JP5592490B2 - Cyanovirin-N mutant, modified derivative thereof and application thereof - Google Patents

Description

  The present invention falls within the biopharmaceutical field and specifically relates to cyanovirin-N (CVN) variants, PEG-modified derivatives thereof and their pharmaceutical applications.

  Cyanovirin-N (Cynovirin-N, CVN) is a protein having anti-HIV activity that is eluted from cyanobacteria by US scientists BODY and others. CVN binds specifically and with high affinity to gp120, which is the envelope glycoprotein of human immunodeficiency virus HIV-1, and exhibits antiviral activity. At the same time, it has features such as broad spectrum antiviral properties and stability. It shows that CVN protein can be a valuable antiviral drug. [Non-Patent Document 1: BOYD MR, GUSTAFSON KR, MCMAHON JB, et al. Discovery of cyanovirin-N, a novel human immuno-deficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: Potential applications to microbicide development [J] Antimicrobial Agents and Chemotherapy, 1997, 41 (7): 1521-30.]. However, since the molecular weight of the protein is relatively small and the molecule has two disulfide bonds, it is difficult to express the protein in Escherichia coli and the production amount is small. As a low molecular weight protein drug derived from prokaryotes, it has drawbacks such as short half-life, cytotoxicity, induction of immune response and the like.

  Polyethylene glycol (PEG) is a non-toxic, non-immune, water-soluble polymer that modifies proteins through covalent bonds. This PEG modification is an effective method used to solve or ameliorate the problems of proteins and peptides having poor stability and short half-life in pharmaceutical processes. (Nonpatent literature 2: IANG ZY, XUSW, WANGYQ. Chemistry for pegylation of protein and peptide molecules [J]. Chinese Journal of Organic Chemistry, 2003, 23 (12): 1340-7.). The modification of CVN under acidic conditions using PEG-maleimide has already been reported in the literature. Zappe et al. Selected a mutant (CVN (Q62C)) in which the 62nd glutamine was replaced with cysteine and modified it with mPEG-maleimide (mPEG-MAL) under neutral PH conditions. (Non-patent Document 3: ZAPPE H, SNELL ME, BOSSARD M J. PEGylation of cyanovirin-N, an entry inhibitor of HIV [J]. Advanced Drug Delivery Reviews, 2008, 60 (1): 79-87.).

  Specifically, Zappe et al.'S modification strategy selected 20 KD and 30 KD mPEG-maleimide in mutants in which the 62nd and 14th glutamine were replaced with cysteine, respectively, under PH conditions approaching neutral PH and alkalinity. , It has been modified. As a result, the modification efficiency of the mutant in which the 14th glutamine was replaced with cysteine (CVN (Q14C)) was very low, whereas the mutant in which the 62nd glutamine was replaced with cysteine (CVN (Q62C)). Is a neutral PH, and the modification rate is relatively high under the condition where the molar ratio of CVN mutant to mPEG-MAL is 1: 3. However, since the anti-HIV activity of CVN (Q62C) is lower than that of unmutated CVN, and the selected modification site is inside CVN, mutation through the modification of mPEG-maleimide 30KDa (mPEG-MAL-30KDa) The body had almost lost anti-HIV activity.

BOYD MR, GUSTAFSON KR, MCMAHON JB, et al. Discovery of cyanovirin-N, a novel human immuno-deficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: Potential applications to microbicide development [J]. Antimicrobial Agents and Chemotherapy, 1997, 41 (7): 1521-30. IANG Z Y, XU SW, WANG Y Q. Chemistry for pegylation of protein and peptide molecules [J]. Chinese Journal of Organic Chemistry, 2003, 23 (12): 1340-7. ZAPPE H, SNELL ME, BOSSARD M J. PEGylation of cyanovirin-N, an entry inhibitor of HIV [J]. Advanced Drug Delivery Reviews, 2008, 60 (1): 79-87.

  In order to overcome the deficiencies and disadvantages existing in the prior art, an object of the present invention is to provide a CVN mutant that is easier to express in the host, easier to purify, and advantageous for further modification. .

  Another object of the present invention is to provide a modified derivative of a CVN mutant that can be adapted to more applications by reducing the cytotoxicity and immunogenicity of the protein itself.

  Another object of the present invention is to use the mutants and modified derivatives for the manufacture of a drug for preventing and / or treating AIDS.

  In order to achieve the above object, the present invention provides the following techniques.

The amino sequence consists of an A sequence and a B sequence, the A sequence is located at the N-terminus of the B sequence,
A array is
a. Sequence table SEQ ID NO: 1,
b. SEQ ID NO: 1 in the sequence table, which has hydrophilic and flexible properties obtained by substituting, deleting, or inserting one or several amino acids for the amino acid residue sequence of 1.
Any one of
B array is
c. SEQ ID NO of sequence table: 2,
d. Specificity obtained by substituting, deleting, or inserting one or several amino acids in the amino acid sequence of SEQ ID NO: 2 in the sequence table without changing the three residues before the N-terminus Having typical anti-HIV virus activity,
A cyanovirin-N mutant characterized by being any one of the above.

