WO2011026351A1 - A cyanovirin n mutant, modified derivative and uses thereof - Google Patents

Abstract

A cyanovirin N (CVN) mutant, its PEG modified derivative and their uses in preparing drugs. The said cyanovirin N mutant is composed of sequence A and sequence B: sequence A is a linker peptide with hydrophilicity and flexibility, sequence B is a codon optimized sequence of cyanovirin N. The cyanoviein N mutant is modified by PEG to obtain derivative. The mutant and the derivative both exhibit good anti-HIV activities, and have prospect of being used in preparing drugs for anti-HIV and other viral microorganism.

Description

 Cyanobacteria virus protein N mutant, modified derivative thereof and application thereof

 Technical field

 The invention belongs to the field of biomedicine, and particularly relates to a cyanobacterial virus protein N (CVN) Mutants, their PEG-modified derivatives and their use in pharmaceuticals.

 Background technique

 Cyanovirin-N (CVN) is an American scientist BOYD A protein that has anti-HIV activity that can be extracted from cyanobacteria. CVN binds specifically and highly affinity to the human immunodeficiency virus HIV-1 capsid protein gp120 It exerts antiviral activity, which makes it immune to virus mutation. It also has the characteristics of wide antiviral spectrum and stable nature, which makes CVN protein a valuable antiviral drug. 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 gp120: Potential applications to microbicide development [J]. Antimicrobial Agents and Chemotherapy, 1997, 41(7): 1521-30.]. But because the protein has a lower molecular weight And there are two disulfide bonds in the molecule, making the protein difficult to express in E. coli and the yield is low. At the same time, as a prokaryotic source of small molecular protein drugs, it has a short half-life, cytotoxicity and Causes shortcomings such as immune response.

 Polyethylene glycol (PEG) It is a non-toxic, non-immunogenic, water-soluble polymer substance that can be modified by covalent binding. Polyethylene glycol modification is used to solve or alleviate the poor stability of proteins and peptides in the medicinal process. An effective way to solve problems such as short half-life ( IANG Z Y, XU S W, WANG Y Q. Chemistry for pegylation of protein and peptide Molecular [J]. Chinese Journal of Organic Chemistry, 2003, 23 ( 12 ) : 1340-7. Using PEG-maleimide to modify CVN under acidic conditions It has been reported in the literature that Zappe et al. selected a mutant in which 62 glutamine was replaced by a semi-leucine (CVN(Q62C)). Modification of mPEG-maleimide (mPEG-MAL) under neutral pH conditions has effectively improved its drug-forming properties (ZAPPE H, SNELL M E, BOSSARD M J. PEGylation of cyanovirin-N, an entry inhibitor of HIV [J]. Advanced Drug Delivery Reviews, 2008, 60 ( 1 ) : 79-87. ).

 The specific modification strategy of Zappe et al. is that the molecular weights of the mutants in which 62 and 14 glutamines are replaced by hemi-amino acid are respectively 20KD and 30KD of mPEG-maleimide (mPEG-MAL) were modified under neutral pH and alkaline pH. Results 14 The mutant with glutamine replaced by hemi-amino acid (CVN(Q14C)) has a very low modification efficiency, while the mutant with 62 glutamine replaced by hemi-amino acid (CVN(Q62C)) is neutral pH. The modification rate of the CVN mutant with mPEG-MAL molar ratio of 1:3 was higher. However, CVN (Q62C) is more active against HIV than unmutated CVN. To be low, and since the selected modification site is inside the CVN, the body variant modified by mPEG-maleimide 30KDa (mPEG-MAL-30KDa) is anti-HIV The activity is almost lost.

 Summary of the invention

 In view of the shortcomings and deficiencies of the prior art, the object of the present invention is firstly to provide a CVN. Mutants, which are easier to express in the host, are easier to purify, and facilitate further modification.

 Another object of the present invention is to provide the above CVN Modified derivatives of mutants to reduce the cytotoxicity and immunogenicity of the protein itself, making it more suitable for use.

 A further object of the present invention is to use the above mutants and modified derivatives for the preparation of a medicament for the prevention and/or treatment of AIDS.

 To achieve the above object, the present invention provides the following technical solutions:

 A cyanobacterial virus protein N mutant having an amino acid sequence consisting of sequence A and sequence B, and sequence A is located in sequence B of N End,

 Sequence A is one of the following:

 a. SEQ ID NO: 1 in the sequence listing;

 b. SEQ ID NO: 1 in the sequence listing The amino acid residue sequence is substituted, deleted or added with one or several amino acids, which is hydrophilic and flexible;

 Sequence B is one of the following:

 c. SEQ ID NO: 2 in the sequence listing;

 d. SEQ ID NO: 2 in the sequence listing The amino acid residue sequence is substituted, deleted or added with one or several amino acids, but does not change the first three residues at the N-terminus, and it has specific anti-HIV activity;

 The present invention also provides a nucleotide sequence encoding a cyanobacterial virus protein N mutant.

 Preferably, the above-described coding nucleotide sequence comprises one of the following sequences:

 e. SEQ ID NO: 3 in the sequence listing;

 f. DNA that is compatible with SEQ ID NO: 3 in the Sequence Listing under high stringency conditions The nucleotide sequence of the sequence hybridization.

