CN101638435B - Blue-green algal virus protein N mutant, modified derivative and application thereof - Google Patents

Abstract

The invention belongs to the field of biomedicine, and in particular relates to a cyanobacteria viral protein N mutant, its PEG-modified derivative and their application in pharmacy. The cyanobacteria virus protein N mutant of the present invention is composed of two sequences, one is a connecting peptide with hydrophilic flexibility, and the other is a codon-optimized sequence of the cyanobacteria virus protein N. The cyanovirus protein N mutant of the present invention is modified by PEG to obtain a derivative. The mutant and its derivatives all show good anti-HIV activity. It has the prospect of being applied to the preparation of drugs against HIV or other viral microorganisms.

Description

A kind of cyanobacteria virus protein N mutant, its modified derivative and application

technical field

The invention belongs to the field of biomedicine, and in particular relates to a cyanobacteria viral protein N (CVN) mutant, its PEG-modified derivative and their application in pharmacy.

Background technique

Cyanovirin-N (CVN) is a protein with anti-HIV activity that can be extracted from cyanobacteria by American scientists BOYD et al. CVN can specifically bind to the capsid protein gp120 of human immunodeficiency virus HIV-1 with high affinity to exert antiviral activity. Broad spectrum and stable properties make CVN protein a valuable antiviral drug [BOYD M R, GUSTAFSON K R, MCMAHON J B, et al. virus-inactivating protein that binds viral surface envelope glycoprotein gp 120: Potential applications to microbicide development [J]. Antimicrobial Agents and Chemotherapy, 1997, 41(7): 1521-30.]. However, because the molecular weight of the protein is small and there are two disulfide bonds in the molecule, it is difficult to express the protein in Escherichia coli and the yield is low. At the same time, as a small molecule protein drug derived from prokaryotic organisms, it has shortcomings such as short half-life, cytotoxicity, and immune response.

Polyethylene glycol (PEG) is a non-toxic, non-immunogenic, water-soluble polymer substance that can modify proteins through covalent bonding. Poor stability, short half-life and other problems in the process (IANG Z Y, XU S W, WANG Y Q. Chemistry for pegylation of protein and peptide molecules [J]. Chinese Journal of Organic Chemistry, 2003, 23 (12) : 1340-7.) The use of PEG-maleimide to modify CVN under acidic conditions has been reported in the literature. Zappe et al. selected a mutant in which glutamine at position 62 was replaced by cysteine (CVN(Q62C)) Using mPEG-maleimide (mPEG-MAL) to modify it under neutral pH conditions effectively improved its druggability (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: select the mPEG-maleimide (mPEG-MAL) of 20KD and 30KD in the neutral pH and It was modified under alkaline pH conditions. As a result, the modification efficiency of the mutant (CVN(Q14C)) in which glutamine at position 14 was replaced by cysteine was very low, while glutamine at position 62 was replaced by cysteine The mutant (CVN(Q62C)) has a higher modification rate under the condition of neutral pH and the molar ratio of CVN mutant to mPEG-MAL of 1:3. However, the anti-HIV activity of CVN (Q62C) is lower than that of unmutated CVN, and since the selected modification site is inside CVN, the body modified by mPEG-maleimide 30KDa (mPEG-MAL-30KDa) The activity of the variant against HIV was nearly lost.

Contents of the invention

In view of the shortcomings and deficiencies in the prior art, the purpose of the present invention is to provide a CVN mutant that is easier to express in the host, easier to purify, and conducive to further modification.

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

Another object of the present invention is to use the above mutants and modified derivatives for the preparation of medicaments for preventing and/or treating AIDS.

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

A kind of cyanobacteria virus protein N mutant, its amino acid sequence is made up of sequence A and sequence B, and sequence A is positioned at the N terminal of sequence B,

Sequence A is one of the following:

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

b. Substituting, deleting or adding one or several amino acids to the amino acid residue sequence of SEQ ID NO: 1 in the sequence listing, which has the characteristics of hydrophilic flexibility;

Sequence B is one of the following:

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

d. Substitute, delete or add one or several amino acids to the amino acid residue sequence of SEQ ID NO: 2 in the sequence list, but do not change the first three residues at the N-terminus, and it has specific anti-HIV virus active;

The invention also provides the coding nucleotide sequence of the cyanovirus protein N mutant.

Preferably, the above coding nucleotide sequence includes one of the following sequences:

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

f. A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID NO: 3 in the sequence listing under high stringency conditions.

Here, the high stringency conditions refer to low-salt and high-temperature hybridization conditions, such as 0.1×SSC, 0.1% SDS and 65° C. temperature.

The above-mentioned nucleotide sequence can be mainly used to express the target protein in the host, and the expression vector, cell line, host bacteria, etc. containing the above-mentioned nucleotide sequence can be obtained by conventional technical means. Conventional methods can also be used for protein purification from hosts.

On the basis of obtaining the purified protein, the present invention also provides a modified derivative of the CVN mutant, which is to modify the N-terminus of the above-mentioned CVN mutant with PEG, and the modification site is generally aimed at the α-amino group of the N-terminal glycine residue.

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

The above-mentioned mutants and modified derivatives can be used in the preparation of drugs for the prevention and/or treatment of AIDS. Based on the same antiviral mechanism, the mutants and modified derivatives of the present invention can be applied to the preparation of drugs for the prevention and/or treatment of other diseases. Drugs for diseases caused by viral microorganisms.