The present invention also provides the encoded nucleotide sequence of the cyanovirin-N variant.
The encoded nucleotide sequence is:
e. SEQ ID NO of sequence table: 3,
f. A nucleotide sequence capable of hybridizing with a DNA sequence limited to SEQ ID NO: 3 in the sequence table under stringency conditions;
It is preferable to contain any one of the sequences.
Here, the stringency condition refers to a hybridization condition with a low salt concentration and a high temperature, for example, 0.1 × SSC, 0.1% SDS, and a temperature of 65 ° C.

  The nucleotide sequence is mainly used for the expression of the target protein in the host, and the expression vector, cell line, host fungus and the like containing the nucleotide sequence can be obtained through ordinary technical means. The process in which the protein is purified from the host may also use a normal method.

  In addition to obtaining a purified protein, the present invention also performs PEG modification on the N-terminus of the CVN mutant, and the modification site is generally a site for the α-amino group of the N-terminal glycine residue. CVN variant modified derivatives are provided.

  As the modifier for PEG modification, mPEG-ALD (monomethoxypolyethyleneglycolaldehyde) is preferably used. The molecular weight of the mPEG-ALD is preferably 10 KD to 20 KD.

  Both the mutant and the modified derivative can be applied to the manufacture of a drug for preventing and / or treating AIDS. Based on similar antiviral mechanisms, the variants and modified derivatives of the present invention can be used in the manufacture of drugs for the prevention and / or treatment of diseases caused by other viral microorganisms.

  In contrast to the prior art, the present invention has the following beneficial effects.

  The CVN mutant and its modified derivative provided by the present invention are for realizing the antiviral function of recombinant CVN in vitro, and the anti-HIV activity of recombinant L-CVN is improved (WST method and cell Fusion method). Since the hydrophilic and flexible peptide sequence was introduced into the LCVN terminal produced in the present invention, amino group modification at the N-terminal defined site can be performed. Our experimental results demonstrated that LCVN not only obtained an active modification after the N-terminal defining site was modified with an amino group, but further improved the anti-viral activity of the modification.