 The high stringency conditions here refer to low salt high temperature hybridization conditions such as 0.1 × SSC, 0.1% SDS and 65 °C. Temperature.

The above nucleotide sequence can be mainly used for expressing a target protein in a host, and an expression vector, a cell line, a host strain or the like containing the above nucleotide sequence can be obtained by a conventional technical means. The purification process of the protein from the host can also be carried out by a conventional method.

 Based on the obtained purified protein, the present invention also provides a CVN mutant modified derivative which is the N of the above CVN mutant. The ends are PEG-modified, and the modification sites are generally directed to the α-amino group of the N-terminal glycine residue.

 Preferably, the PEG-modified modifier uses mPEG-ALD (monomethoxy ether PEG-propionaldehyde), The molecular weight of mPEG-ALD is preferably 10KD-20KD.

 Both the above mutants and modified derivatives can be used in the preparation of prophylaxis and / Or the use in the treatment of AIDS drugs, based on the same antiviral mechanism, the mutants and modified derivatives of the present invention can be applied to the preparation of a medicament for preventing and/or treating diseases caused by other viral microorganisms.

 Compared with the prior art, the present invention has the following beneficial effects:

 The CVN mutant and the modified derivative thereof provided by the invention are for better realization of the antiviral function of the recombinant CVN in vitro, and the modified L-CVN has enhanced anti-HIV activity (WST method and cell fusion method). Since the LCVN end prepared by the present invention introduces a hydrophilic and flexible polypeptide sequence, N can be performed. Fixed-point amino modification at the end. Our experimental results show that LCVN can not only obtain active modified products after N-terminal amino group modification, but also enhance the antiviral activity of the modified products.

 DRAWINGS

 Figure 1 is a diagram showing the recombinant plasmid pET3c-6His-SUMO-LCVN.

Figure 2 is a recombinant pET3C-6His-SUMO-LCVN plasmid restriction enzyme and PCR analysis, wherein M: DL2000 DNA marker; Lane 1: pET3C-6His-SUMO-LCVN / Nde I+ BamH I; Lane 2: PCR.

 Figure 3 is an SDS-PAGE electrophoresis analysis of BL21/ pET3c-6His-SUMO-LCVN The expression characteristics of the protein were as follows: from left to right, the lanes were sequentially induced by IPTG without induction, IPTG induction for 20 hours, induced expression of disrupted centrifugal supernatant, and induction of fragmentation and centrifugation.

 Figure 4 shows the purified target protein LCVN, wherein: from left to right, each lane is Sumo-LCVN fusion protein, Sumo-LCVN fusion protein was digested with Sumo protease, LCVN protein.

 ( * ) indicates Sumo-LCVN fusion protein

 ( ** ) indicates the LCVN protein.

 Figure 5 shows the purity of recombinant LCVN analyzed by RP-HPL.

 Figure 6 shows the 10K mPEG-ALD modified LCVN at different pH and feed ratios. Tricine-SDS-PAGE electrophoresis map, where: from left to right, lane 1 is protein Maker, lanes 2, 3, and 4 are PH3.5, LCVN and 10K mPEG-ALD molar ratio is 1:1, 1:3, 1:5 modified product electrophoresis bands; lanes 5, 6, and 7 are PH4.0, LCVN and 10K mPEG-ALD molar ratio is 1:1, 1:3, 1:5 modified product electrophoresis bands; lanes 8, 9, 10 are PH5.0, LCVN and 10K mPEG-ALD molar ratio is 1:1, 1:3, 1:5 modified product electrophoresis bands; lanes 11, 12, 13 are PH6.0, LCVN The electrophoresis bands of the modified products with the molar ratio of 10K mPEG-ALD were 1:1, 1:3, 1:5; the lanes 14, 15, and 16 were PH7.0, respectively. The electrophoresis bands of the modified products when the molar ratio of LCVN to 10K mPEG-ALD were 1:1, 1:3, 1:5, respectively.

 Figure 7 is a comparison of the modification rates of 10K mPEG-ALD modified LCVN under different pH and feed ratio conditions.

 Figure 8 shows the 20K mPEG-ALD modified LCVN at different pH and feed ratios. Tricine-SDS-PAGE electrophoresis map, wherein: from left to right, lane 1 is protein Maker, lanes 2, 3, and 4 are PH3.5, LCVN and 20K, respectively. The mPEG-ALD molar ratio is 1:1, 1:3, 1:5 for the modified product electrophoresis bands; lanes 5, 6, and 7 are PH4.0, LCVN and 20K, respectively. The mPEG-ALD molar ratio is 1:1, 1:3, 1:5 for the modified product electrophoresis bands; lanes 8, 9, and 10 are PH5.0, LCVN and 20K, respectively. The mPEG-ALD molar ratio is 1:1, 1:3, 1:5 for the modified product electrophoresis bands; lanes 11, 12, and 13 are PH6.0, LCVN and 20K mPEG-ALD molar ratio is 1:1, 1:3, 1:5 modified product electrophoresis bands; lanes 14, 15, 16 are PH7.0, LCVN The electrophoresis band of the modified product with a molar ratio of 20K mPEG-ALD of 1:1, 1:3, 1:5, respectively.

