-04 | 2011-11-16 | 暨南大学 | Blue-green algal virus protein N mutant, modified derivative and application thereof |
| UA116431C2 (en) * | 2013-02-05 | 2018-03-12 | Агрікалчерал Текнолоджи Рісьоч Інстітьют | COMPOSITION FOR PREVENTION OF INFECTION MYCOPLASMA SPP. |
| CN110903363A (en) * | 2019-11-26 | 2020-03-24 | 中国药科大学 | Preparation method and application of blue algae antiviral protein N |
| CN111494604B (en) * | 2020-05-11 | 2022-05-06 | 中国药科大学 | Application of blue algae antiviral protein N in preparation of anti-inflammatory drugs |
| US20230355708A1 (en) * | 2020-09-29 | 2023-11-09 | Drexel University | Compositions for inhibiting viral entry and methods using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5843882A (en) * | 1995-04-27 | 1998-12-01 | The United States Of America As Represented By The Department Of Health And Human Services | Antiviral proteins and peptides |
| EP1488804A4 (en) * | 2002-03-22 | 2006-10-25 | Bipha Corp | Immunoglobulin/hydrophilic peptide complexes |
| AU2003301122B2 (en) * | 2002-12-19 | 2009-11-26 | Nektar Therapeutics | Cyanovirin variant-polymer conjugates |
| CN101705241B (en) * | 2008-09-28 | 2012-06-06 | 暨南大学 | Method for preparing recombined blue algae antiviral protein and application thereof |
| CN101638435B (en) * | 2009-09-04 | 2011-11-16 | 暨南大学 | Blue-green algal virus protein N mutant, modified derivative and application thereof |
-
2009
- 2009-09-04 CN CN2009101920630A patent/CN101638435B/en not_active Expired - Fee Related
-
2010
- 2010-05-24 WO PCT/CN2010/073124 patent/WO2011026351A1/en active Application Filing
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- 2010-05-24 JP JP2012527184A patent/JP5592490B2/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| Gao X.L. et al.Soluble cytoplasmic expression, rapid purification, and characterization of cyanovirin-N as a His-SUMO fusion.《Applied Microbiology and Biotechnology》.2009,第85卷(第4期), * |
| 吕锐 等.蓝藻抗病毒蛋白N基因的原核表达及重组蛋白的纯化和复性.《医学研究生学报》.2007,第20卷(第11期), * |
| 庞峰等.蓝藻抗病毒蛋白N的研究进展.《中国海洋药物杂志》.2006,第25卷(第6期), * |
| 黄小花等.蓝藻抗病毒蛋白-N在大肠杆菌中的表达及其抗体的制备.《中国免疫学杂志》.2007,第23卷(第12期), * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2011026351A1 (en) | 2011-03-10 |
| JP2013503609A (en) | 2013-02-04 |
| JP5592490B2 (en) | 2014-09-17 |
| IN2012DN02837A (en) | 2015-07-24 |
| CN101638435A (en) | 2010-02-03 |
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