It is a pET3c-6His-SUMO-LCVN recombinant plasmid figure. It is a restriction enzyme treatment and PCR analysis result of the recombinant pET3c-6His-SUMO-LCVN plasmid. Here, M: DL2000 DNA marker, Lane 1: pET3c-6His-SUMO-LCVN / Nde I + BamHI; Lane 2: PCR product. The protein expression characteristic result of BL21 / pET3c-6His-SUMO-LCVN of SDS-PAGE electrophoresis. Here, in each lane, from left to right, IPTG is not added, IPTG induction 20h, centrifugal supernatant of the induced expression pulverized product, and centrifugal precipitation of the induced expression pulverized product. It is an analysis result of extracted protein LCVN. Each lane is, sequentially from left to right, Sumo-LCVN fusion protein, Sumo-LCVN fusion protein after treatment with Sumo protease, and LCVN protein. (*) Sumo-LCVN fusion protein. (**) Indicates LCVN protein. RP-HPLC analysis for purity of recombinant LCVN. Tricine-SDS-PAGE electrophoresis pattern of 10K mPEG-ALD modified LCVN under different pH and mixing ratio conditions. Here, from left to right, lane 1 is a protein marker, lanes 2, 3, and 4 are PH 3.5, respectively, and the molar ratio of LCVN to 10K mPEG-ALD is 1: 1, 1: 3, and 1: 5. Electrophoretic pattern of the modified product, lanes 5, 6, and 7 are PH 4.0 and the modified product when the molar ratio of LCVN to 10K mPEG-ALD is 1: 1, 1: 3, and 1: 5, respectively. Electrophoresis patterns, lanes 8, 9, and 10 are PH 5.0, and the modified electrophoresis patterns and lanes when the molar ratio of LCVN to 10K mPEG-ALD is 1: 1, 1: 3, and 1: 5, respectively. 11, 12, and 13 are PH 6.0, and the electrophoretic pattern of the modified product when the molar ratio of LCVN to 10K mPEG-ALD is 1: 1, 1: 3, 1: 5, lanes 14, 15, 16 Are electrophoretic patterns of the modified products when the pH is 7.0 and the molar ratio of LCVN to 10K mPEG-ALD is 1: 1, 1: 3, and 1: 5, respectively. It is a comparison figure of the modification rate of 10K mPEG-ALD modification LCVN under different PH and mixing ratio conditions. Tricine-SDS-PAGE electrophoresis pattern of 20K mPEG-ALD modified LCVN under different PH and mixing ratio conditions. Here, from left to right, lane 1 is the protein marker, lanes 2, 3, and 4 are PH 3.5, respectively, and the molar ratio of LCVN to 20K mPEG-ALD is 1: 1, 1: 3, 1: 5. Electrophoretic pattern of the modified product at the time of lanes 5, lanes 5, 6 and 7 are PH 4.0 and the modified product when the molar ratio of LCVN to 20K mPEG-ALD is 1: 1, 1: 3, 1: 5, respectively. Electrophoretic patterns of lanes 8, 9, and 10 are PH 5.0 and the electrophoretic patterns of the modified products when the molar ratio of LCVN to 20K mPEG-ALD is 1: 1, 1: 3, and 1: 5, Lanes 11, 12, and 13 are PH 6.0 and the electrophoresis pattern of the modified product when the molar ratio of LCVN to 20K mPEG-ALD is 1: 1, 1: 3, and 1: 5, and lanes 14, 15, 16 are the electrophoretic patterns of the modified products when PH is 7.0 and the molar ratio of LCVN to 20K mPEG-ALD is 1: 1, 1: 3, 1: 5. It is a comparison figure of the modification rate of 20K mPEG-ALD modification LCVN under different PH and mixing ratio conditions. Tricine-SDS-PAGE electrophoresis pattern of 10K mPEG-ALD modified LCVN at different reaction times. Here, lane 1 is a protein marker, and lanes 2-8 are sample electrophoresis results 1 h, 3 h, 5 h, 7 h, 9 h, 12 h and 24 h after the reaction. Lane 9 is unmodified LCVN. Tricine-SDS-PAGE electrophoresis pattern of 20K mPEG-ALD modified LCVN at different reaction times. Here, lane 1 is a protein marker, and lanes 2-8 are sample electrophoresis results 1 h, 3 h, 5 h, 7 h, 9 h, 12 h and 24 h after the reaction, respectively. Lane 9 is unmodified LCVN. SP-Sepharose elution curve for separation and purification of 10K mPEG-ALD modified LCVN. Tricine-SDS-PAGE electrophoresis pattern of 10K mPEG-ALD modified LCVN of each composition after SP-Sepharose separation and purification. Here, lane 1 is a protein marker, and lanes 2-5 are a modification reaction mixture, a penetration peak, an elution peak of buffer A containing 80 mM NaCl, and an elution peak of buffer A containing 400 mM NaCl, respectively. Elution curve of 20K mPEG-ALD modified LCVN modified through SP-Sepharose separation and purification. 20K mPEG-ALD modified LCVN is a Tricine-SDS-PAGE electrophoresis pattern of each composition after SP-Sepharose separation and purification. Lane 1 is a protein marker, Lane 2-5 is a mixture of modification reaction, penetration peak, The elution peak of buffer A containing 70 mM NaCl and the elution peak of buffer A containing 400 mM NaCl. MT-4 cell survival rate (%) after treatment with different concentrations of LCVN and its modifications. MT-4 cell survival rate (%) after treatment with different concentrations of LCVN and its modifications. MT-4 cell survival rate (%) after treatment with different concentrations of LCVN and its modifications. MT-4 cell survival rate (%) after treatment with different concentrations of LCVN and its modifications. It is the HIV-1 / IIIB growth inhibition rate (%) of CVN and LCVN. It is the HIV-1 / IIIB growth inhibition rate (%) of CVN and LCVN. 1 shows the anti-human immunodeficiency virus activity of LCVN and its PEG-modified products. Here, (a) is a phase-contrast micrograph of (1) MOLT-4 cells, (2) MOLT-4 / IIIB cells, (3) MOLT-4 and MOLT-4 / IIIB Cultured cells, (4) mock-treated (MOCK) co-cultured cells, (5)-(8) are co-cultured cells after treatment with CVN, LCVN, 10 kPEG-LCVN, 20 kPEG-LCVN at a concentration of 113 nM. The black arrows show the fused giant cells seen with HIV induction. (b) is each fusion inhibitory activity of CNV, LCVN, 10 kPEG-LCVN, and 20 kPEG-LCVN, and all experiments are statistical processing results after at least three independent experiments. It is a CPE observation result regarding the anti-HSV-1 activity of LCVN and its PEG modification product. A, normal control, B, virus control, C, positive drug ACV control (1 μg / ml), D, L-CVN sample (1.562 μg / ml), E, SUMO-L-CVN sample (3.125 μg / ml) ), F, mPEG-ALD-10 kDa-L-CVN (3.125 μg / ml), G, mPEG-ALD-20 kDa-L-CVN (3.125 μg / ml).

  Hereinafter, in order to better understand the present invention, some preferred examples of the present invention are given, but the embodiments of the present invention are not limited thereto.

The main materials related to the examples of the present invention are as follows. The host bacteria E. coli BL21 (DE3) (purchased from Novagen), plasmid pET3c (purchased from Novagen), and pET3c-SUMO-CVN are stored in this room. The title of the invention is “Production method and application of recombinant cyanobacterial antiviral protein”, and its application number is 200810198926.0 ”. The construction of the plasmid may be performed by a known genetic engineering method, and its basics. The concept is that the SUMO-CVN fusion sequence was first obtained through the PCR method and ligated with the pET3c vector, and then the plasmid pET3c-SUMO-CVN was obtained.), SUMO protease (purchased from Marine Chemicals Co., Ltd.), Taq polymerase, T4 DNA ligase, DNA molecular weight standard, various restriction enzymes are purchased from Takara, protein molecular weight standard (purchased from Polylab Biotech Co., Ltd.), primers are Shanghai Purchased from biotech company, Ni ²⁺ Sepharose Fast Flow, SP Sepharose Fast Flow purchased from GE Healthcare company, MTT, WST purchased from SIGMA, USA Herpes simplex virus type 1 (HSV-1) F strain is Wuhan University virus Purchased from the laboratory (CGMCC No. 0396), Vero cells (CCL-81 ™), MOLT-4 cells (CRL-1582 ™), MT-4 cells (CRL-1942 ™), HIV- I / IIIB virus (CRL-1973 (trademark)), etc. were purchased from ATCC, and mPEG-ALD (10K) and mPEG-ALD (20K) were purchased from Beijing Zhengzheng Biological Process Development Co., Ltd.