 Figure 9 is a comparison of the modification rates of 20K mPEG-ALD modified LCVN under different pH and feed ratio conditions.

 Figure 10 shows the 10K mPEG-ALD modified LCVN at different times. Tricine-SDS-PAGE electrophoresis map, where lane 1 is protein Maker and lanes 2-8 are 1h, 3h, 5h, 7h, 9h, After 12h and 24h sample electrophoresis results, lane 9 is unmodified LCVN.

 Figure 11 shows the 20K mPEG-ALD modified LCVN at different times. Tricine-SDS-PAGE electrophoresis map, where lane 1 is protein Maker and lanes 2-8 are 1h, 3h, 5h, 7h, 9h, After 12h and 24h sample electrophoresis results, lane 9 is unmodified LCVN.

 Figure 12 is 10K mPEG-ALD modified LCVN via SP-Sepharose The purified elution curve was isolated.

 Figure 13 shows the separation and purification of each component by 10K mPEG-ALD modified LCVN by SP-Sepharose Tricine-SDS-PAGE electropherogram, where lane 1 is protein Maker, lane 2-5 is a mixture of modification reactions, loading peaks, containing 80 mM NaCl The elution peak of buffer A; the elution peak of buffer A containing 400 mM NaCl.

 Figure 14 is 20K mPEG-ALD modified LCVN via SP-Sepharose The purified elution curve was isolated.

 Figure 15 is a 20K mPEG-ALD modified LCVN separated and purified by SP-Sepharose Tricine-SDS-PAGE electrophoresis map, wherein: lane 1 is protein Maker, lanes 2-5 are mixtures of modification reactions, loading breakthrough peaks, containing 70 mM NaCl The elution peak of buffer A; the elution peak of buffer A containing 400 mM NaCl.

 Figure 16 shows the cell viability (%) of MT-4 after treatment with different concentrations of LCVN and its modified products.

 Figure 17 shows the inhibition rate (%) of CVN and LCVN on HIV-1/IIIB proliferation.

 Figure 18 is an anti-human immunodeficiency virus activity of LCVN and its PEG-modified product, wherein (1) MOLT-4 cells, (2) MOLT-4/IIIB cells, and (3) cells after co-culture, observed under phase contrast microscopy, (4) Pseudo-treated co-cultured cells, (5-8) CVN/LCVN/10kPEG-LCVN/20kPEG-LCVN at a concentration of 113 nM Co-cultured cells after treatment, indicated by black arrows, are fused large cells. Figure (b) CNV, LCVN, 10kPEG-LCVN, 20kPEG-LCVN The fusion inhibition activity, all experiments are at least 3 statistical results after independent experiments.

 Figure 19 shows the CPE of LCVN and its PEG modified product against HSV-1 activity. Observation results. among them:

 A, normal control; B, virus control; C, positive drug ACV control (1μg/ml); D, L-CVN sample ( 1.562 μg/ml); E, SUMO-L-CVN sample ( 3.125 μg/ml); F , mPEG-ALD-10kDa-L-CVN ( 3.125 μg/ml ); G , mPEG-ALD-20kDa-L-CVN ( 3.125 μg/ml ).

 detailed description

 The preferred embodiments of the invention are listed below to assist in further understanding of the invention, but embodiments of the invention are not limited thereto.

The main materials involved in the examples of the present invention are as follows: Host strain Escherichia coli BL21 (DE3) (purchased from Novagen), plasmid pET3c (purchased from Novagen), pET3c-SUMO-CVN is preserved by the laboratory (the construction method has been patented) The preparation method and application of recombinant cyanobacterial antiviral protein, application number: 200810198926.0", the construction of the plasmid can also adopt the well-known genetic engineering method, the basic idea is to first obtain the SUMO-CVN fusion sequence by PCR, and then connect to the pET3c vector. Plasmid pET3c-SUMO-CVN); SUMO protease (purchased from Haiji Biotechnology Co., Ltd.); Taq enzyme, T4 DNA ligase, DNA molecular weight standard, various restriction enzymes purchased from Dalian Bao Biotech Co., Ltd.; Standards (purchased from Juyan Biotechnology Co., Ltd.), primers purchased from Shanghai Shenggong Biotech Co., Ltd.; Ni ^{2 +} Sepharose Fast Flow, SP Sepharose Fast Flow from GE Healthcare; MTT and WST from SIGMA, USA; Herpes simplex Virus type 1 ( HSV-1 ) F strain from Wuhan University Virus Research Institute ( CGMCC No.0396 ); Vero cells CCL-81TM), MOLT-4 cells (CRL-1582TM), MT-4 cells (CRL-1942TM), HIV-I/IIIB virus (CRL-1973TM), etc. from the American Type Culture Collection (ATCC) mPEG-ALD (10K) and mPEG-ALD (20K) were purchased from Beijing Kaizheng Bioengineering Development Co., Ltd.