  NTA-0 buffer (20 mmol / L Tris-HCl, pH 8.0, 0.15 mol / L NaCl), NTA-20 buffer (20 mmol / L Tris-HCl, pH 8.0, 0.15 mol / L NaCl, 20 mmol) / L imidazole), NTA-250 buffer (20 mmol / L Tris-HCl, pH 8.0, 0.15 mol / L NaCl, 250 mmol / L imidazole), digestion buffer (20 mmol / L Tris-HCl, pH 8.0) 0.15 mol / L NaCl), buffer A (20 mmol / L NaAc-Ac, pH 4.0).

Example
1. Construction of recombinant plasmid pET3c-6His-SUMO-LCVN
The construction and synthesis of SUMO-L-CVN gene is divided into two steps. First, L-CVN gene is synthesized through two rounds of PCR. In the first round, pET3c-SUMO-CVN plasmid is used as a template and F1- Use CVN and R-CVN as upstream and downstream primers. The reaction system is 1 ng of template, the upstream and downstream primers are 1 μM each, 20 μl Taq PCR MasterMix, water is added to 40 μl, the reaction mixture is denatured at 94 ° C. for 1 min, annealed to 55 ° C., kept for 1 min, 72 ° C. 1 min, and 29 cycles were performed. The reaction product is subjected to 1% agarose gel electrophoresis, the target fragment is recovered from the gel, and used as the next PCR template. The second round PCR uses the previous PCR product as a template, and F2-CVN and R-CVN are upstream / downstream primer pairs (of which the F2-CVN primer is a flexible peptide with 15 amino acid residues encoded) L-CVN full-length sequence was synthesized. The SUMO full-length sequence was synthesized from pET3c-SUMO-CVN through the PCR method. PCR is carried out using the 26 bp overlapping complementary sequence at the end of the SUMO sequence and the front end of the L-CVN sequence, the SUMO sequence and the L-CVN sequence are used as extension templates, and F-SUMO and R-CVN are the upstream and downstream primers. Under the PCR conditions, the reaction was carried out, the reaction product was subjected to 1% agarose gel electrophoresis, and the gel was recovered to obtain the full-length SUMO-L-CVN sequence.

Each plasmid pET3C and 6His-SUMO-LCVN full length sequence, is digested with the two enzymes Nde I and BamH I, the digestion product was electrophoresed on a 1% agarose gel, recovered its digestion product, with T ₄ DNA ligase The product was ligated, incorporated into E. coli JM109 competent cells, spread on LB plates containing ampicillin, cultured overnight at 37 ° C., the plasmid was eluted, amplified by PCR, then Nde I and BamH I two enzymes After digestion with, identify and send the positive plasmid to Invitrogen and sequence.

2. Flask shaking culture, protein purification and purity measurement of LCVN process bacteria (recombinant bacteria)
The correct sequencing plasmid is transformed into E. coli BL21 (DE3) to obtain the process bacterium BL21 [pET3c-6His-SUMO-LCVN], single cell clones are selected and cultured to induce expression, and the cells are collected. The protein was analyzed by SDS-PAGE electrophoresis. As a result, after inducing a BL21 [pET3c-6His-SUMO-LCVN] -positive clonal strain, a fusion protein having a size of about 28 kDa was expressed. After pulverizing the cells with ultrasound, the fusion protein is present in the supernatant and becomes soluble, and after gel light density scanning, the soluble target protein is 28.3 ± 3.4 of the total supernatant protein. % (FIG. 3).

  Highly expressing strains are selected and inoculated into 1 L of ampicillin-containing (100 mg / L) LB medium, cultured at 37 ° C. and 180 rpm to OD600 = 0.6 to 1.0, the temperature is lowered to 20 ° C., and the final concentration IPTG was added to induce expression for 24 hours. The cells are collected by centrifugation at 4 ° C., 6000 × g for 10 min, and once frozen and thawed, the cells are precipitated, suspended again in NTA-10 buffer at a 1:10 ratio, and pulverized with ultrasonic waves (working time) The supernatant was collected by centrifugation at 4 ° C., 25000 × g, 30 min.

  The supernatant is put into Ni-NTA affinity chromatography with a column volume of 20 ml, washed to the baseline with NTA-0 buffer at a flow rate of 0.6 ml / min, and NTA-20 buffer at a flow rate of 1 ml / min. The unnecessary protein was washed and the target protein was eluted with NTA-250 buffer. After purification, target protein 6His-SUMO-LCVN removed imidazole with Sephadex G-25 molecular sieve, and then digested with SUMO protease to remove SUMO fusion protein.

  6His-SUMO-LCVN was adjusted so that the concentration was 1 mg / ml, 1 U SUMO protease / mg was added, the protein was fused, and digested at 30 ° C. for 1 h. Since both 6His-SUMO labeling and SUMO protease contain 6xHis labeling, the digested sample is again put into Ni-NTA affinity chromatography and purified, 6His-tagged SUMO, undigested 6His-SUMO -Remove LCVN and SUMO protease, obtain non-fusion target protein LCVN, remove salt through G-25 molecular sieve column, freeze-dry to obtain LCVN product, measure physical and scientific properties, Used for activity measurement or PEG modification.

  Fig. 3 is a protein expression diagram after the IPTG induction of the process fungus BL21 [pET3c-6His-SUMO-LCVN]. From the figure, a clear thick protein band appears in the place where the molecular weight is 28 kD after the induction. (Lane 3), which matches the logical molecular weight of 6His-SUMO-LCVN, and after pulverization with ultrasonic waves, the target protein is located in the supernatant after pulverization of the cell, and the content is 40 of the soluble protein of the cell. % (Band 2). FIG. 4 shows the purification process of SDS-PAGE analysis LCVN, in which the location indicated by * is the 6His-SUMO-LCVN fusion protein and the location indicated by ** is the LCVN protein. Fig. 5 shows the results of LCVN product reverse phase high performance liquid chromatography (RP-HPLC) purity analysis. The column type is a C-18 reverse column, the measuring instrument wavelength is 280 nm, and the fluid phase A is 0.1% trifluoro. Gradient elution was performed with acetic acid (TFA) -containing ultrapure water, fluid phase B: acetonitrile, 40% -60% B, and an elution peak of LCVN was obtained with a retention time of 4-6 min.

3. Medium scale fermentation and protein production of LCVN process bacteria
A single cell clone was selected from the process bacterium BL21 [pET3c-6His-SUMO-LCVN], cultured, induced and expressed in a test tube, and a high-expressing strain was selected and subjected to medium-scale fermentation. In each Erlenmeyer flask, one and two species were cultured to an appropriate concentration and inoculated into 15 L of fermentation medium at a 10% inoculum. The temperature was automatically controlled at 37 ° C, and the rotation speed and air flow rate were adjusted as appropriate so as to keep a moderate dissolved oxygen level, and adjusted with NaOH and HCl so that the pH was 7.0 to 7.2. . When the cell density (OD ₆₀₀ ) reached about 12, the temperature was lowered to 20 ° C., IPTG was added so that the final concentration was 0.5 mM, and expression was induced for 20 hours. The cells were collected by continuous centrifugation at 9000 g at 100 ml / min, and about 550 g of wet cells could be obtained with 15 L of fermentation medium, and the collected cells were stored in a freezer at −20 ° C.

  Suspension of bacterial cell suspension in NTA-0 buffer at a ratio of 1:10 and pulverization with ultrasonic waves (working time 5s, intermittent time 5s, 99 cycles for 1 cycle, 3 cycles overlapped), 4 ° C, 25000xg The supernatant was collected by centrifugation at 30 min. The supernatant is put into a Ni-NTA packing with a column volume of 20 ml, washed at the flow rate of 1 ml / min to the baseline with NTA-0 buffer, and the flow rate of 1 ml / min is not required with NTA-20 buffer. The protein was washed and the target protein was eluted with NTA-250 buffer. The purified target protein 6His-SUMO-LCVN removed imidazole with Sephadex G-25 molecular sieve and then digested with SUMO protease to remove SUMO fusion protein.

  6His-SUMO-LCVN was adjusted so that the concentration was 1 mg / ml, 1 U SUMO protease / mg was added, the protein was fused, and digested at 30 ° C. for 1 h. Since both 6His-SUMO labeling and SUMO protease contain 6xHis labeling, the digested sample is again put into Ni-NTA affinity chromatography and purified, 6His-tagged SUMO, undigested 6His-SUMO -LCVN and SUMO protease were removed, non-fusion target protein LCVN was obtained, buffer solution was exchanged through G-25 molecular sieve column, LCVN protein was concentrated using ultrafiltration with a restriction amount of 3KD, Used for physicochemical property measurement, activity measurement or PEG modification.

4). PEG modification of LCVN protein
Performing PEG modifications to medicinal proteins is an effective way to improve many types of pharmaceutical properties such as their pharmacokinetic properties, stability and immunogenicity. Sites that proteins can provide for PEG modification contain many types such as side chain amino group, N-terminal amino group, side chain carboxy group, C-terminal carboxy group, side chain mercapto group and the like. In the prior art carboxy group modification technology, non-specific cross-linking reaction is likely to occur, so amino group modification is often used, and its technological development is more mature. The results of Zappe et al. Show that modifications to the side chain amino group or N-terminal amino group of wild-type CVN disrupted the activity of the protein, so the authors first performed site-specific mutations on CVN. Thus, a Q62C mutant was obtained, and Cys introduced at 62 site was modified with a side chain mercapto group to obtain an active protein. Natural CVN already forms two sets of disulfide bonds between the four Cys residues present, and in solution the protein disulfide bonds are placed in dynamic isomerism and balance, thus After introducing the Q62C mutation, it is difficult to avoid that the introduced Cys does not interfere with the correct formulation of the disulfide bond, or it becomes a non-62 site Cys modification, and the author's last experimental result is also the CVN Q62 mutant and its modification All the activity of the product was lower than that of wild type CVN.

  In the present invention, since a hydrophilic and flexible 15 peptide sequence is introduced into the produced LCVN terminal, site-specific amino group modification at the N terminal may be attempted. Our experimental results have demonstrated that LCVN not only obtains active modification after N-terminal site-specific amino group modification, but also further improves the antiviral activity of the modification.