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

 Example

 1. Construction of recombinant plasmid pET3c-6His-SUMO-LCVN

 The construction of the SUMO-L-CVN gene was synthesized in two steps. First, the L-CVN gene was synthesized by two PCRs, the first time. PCR uses pET3c-SUMO-CVN plasmid as template and F1-CVN and R-CVN as upstream and downstream primers. The reaction system is 1 ng for the template and 1 μM for each of the upstream and downstream primers. 20μl Taq PCR MasterMix, add water to 40μl, the reaction mixture is denatured at 94 °C for 1min, annealed to 55 °C for 1 min, 72 °C Extend 1 min for 29 cycles. The reaction product was subjected to 1% agarose gel electrophoresis, and the target fragment was recovered by gel as a template for the next round of PCR. Second PCR above one round of PCR The product is a template, F2-CVN and R-CVN are upstream and downstream primer pairs (where the F2-CVN primer contains a flexible polypeptide encoding 15 amino acid residues), and the full-length sequence of L-CVN is synthesized. The SUMO full-length sequence was synthesized from pET3c-SUMO-CVN by PCR. Using the 26 bp overlapping complementary sequence at the end of the SUMO sequence and the front end of the L-CVN sequence PCR: using SUMO sequence and L-CVN sequence as extension template, F-SUMO and R-CVN are upstream and downstream primers, and the reaction was carried out under normal PCR conditions, and the reaction product was subjected to 1%. Electrophoresis on agarose gel and gel recovery gave the full length sequence of SUMO-L-CVN.

The full-length sequences of plasmid pET3C and 6His-SUMO-LCVN were digested with Nde I and BamH I respectively, and the digested product was subjected to 1% agarose gel electrophoresis, and the digested product was recovered, T ₄ DNA ligase, and the ligation product was transformed into the large intestine. Bacterial JM109 competent cells were plated on LB plates containing ampicillin and cultured overnight at 37 °C. Plasmids were extracted, amplified by PCR and identified by double digestion with Nde I and BamH I. The positive plasmids were sent to Yingjun for sequencing.

 Table 1 Primers used to synthesize the full length sequence of SUMO-LCVN

Primer name	sequence
F2-CVN	5' -CAGATTGGTGGTGGTGGCGGAGGGAGCGGTGGAGGGGGCAGTGGCGGAG-3'
F1-CVN	5' -GGAGGGGGCAGTGGCGGAGGAGGTAGCCTTGGTAAATTCTCCCAG-3'
R-CVN	5 '-AGA GGATCC TCATCATTCGTATTTCAGGGTAC-3'
F-SUMO	5' -CAG CATATG CATCATCATCATC-3'
R-SUMO	5' -CTCCCTCCGCCACCACCACCAATCTGTTCTCTG-3'

 2, LCVN engineering bacteria shake flask culture, protein purification and purity determination

 Sequencing the correct plasmid into E. coli BL21 (DE3) BL21[pET3c-6His-SUMO-LCVN], monoclonal clones were selected for culture and induced expression, and bacterial proteins were collected for SDS-PAGE electrophoresis analysis. The result shows that BL21 The [pET3c-6His-SUMO-LCVN] positive clone strain will express a fusion protein of about 28kDa after induction. The uninduced control group showed no macroscopic expression at the corresponding position. After the bacteria were disrupted by sonication, the fusion protein was found to be soluble in the supernatant, and the soluble target protein accounted for the total protein of the supernatant. 28.3 ± 3.4 % (Figure 3).

 High-expression strains were selected and inoculated into 1L LB medium containing ampicillin (100 mg/L), 37 ° C, 180 When rpm was cultured to OD600 =0. 6 ～ 1. 0, the temperature was lowered to 20 °C and IPTG was added to a final concentration of 0.5 mM to induce expression for 24 h. 4 °C, 6000×g The cells were collected by centrifugation at 10 min, frozen and thawed, and then the cells were resuspended in NTA-10 buffer at a ratio of 1:10, sonicated (working time 5 s, intermittent time) 5s, 99 times, repeated 3 times), the supernatant was collected by centrifugation at 4 ° C, 25000 × g, 30 min.

 The supernatant was loaded with a 20 ml Ni-NTA affinity column at a flow rate of 0.6 ml/min, NTA-0. The buffer was washed back to the baseline at a flow rate of 1 ml/min, NTA-20 buffer washed protein, and NTA-250 buffer eluted the target protein. Purified target protein 6His-SUMO-LCVN was subjected to deamidation by Sephadex G-25 molecular sieve and then subjected to SUMO protease digestion to remove the SUMO fusion protein.

 6His-SUMO-LCVN Adjust the concentration to 1mg/ml and add 1 U SUMO protease /mg Fusion protein, digested at 30 ° C for 1 h. Because the 6His-SUMO tag and SUMO protease both contain a 6×His tag, the digested sample is loaded again. Ni-NTA The affinity column was purified to remove the 6His-tagged SUMO, un-cut 6His-SUMO-LCVN and SUMO protease to obtain the non-fused target protein LCVN. The G-25 molecular sieve column is desalted and freeze-dried to obtain LCVN products for subsequent physical and chemical properties determination, activity determination or PEG modification.