  There are many PEG modification methods for amino groups, and many modifying agents can be selected. In the present invention, mPEG-ALD (10K) and mPEG-ALD (20K) are selected as the modifying agents, and the ratio of substrate: modifying agent, The optimal PEG modification conditions for LCVN were selected from three aspects, including modification pH and modification reaction time.

Selected pH 3.5, 4.0, 5.0, 60, 7.0, Na-Ac buffer with ionic strength 20 mM, LCVN concentration in reaction system 5 mg / ml, LCVN and mPEG-ALD ( 10K) molar ratios of 1: 1, 1: 3, 1: 5 are selected, respectively, and the final concentration of NaCNBH ₃ is 5 mg / ml. At room temperature, the reaction was vibrated with a vibrator. After 3 hours of reaction, sampling was performed, and the effect of the modification reaction was observed by electrophoresis. A simple modified product of LCVN modified with mPEG-ALD (10K) has an expression molecular weight of about 30 KD on SDS-PAGE, and the analysis result of SDS-PAGE shows that the molar ratio of LCVN to mPEG-ALD (10K) is 4.0. In the condition of 1: 3, the simple modification rate was the highest (FIG. 6).

  In a similar manner, the optimal modification pH and feed ratio of LCVN modification of mPEG-ALD (20K) was studied. After modifying LCVN with mPEG-ALD (20K), the simple modified product has an expressed molecular weight of about 50 KD on SDS-PAGE. The experimental result shows that the molar ratio of LCVN to mPEG-ALD (20K) is 1 It shows that the simple modification rate is the highest under the condition of: 1 (FIG. 18).

  After determining the optimal modification pH and feed ratio, the LCVN modifications of mPEG-ALD (10K) and mPEG-ALD (20K) were sampled at reactions 1h, 3h, 5h, 7h, 9h, 12h and 24h, respectively. The optimal reaction time of Tricine-SDS-PAGE electrophoresis was observed. The results of SDS-PAGE analysis show that the reaction time has no significant effect on the LCVN modification reaction of mPEG-ALD (10K) and mPEG-ALD (20K). It was shown that reaction 2h is preferable by room temperature vibration (FIGS. 10 and 11).

5. Separation and purification of PEG-modified products of LCVN
After LCVN is modified with PEG-ALD, the reaction mixture mainly contains unmodified LCVN, excess PEG, LCVN modified with a large amount of PEG, and LCVN with simple PEG modification. In addition to AKTA prime, a simple purification mPEG-ALD-LCVN is separated and purified by SP sepharose chromatography using a separation and purification system to obtain a Na-Ac buffer (buffer A) with a fluid phase of 20 mM, and a sample is added. Prior to this, the column was first balanced with 5 times the column volume of bufferA, a sample was added, the penetration peak was collected, and elution was carried out with different concentrations of NaCl-containing bufferA to collect each elution peak. The flow rate is 1 ml / min and the measurement wavelength is 280 nm. Collected samples were measured by SDS-PAGE gel electrophoresis.

After the LCVN-modified mixture with mPEG-ALD (10K) was purified by SP sepharose cation chromatography, the modifier mPEG-ALD and the over-modified product did not bind to SP sepharose and penetrated, 80 mM NaCl. The elution product of buffer A containing simple modified mPEG-ALD _(10K) -LCVN, and the elution product of buffer A containing 400 mM NaCl is unmodified LCVN. FIG. 12 is an elution curve of separation and purification of mPEG-ALD _(10K) -LCVN. Peak 1 is a modifying agent and a product that has been modified a lot, Peak 2 is a simple modified product, and Peak 3 is an unmodified substrate. FIG. 13 is an analysis of each elution composition by SDS-PAGE. Lane M is the protein molecular weight standard, Lane A is the reaction sample, and unmodified substrate, simple modified product, and more modified product are seen. Lane 1 Is the penetration peak, lane 2 is a simple modification, in other words, the target protein mPEG-ALD _(10K) -LCVN, and lane 3 is an unmodified product.

The mixture of LCVN modified with mPEG-ALD (20K) was purified by SP sepharose cation chromatography, and then the product modified with a large amount of modifier mPEG-ALD was also present in the penetration peak of the sample. The eluted product of buffer A containing 400 mM NaCl is unmodified LCVN. The modified product is simple modified mPEG-ALD _(20K) -LCVN.

FIG. 14 is an elution curve of separation and purification of mPEG-ALD _(20K) -LCVN, where peak 1 is a modifying agent and a product that has been modified a lot, peak 2 is a simple modified product, and peak 3 is an unmodified substrate. FIG. 15 is an analysis of each elution composition by SDS-PAGE. Lane M is the protein molecular weight standard, Lane A is the reaction sample, and unmodified substrates, simple modifications, and more modified products are seen. 1 through peak, lane 2 is a simple modification products, in other words, a desired protein mPEG-ALD _(10K) -LCVN, lane 3 is the elution the composition of 400 mM NaCl, although unmodified product was observed, partial Simple modified products are also included.