 Figure 3 is the engineering strain BL21[ pET3c-6His-SUMO-LCVN] by IPTG The protein expression profile after induction showed that after induction, a significantly thickened protein band (lane 3) appeared at a molecular weight of 28 kD, and 6His-SUMO-LCVN The theoretical molecular weight is consistent. After sonication, the target protein is located in the supernatant of the broken cell, which accounts for 40% of the soluble protein of the bacteria (lane 2). Figure 4 is an SDS-PAGE analysis of LCVN Purification process, where * is shown as 6His-SUMO-LCVN fusion protein, and ** is shown as LCVN protein. Figure 5 is a reverse high performance liquid chromatography of LCVN products ( RP-HPLC) Purity analysis results, the plant type is a C-18 reverse column with a detector wavelength of 280 nm, mobile phase A: ultrapure water containing 0.1% trifluoroacetic acid (TFA), mobile phase B : Ethyl cyanide, gradient elution with 40% -60% B, and elution peak of LCVN with retention time between 4-6 min.

 3. Pilot fermentation and protein preparation of LCVN engineering bacteria

The engineered strain BL21[pET3c-6His-SUMO-LCVN] was selected and cultured and induced to express in a test tube, and a high expression strain was selected for pilot fermentation. Primary and secondary seeds were cultured in conical flasks, secondary seeds were incubated to appropriate concentrations, and inoculated into 15 L of fermentation medium at 10% inoculum. The temperature is automatically controlled at 37 °C, and the speed and ventilation are adjusted in time to maintain a proper dissolved oxygen level. The pH is adjusted to 7.0 to 7.2 with NaOH and HCl. When the cell density (OD ₆₀₀ ) was about 12, the temperature was lowered to 20 °C, and IPTG was added to a final concentration of 0.5 mM to induce expression for 20 h. 100 ml/min, 9000 g, the cells were collected by continuous flow centrifugation, and about 550 g of wet cells were harvested in a 15 L fermentation medium, and the collected cells were stored in a refrigerator at -20 °C.

 The cell pellet was suspended in NTA-0 buffer at a ratio of 1:10, sonicated (working time 5s, intermittent time) 5s, 99 times, repeated 3 times), the supernatant was collected by centrifugation at 4 ° C, 25000 × g, 30 min. The loading of Ni-NTA packing with a volume of 20 ml is 250ml supernatant, flow rate 1ml/min, NTA-0 buffer wash back to baseline; flow rate 1ml/min, NTA-20 buffer wash protein, NTA-250 Buffer elutes the protein of interest. The purified target protein 6His-SUMO-LCVN is subjected to SUMO protease digestion by removing the imidazole from Sephadex G-25 molecular sieve. SUMO fusion protein.

 6His-SUMO-LCVN Adjust the concentration to 1mg/ml and add 1 U SUMO protease /mg Fusion protein, digested at 30 ° C for 1 h. Because the 6His-SUMO tag and SUMO protease both contain a 6×His tag, the digested sample is loaded again. Ni-NTA The affinity column was purified to remove the 6His-tagged SUMO, un-cut 6His-SUMO-LCVN and SUMO protease to obtain the non-fused target protein LCVN. Replace the buffer with G-25 molecular sieve column and concentrate the LCVN protein with a 3KD ultrafiltration tube for subsequent physical and chemical determination, activity determination or PEG modification.

 4, PEG modification of LCVN protein

 PEG modification of pharmaceutical proteins is an effective method to improve various drug properties such as pharmacokinetic properties, stability and immunogenicity. Protein available PEG-modified sites include side chain amino groups, N-terminal amino groups, side chain carboxyl groups, C A terminal carboxyl group, a side chain thiol group, and the like. Existing carboxyl modification techniques are prone to non-specific cross-linking reactions, so amino modification is more common and technological development is more mature. Zappe et al.'s findings indicate that wild-type CVN The modification of the side chain amino or N-terminal amino group will destroy the activity of the protein. Therefore, the author first performs site-directed mutagenesis of CVN to obtain the Q62C mutant, and then introduced the Cys introduced at position 62. The side chain thiol modification was carried out to obtain an active protein. Due to the formation of 4 Cys residues in the native CVN 2 For disulfide bonds, and in solution, the disulfide bond of the protein is in a dynamic isomeric and equilibrium state. Therefore, after introducing the Q62C mutation, it is difficult to prevent the introduced Cys from interfering with the correct matching of the disulfide bond, or non-62 position. Cys modification, the authors' final experimental results also showed that the activity of CVN Q62C mutant and its modified products were lower than wild-type CVN.

 Since the LCVN end prepared by the present invention introduces a hydrophilic and flexible 15-peptide sequence, an attempt can be made to N. Fixed-point amino modification at the end. Our experimental results show that LCVN can not only obtain active modified products after N-terminal amino group modification, but also enhance the antiviral activity of the modified products.

 There are many PEG modification methods for amino groups, and there are many alternative modifiers. The present invention selects mPEG-ALD (10K) and mPEG-ALD (20K) was used as a modifier to screen the optimal PEG modification conditions for LCVN from three aspects: substrate ratio, modifier ratio, modification pH and modification reaction time.

Select Na-Ac buffer with pH of 3.5, 4.0, 5.0, 60, 7.0 and ionic strength of 20 mM. The concentration of LCVN in the reaction system is 5 mg/ml, and the molar ratio of LCVN to mPEG-ALD (10K) is selected as 1 respectively. :1 , 1:3 , 1:5 ; The final concentration of NaCNBH ₃ is 5 mg/ml. The reaction was shaken on a shaker at room temperature, and after 3 hours of reaction, the effect of the modification reaction was identified by electrophoresis. The apparent molecular weight of the single modified product of mPEG-ALD (10K) modified LCVN was about 30KD on SDS-PAGE. The SDS-PAGE analysis showed that the molar ratio of LCVN to mPEG-ALD (10K) was 1: at pH 4.0. Under the conditions of 3, the single modification rate was the highest (Fig. 6).

 A similar method was used to study the optimal pH and feed ratio of mPEG-ALD (20K) modified LCVN. After mPEG-ALD (20K) modification of LCVN, the apparent molecular weight of the single modified product on SDS-PAGE is about 50KD. The experimental results show that at pH5.0, LCVN The single modification rate was the highest with a molar ratio of mPEG-ALD (20K) of 1:1 (Fig. 18).

 mPEG-ALD (10K) and mPEG-ALD (20K) modification at the optimum pH and feed ratio determined LCVN was sampled at 1h, 3h, 5h, 7h, 9h, 12h and 24h, and the optimal reaction time was identified by Tricine-SDS-PAGE electrophoresis. The results of SDS-PAGE analysis indicated that the reaction time was modified by mPEG-ALD (10K) and mPEG-ALD (20K). The reaction did not have a significant effect, considering the time cost, preferably the optimal modification reaction time was 2 h at room temperature (Fig. 10 and Fig. 11).

 5, separation and purification of PEG modified products of LCVN

 After LCVN is modified by PEG-ALD, the reaction mixture mainly contains unmodified LCVN and the remaining PEG. PEG multi-modified LCVN, PEG single-modified LCVN. AKTA prime plus separation and purification system, SP sepharose column The single modified mPEG-ALD-LCVN was isolated and purified, and the mobile phase was 20 mM Na-Ac buffer (buffer A), and 5 column volumes were used before loading. bufferA equilibrate the column, collect the breakthrough peak after loading, and then elute with bufferA containing different concentrations of NaCl to collect each elution peak. The flow rate is 1ml/min and the detection wavelength is 280nm. The collected samples were detected by SDS-PAGE gel electrophoresis.

mPEG-ALD (10K) modified LCVN mixture, after purification by SP sepharose cation chromatography, the modifier mPEG-ALD and multi-modification products were not penetrated by SP sepharose, and the buffer A elution product containing 80 mM NaCl was single. Modified mPEG-ALD _(10K) -LCVN; buffer A eluting product containing 400 mM NaCl is unmodified LCVN. Figure 12 shows the elution curve of mPEG-ALD _(10K) -LCVN separation and purification. Peak 1 is the modifier and multi-modification product, peak 2 is the single modification product, and peak 3 is the unmodified substrate. Figure 13 shows the elution components by SDS-PAGE. Lane M is the protein molecular weight standard; Lane A is the sample, showing unmodified substrate, single modified product and multiple modified products; Lane 1 is the breakthrough peak; Lane 2 It is a single modified product, that is, the target protein mPEG-ALD _(10K) -LCVN; Lane 3 is an unmodified product.

mPEG-ALD (20K) modified LCVN mixture, after purification by SP sepharose cation chromatography, the modifier mPEG-ALD and multi-modification products were also present in the loading peak, and the buffer A eluting product containing 70 mM NaCl was single. Modified mPEG-ALD _(20K) -LCVN; buffer A eluting product containing 400 mM NaCl is unmodified LCVN.

Figure 14 shows the elution curve of mPEG-ALD _(20K) -LCVN separation and purification. Peak 1 is the modifier and multi-modification product, peak 2 is the single modification product, and peak 3 is the unmodified substrate. Figure 15 shows the elution components by SDS-PAGE. Lane M is the protein molecular weight standard; Lane A is the sample, showing unmodified substrate, single modified product and multiple modified products; Lane 1 is the breakthrough peak; Lane 2 Is a single modified product, that is, the target protein mPEG-ALD _(10K) -LCVN; Lane 3 is a 400 mM NaCl elution component, showing unmodified products, but also

 There are some single modified products.

 Application Example 1 Determination of cytotoxicity of LCVN on T cells by WST method

CVN, LCVN and PEG modified products were serially diluted in RPMI-1640 medium and added to 96-well cell culture plates at 50 μl per well. CVN and LCVN were diluted from 10 μg/ml to 0.04 μg/ml. PEG-modified LCVN The dilution range is from 50 μg/ml to 0.19 μg/ml. The MT-4 cells were adjusted to a concentration of 1×10 ⁵ /ml, 100 μl per well, mixed, and cultured in a 37 ° C, 5% CO ₂ cell incubator, with a cell control and a positive drug control (63 nM azidothymidine, AZT), 3 replicate wells were determined in parallel for each sample. After 4 days, 10 μl of WST-1 (water-soluble tetrazolium, 5 mmol/L) solution was added to each well of the culture plate, and the culture was continued for 4 hours. The absorbance (A) was read on the plate reader, and the wavelength was calculated at 450/650 nm. Cell viability ( RP , % ) and 50% toxic concentration (CC ₅₀ ) based on RP.

 Cell viability (%) = drug treatment group A value / cell control group A Value ×100%.