Application Example 1 Measurement of LCVN cytotoxicity against T cells by WST method
CVN, LCVN, and PEG-modified products are RPMI-1640 medium diluted 5-fold with RPMI-1640 medium, put into a 96-well cell culture plate, 50 μl per well, and 10 μg of CVN and LCVN dilution range. / ml to 0.04 μg / ml and PEG modification to reduce the LCVN dilution range from 50 μg / ml to 0.19 μg / ml. The MT-4 cell concentration is adjusted to 1 × 10 ⁵ / ml, homogenized with 100 μl per well, then allowed to stand at 37 ° C. and cultured in a 5% CO ₂ cell culture box, simultaneously with cell control and A positive drug control (63 nM azidothymidine, AZT) was provided and three duplicate wells were measured in parallel for each sample. After 4 days, 10 μl WST-1 (water-soluble tetrazole, 5 mmol / L) solution was added to each well of the culture plate, followed by culturing for 4 hours, placing in a plate reader, reading the absorbance (A), at a wavelength of 450/650 nm, Cell survival rate (Relative percentage, RP,%) was calculated, and 50% toxic concentration (CC ₅₀ ) was calculated based on RP.

Cell survival rate (Relative percentage,%) = drug treatment group A value / cell control group A value × 100%
FIG. 16 is a diagram showing the cell survival rate after treating MT-4 with four types of proteins such as CVN, LCVN, and modified products thereof at different concentrations. In the figure, NC is an untreated control cell. And the density of control cells is 100%. AZT is a positive control drug, 63 nM azidothymidine. The calculated 50% toxic concentrations are shown in Table 2.

Application Example 2 Measurement of anti-human immunodeficiency virus activity of LCVN by WST method
CVN, LCVN, and PEG-modified products were used as RPMI-1640 culture solutions diluted in 5-fold series, placed in a 96-well cell culture plate, and 50 μl per well. The MT-4 cell concentration is adjusted to 1 × 10 ⁵ / ml, homogenized with 100 μl per well, 50 μl of HIV-1 / IIIB virus suspension solution is added, and the titer is 100 TCID50. The cells were allowed to stand at 37 ° C. and cultured in a 5% CO ₂ cell culture box. At the same time, cell control, virus control and positive drug control (azidothymidine, AZT) were provided, and three overlapping pores were measured in parallel for each sample. . Four days later, 10 μl WST-1 (water-soluble tetrazole, 5 mmol / L) solution was added to the culture plate for each well, followed by culturing for 4 hours, placing on a plate reader, reading the absorbance (A), and the wavelength being 450/650 nm. is there.

Cell survival rate (Relative percentage,% = drug treatment group A value / cell control group A value x 100%
Virus inhibition rate (%) = (Drug treatment group A ₄₅₀ / _650- Virus control group A _450/650 ) / (Cell control group A ₄₅₀ / ₆₅₀ -Virus control group A _450/650 ) x 100%
The 50% inhibitory concentration (IC50) was calculated by the Reed-Muench method, and the selectivity index (TI) was calculated based on the 50% toxic concentration (CC50) of Example x. TI = CC50 / IC50.

As can be seen from Table 3, the positive drug AZT has higher inhibitory activity of CVN and LCVN on the proliferation of HIV-1 / IIIB.

Application Example 3 Measurement of anti-human immunodeficiency virus activity of LCVN and its PEG-modified products by fusion inhibition method
Previous studies have shown that HIV infection that propagates between T cells occurred by binding to receptors on uninfected cells, mainly through the intervention of infected cell surface expressed gp120, to form fused cells. Has been. CVN specifically binds to gp120, suppresses fusion between infected and normal cells and prevents viral spread. Therefore, we apply cell fusion, apply an inhibition model, and measure the antiviral activity of LCVN and its derivatives [Tochikura TS, Nakashima H, Tanabe A, Yamamoto N. Human immunodeficiency virus (HIV)- induced cell fusion: quantification and its application for the simple and rapid screening of anti-HIV substances in vitro. Virology, 1988, 164 (2): 542-546].

Prepare until the density of MOLT-4 cells and MOLT-4 / IIIB cells grown to logarithmic phase is 1 × 10 ⁶ / ml, take 250 μl of each cell suspension, mix in equal volumes, and prepare 24 well cells culture plates (total number of cells of 5 × a 10 ^5/500 [mu] l / well) was added to, and CVN, a LCVN and fetal bovine serum and antibiotics RPMI-1640 medium containing that PEG-modified product was 4-fold series dilution, receiving For each drug type, select three concentrations (452nM, 113nM, 28nM) and add them to the cell suspension solution in equal volumes. At the same time, set up various controls, and make all samples in duplicate wells in parallel. The cells were cultured at 37 ° C. and 5% CO ₂ for 24 hours. After 24 hours, the cell suspension was taken out, stained with trypan blue, and placed in a cell count plate, and the number of MOLT-4 cells that survived was counted with a microscope. Since MOLT-4 / IIIB cells are not able to tolerate HIV-I / IIIB virus granules, and by fusion with normal MOLT-4 cells, large multinucleated cells (syncytia) are formed, and normal host cells are Since the fused cells do not enter the count plate when the cells are infected and counted, the number of normal size MOLT-4 or MOLT-4 / IIIB cells in the cell count plate is counted, and the formed syncytium The number of cells could be estimated and the fusion index (FI) was calculated by comparing the number of cells in the dosing co-culture group and the number of MOLT-4 cells that were not co-cultured.
FI = 1- (number of cells in co-culture cell well / number of cells in control well containing only MOLT-4 cells)
The fusion index of the medicated co-cultured cells and the non-medicated co-cultured cells were compared, and the fusion inhibition rate (FIR) was calculated.
FIR (%) = [1- (FI _T / FI _C )] × 100, where FI _T is the fusion index of the administered sample, and FI _C is the fusion index of the untreated co-cultured cells.