 Figure 16 shows cell viability after treatment with four different proteins, CVN, LCVN and their modified products. In the figure, N.C. refers to an untreated cell control with a cell control density of 100%. AZT is a positive control drug, 63 nM azidothymidine. The calculated 50% toxic concentration is shown in Table 2. Shown.

Table 2 50% toxic concentration of LCVN and its PEG modified products

Sample	Cytotoxicity (CC50, μM)
AZT	> 4.482
CVN	0.160±0.009
LCVN	0.658±0.277
mPEG-ALD (10K) -LCVN	4.519±0.191
mPEG-ALD (20K) -LCVN	3.629±0.137

 Application Example 2 Determination of anti-human immunodeficiency virus activity of LCVN by WST method

CVN, LCVN and PEG modified products were serially diluted in RPMI-1640 medium and added to 96-well cell culture plates at 50 μl per well. Adjust the concentration of MT-4 cells to 1 × 10 ⁵ /ml, 100 μl per well, mix, and then add 50 μl of HIV-1/IIIB virus suspension with a titer of 100 TCID50. The cells were cultured in a 37 ° C, 5% CO ₂ cell incubator, and a cell control, virus control, and positive drug control (azidothymidine, AZT) were set. Three replicate wells were determined in parallel for each sample. After 4 days, 10 μl of WST-1 (water-soluble tetrazolium, 5 mmol/L) solution was added to each well of the culture plate, and incubation was continued for 4 hours. The absorbance (A) was read on a plate reader at a wavelength of 450/650 nm.

 Cell viability (%) = drug treatment group A value / cell control group A Value ×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 ) × 100%

 The Reed-Muench method calculates the 50% inhibitory concentration (IC50) and is 50% according to the example × Toxicity concentration (CC50), and the selection index (TI), TI=CC50/IC50.

 As can be seen from Table 3, for the positive drug AZT, CVN, LCVN vs HIV-1/IIIB The inhibitory activity of proliferation is stronger.

Table 3 Inhibitory activity of CVN and LCVN on HIV-1/IIIB proliferation

Sample	IC50 (nM)	CC50 (μM)	SI
AZT	36.55±5.64	> 4.482	122
CVN	21.83±2.79	0.160±0.009	7.3
LCVN	14.17±6.43	0.658±0.277	46.4

 Application Example 3 Determination of anti-human immunodeficiency virus activity of LCVN and its PEG modified product by fusion inhibition method

 Existing studies have shown that the spread of HIV between T cells is mainly through the gp120 expressed on the surface of infected cells. It is mediated and binds to receptors on uninfected cells to form fused cells. CVN can specifically bind to gp120 Inhibition of the fusion between infected cells and normal cells blocks the spread of the virus. Therefore, we used the cell fusion inhibition model to determine 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] .

The MOLT-4 cells and MOLT-4/IIIB cells grown to log phase were adjusted to a density of 1×10 ⁶ /ml, and 250 μl of each cell suspension was mixed in equal volumes and added to a 24-well cell culture plate (the total number of cells was 5×10 ⁵ /500μl/well); CVN, LCVN and its PEG-modified products were serially diluted 4 times in RPMI-1640 medium containing fetal bovine serum and antibiotics, and 3 concentrations were selected for each test drug (452 nM, 113 nM). , 28 nM ), an equal volume was added to the cell suspension, and various control groups were set at the same time. All the samples were paralleled with 2 replicate wells, and cultured at 37 ° C, 5% CO _{2 for} 24 hours. After 24 h, the cell suspension was taken, stained with trypan blue, and filled into a cell counting cell, and the number of surviving MOLT-4 cells was counted under a microscope. Since MOLT-4/IIIB cells can continuously produce HIV-I/IIIB virus particles and form large multinucleated cells (synaptosomes) by fusion with normal MOLT-4 cells to infect normal host cells, at the time of cell counting, fusion The cells cannot enter the counting pool. Therefore, by counting the number of normal-sized MOLT-4 or MOLT-4/IIIB cells in the cell counting pool, the number of cells forming the syncytia can be estimated, and the number of cells in the co-culture group is compared and not co-cultured. The number of MOLT-4 cells in the group, calculate the fusion index (FI, fusion index):

 FI=1 - (Number of cells in co-cultured cell wells ÷ Number of cells in control wells containing only MOLT-4 cells)

 Compare the fusion index of the co-cultured cells and the unadministered co-cultured cells to calculate the fusion inhibition rate ( FIR , fusion Inhibition rate ):

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 unadministered co-cultured cells.

Table 4 Inhibition rate of LCVN and its PEG-modified products on cells (%)

	Fusion inhibition rate ( 452nM )	Fusion inhibition rate (113nM)	Fusion inhibition rate (28nM)
CVN	69.97 ± 8.70	56.80 ± 12.79	34.54 ± 8.48
L-CVN	83.18 ± 6.27	79.43 ± 15.40	91.70 ± 1.45
PEG10K-LCVN	91.67 ± 7.66	92.77 ± 10.06	56.03 ± 13.96
PEG20K-LCVN	99.12 ± 2.17	89.90 ± 4.72	29.27 ± 8.51

 As shown in Table 4, the fusion inhibitory activity of LCVN against HIV-1/IIIB was significantly higher than that of the dose group. CVN; In addition, LCVN PEG modified products were more active than unmodified LCVN at medium and high doses.

 Figure 18 (a) shows the (1) MOLT-4 cells after incubation for 24 h under a phase contrast microscope, (2) MOLT-4/IIIB cells, (3) co-cultured cells, (4) sham-treated co-cultured cells, (5-8) at a concentration of 113 nM CVN/LCVN/10kPEG-LCVN/20kPEG-LCVN After the treatment, the co-cultured cells showed large cells fused as indicated by black arrows. No fused cells were observed in the unco-cultured group, and typical fused cells appeared in the co-culture group, while the conjugated cells were not observed in the drug-administered group.

 Figure 18 (b) is CNV, LCVN, 10kPEG-LCVN, 20kPEG-LCVN The fusion inhibition activity, all experiments are at least 3 statistical results after independent experiments. It can be seen that the fusion inhibitory activity of LCVN on HIV-1/IIIB is significantly higher than that of CVN in any dose group. In addition, at medium and high doses, the PEG-modified product of LCVN gradually increased in activity with increasing molecular weight, but in the low-dose group, PEG The activity of the modified product gradually decreases as the molecular weight increases.

 Application Example 4 Determination of anti-herpes simplex virus (HSV-1) activity of LCVN by MTT assay

 Herpes simplex virus type 1 (HSV-I) is a DNA that causes widespread infection in the human population. Virus, human being is the only host, and about 90% of healthy adults are infected with HSV-1. HSV-I can cause various diseases such as cold sore, herpetic keratoconjunctivitis and neonatal encephalitis due to HSV-I It can be latent infection in the ganglion, so the symptoms are easy to relapse. In this experiment, the toxicity of recombinant LCVN and its modified products and wild-type CVN to Vero cells was determined by MTT assay; CPE The method observes the activity of the drug on the cells.

In the single-layered Vero cells, 50 μl of each test substance of different dilutions and 100 TCID50 of HSV-1 virus solution were added, and a normal cell control and a virus control group were set. Incubate with 5% CO _{2 for} 48 h, add 5 μl/ml MTT 10 μl per well, continue to culture for 5 h with 5% CO ₂ , discard the supernatant, add 200 μl DMSO to each well, store at room temperature for 30 min in the dark, shake the plate for about 10 min, and lym. colorimetric reader (wavelength 570nm, reference wavelength of 630nm), the absorbance was measured and calculated 50% cytotoxic concentration of the sample _{(50% cytotoxic concentration, CC 50} ).

 Figure 19 It is the morphology of the cells under a phase contrast microscope after a typical experiment, showing the cytopathic effect of the virus on the cells and the protective effect of the drugs on the cells. The results showed that LCVN and its modified products have good resistance. HSV-1 activity, LCVN and its PEG modified products showed positive control drug ACV under the conditions of close mass concentration and lower molar concentration. Basically close to antiviral activity. The toxicity test results showed that the toxicity of LCVN modified by PEG to Vero cells was also significantly reduced.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and combinations thereof may be made without departing from the spirit and scope of the invention. Simplifications should all be equivalent replacements and are included in the scope of the present invention.

Claims (10)

1. A cyanobacterial virus protein N mutant characterized in that the amino acid sequence consists of sequence A and sequence B, and sequence A is located at the N-terminus of sequence B.

   Sequence A is as follows:

   a. SEQ ID NO: 1 in the sequence listing;

   b. SEQ ID NO in the sequence listing: The amino acid residue sequence of 1 is substituted, deleted or added with one or several amino acids, which has the characteristics of hydrophilic flexibility;

   Sequence B is as follows:

   c. SEQ ID NO: 2 in the sequence listing;

   d. SEQ ID NO in the sequence listing: The amino acid residue sequence of 2 is substituted, deleted or added with one or several amino acids, but does not change the first three residues of its N-terminus, and it has specific anti-HIV virus activity.
2. The nucleotide sequence encoding the cyanobacterial protein N mutant of claim 1.
3. A nucleotide sequence encoding a cyanobacterial protein N mutant according to claim 2, which comprises one of the following sequences:

   e. SEQ ID NO: 3 in the sequence listing;

   f. A nucleotide sequence which hybridizes under high stringency conditions to a DNA sequence as defined by SEQ ID NO: 3 in the Sequence Listing.
4. An expression vector comprising the nucleotide sequence of claim 2 or 3.
5. A host strain comprising the nucleotide sequence of claim 2 or 3.
6. A cyanobacterial virus protein N mutant modified derivative characterized in that the N-terminus of the cyanobacterial protein N mutant of claim 1 is subjected to PEG modification.
7. The cyanobacterial protein N mutant modified derivative according to claim 6, wherein the PEG-modified modifier uses monomethoxy ether PEG-propionaldehyde.
8. The cyanobacterial protein N mutant modified derivative according to claim 6, wherein the mPEG-ALD has a molecular weight of 10 KD to 20 KD.
9. Use of the cyanobacterial protein N mutant of claim 1 for the preparation of a medicament for the prevention and/or treatment of AIDS.
10. Use of a modified derivative of a cyanobacterial protein N mutant according to any one of claims 6-8 for the preparation of a medicament for the prevention and/or treatment of AIDS.

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