From Table 4, the fusion inhibitory activity of LCVN against HIV-1 / IIIB is higher than that of CVN in any combination, and the activity of PEG-modified products of LCVN is higher than that of unmodified LCVN at medium and high doses. I find it expensive.

  Fig. 18 (a) shows the results of (1) MOLT-4 cells, (2) MOLT-4 / IIIB cells, (3) cells after co-culture, (4) co-treatment after 24 h culture using a phase contrast microscope. Cells after culture, (5-8) CVN / LCVN / 10 kPEG-LCVN / 20 kPEG-LCVN-treated co-cultured cells with a concentration of 113 nM were observed. There are no fused cells in the co-culture set, and typical fused cells are found in the co-culture set, but the fused set shows almost no fused cells.

  FIG. 18 (b) shows CNV, LCVN, 10 kPEG-LCVN, and 20 kPEG-LCVN fusion inhibitory activities, and all experiments are the results of statistical processing after at least three independent experiments. The fusion inhibitory activity of LCVN against HIV-1 / IIIB is clearly higher than that of CVN in any combination, and LCVN PEG-modified products are more active with increasing molecular weight at medium and high doses. However, the activity of the PEG-modified product decreases with increasing molecular weight in the low fraction group.

Application Example 4 Measurement of LCVN anti-herpes simplex virus (HSV-1) activity by MTT method
Herpes simplex virus type I (HSV-I) is a kind of DNA virus that causes widespread infections in humans, and human beings are the only host, and about 90% of healthy adults have HSV-1 Infected. HSV-I causes many types of diseases such as cleft lip, herpetic keratoconjunctivitis, neonatal encephalitis, etc. HSV-I can be latently infected in the ganglia, so its symptoms are often repetitive. . In this experiment, the MTT method was used to measure the toxicity of recombinant LCVN and its modified products, wild type CVN to Vero cells, and the drug activity against the cells was observed by the CPE method.

Monolayer Vero cells were supplemented with 50 μl each of different dilutions of drug recipient and 100 TCID50 HSV-1 virus solution, and at the same time a normal cell control and virus control set were provided. Incubate for 48 h in 5% CO ₂ , add 10 μl of 5 mg / ml MTT per well, continue to incubate for 4 h in 5% CO ₂ , discard supernatant, add 200 μl DMSO per well, 30 min at room temperature, protected from light , Shake the culture plate for about 10 min, colorimetrically with a microplate reader (see wavelength 570 nm, wavelength 630 nm), measure absorbance, and determine the 50% cytotoxic concentration (CC ₅₀ ) of the sample. Calculated.

  FIG. 19 shows a form of a cell under a phase contrast microscope after one typical experiment, and a cytopathic effect (B) by a virus on the cell and a protective effect on the cell are seen. The results show that both LCVN and its modifications have good anti-HSV-1 activity, and under conditions where the mass concentration is close and the molar concentration is relatively low, LCVN and its PEG modification are positive control drugs ACV. It can be seen that the antiviral activity is almost the same. From the toxicity measurement results, it can be seen that the toxicity of LCVN to Vero cells was clearly reduced after PEG modification.

  The above examples are preferred embodiments of the present invention. The embodiments of the present invention are not limited to the above-described examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the basic idea and principle of the present invention are equivalent substitution methods. It falls within the protection scope of the invention.

Claims (10)

The amino sequence consists of an A sequence and a B sequence, the A sequence is located at the N-terminus of the B sequence,
A sequence is
SEQ ID NO: 1 amino residue sequence in the sequence table, which has hydrophilic and flexible properties,
B array is
SEQ ID NO: 2 amino acid residue sequence of the sequence table, having a specific anti-HIV virus activity,
Cyanovirin-N mutant characterized by the above. A polynucleotide encoding the cyanovirin-N variant of claim 1. a. SEQ ID NO of sequence table: 3,
b. A nucleotide sequence capable of hybridizing with a DNA sequence limited to SEQ ID NO: 3 in the sequence table under stringency conditions;
Encoded polynucleotide of cyanovirin -N variant according to claim 2, characterized in that it contains any one sequence of. Expression vector characterized by containing the sequence of a polynucleotide according to claim 2 or 3. Host bacterium, characterized in that it contains the sequence of a polynucleotide according to claim 2 or 3.   A modified cyanovirin-N mutant derivative, wherein the N-terminus of the cyanovirin-N mutant according to claim 1 is subjected to PEG modification.   The cyanovirin-N mutant modified derivative according to claim 6, wherein the modifying agent for PEG modification is monomethoxypolyethyleneglycolaldehyde (mPEG-ALD). The cyanovirin-N mutant-modified derivative according to claim 7 , wherein the molecular weight of the mPEG-ALD is 10 KD to 20 KD.   Use of the cyanovirin-N mutant according to claim 1 for the manufacture of a drug for preventing and / or treating AIDS.   Use of the modified derivative of the cyanovirin-N mutant according to any one of claims 6 to 8 for the manufacture of a drug for preventing and / or treating AIDS.