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

The CVN mutants and their modified derivatives provided by the present invention are to better realize the antiviral function of recombinant CVN in vitro, and the modified L-CVN has enhanced anti-HIV activity (WST method and cell fusion method). Since a hydrophilic and flexible polypeptide sequence is introduced into the N-terminus of the LCV prepared in the present invention, site-specific amino modification of the N-terminus can be performed. Our experimental results prove that after LCVN is modified by the N-terminal site-directed amino group, not only active modified products can be obtained, but also the antiviral activity of the modified products is further enhanced.

Description of drawings

Figure 1 is a map of pET3c-6His-SUMO-LCVN recombinant plasmid.

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

Figure 3 is the SDS-PAGE electrophoresis analysis of BL21/pET3c-6His-SUMO-LCVN protein expression characteristics, in which: from left to right, each lane is induced without IPTG, induced by IPTG for 20h, induced by crushed centrifuged supernatant, induced by expression Centrifuge the pellet.

Fig. 4 is the purified target protein LCVN, wherein: from left to right, each lane is Sumo-LCVN fusion protein, the mixture of Sumo-LCVN fusion protein digested by Sumo protease, and LCVN protein.

(*) indicates Sumo-LCVN fusion protein

(**) indicates LCVN protein.

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

Figure 6 is the Tricine-SDS-PAGE electrophoresis of 10K mPEG-ALD modified LCVN under different pH and feed ratio conditions, in which: from left to right, lane 1 is the protein Maker, and lanes 2, 3, and 4 are pH 3.5, respectively. The electrophoresis bands of modified products when the molar ratios of LCVN and 10KmPEG-ALD are 1:1, 1:3, and 1:5 respectively; lanes 5, 6, and 7 are pH 4.0, respectively, and the molar ratios of LCVN and 10KmPEG-ALD are 1 : 1, 1: 3, 1: 5 modified product electrophoresis bands; lanes 8, 9, and 10 are pH 5.0, respectively, and the molar ratios of LCVN and 10KmPEG-ALD are 1: 1, 1: 3, 1: 5 The electrophoresis bands of modified products at 1:1, 1:3, and 1:5 respectively at pH 6.0 and the molar ratios of LCVN and 10KmPEG-ALD at pH 6.0; lane 14 , 15, and 16 are the electrophoresis bands of the modified products when the molar ratios of LCVN and 10K mPEG-ALD are 1:1, 1:3, and 1:5, respectively, at pH 7.0.

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

Figure 8 is the Tricine-SDS-PAGE electrophoresis of 20K mPEG-ALD modified LCVN under different pH and feed ratio conditions, in which: from left to right, lane 1 is the protein Maker, and lanes 2, 3, and 4 are pH 3.5, respectively. The electrophoresis bands of modified products when the molar ratios of LCVN and 20K mPEG-ALD are 1:1, 1:3, and 1:5 respectively; lanes 5, 6, and 7 are pH 4.0 respectively, and the molar ratios of LCVN and 20K mPEG-ALD are respectively Electrophoresis bands of modified products at 1:1, 1:3, and 1:5; lanes 8, 9, and 10 are pH 5.0, respectively, and the molar ratios of LCVN and 20KmPEG-ALD are 1:1, 1:3, and 1: The electrophoresis bands of the modified products at 5 o'clock; lanes 11, 12, and 13 are the electrophoresis bands of the modified products when the molar ratios of LCVN and 20K mPEG-ALD are 1:1, 1:3, and 1:5, respectively, at pH 6.0; Swimming lanes 14, 15, and 16 are the electrophoresis bands of the modified products when the molar ratios of LCVN and 20K mPEG-ALD are 1:1, 1:3, and 1:5, respectively, at pH 7.0.

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

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

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

Figure 12 is the elution curve of 10K mPEG-ALD modified LCVN separated and purified by SP-Sepharose.

Figure 13 is a Tricine-SDS-PAGE electrophoresis image of 10K mPEG-ALD modified LCVN separated and purified by SP-Sepharose, in which: Lane 1 is the protein Maker, and lanes 2-5 are the mixture of the modification reaction and the breakthrough peak of loading , The elution peak of buffer A containing 80mM NaCl; the elution peak of buffer A containing 400mM NaCl.

Figure 14 is the elution curve of 20K mPEG-ALD modified LCVN separated and purified by SP-Sepharose.

Figure 15 is a Tricine-SDS-PAGE electrophoresis image of 20K mPEG-ALD modified LCVN separated and purified by SP-Sepharose, in which: Lane 1 is the protein Maker, and lanes 2-5 are the mixture of the modification reaction and the breakthrough peak of loading , The elution peak of buffer A containing 70mM NaCl; the elution peak of buffer A containing 400mM NaCl.

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

Fig. 17 is the inhibitory rate (%) of CVN and LCVN to HIV-1/IIIB proliferation.

Figure 18 is the anti-human immunodeficiency virus activity of LCVN and PEG modified products thereof, wherein, (1) MOLT-4 cells, (2) MOLT-4/IIIB cells, ( 3) co-cultured cells, (4) sham-treated co-cultured cells, (5-8) co-cultured cells treated with 113nM CVN/LCVN/10kPEG-LCVN/20kPEG-LCVN, indicated by black arrows Fused giant cells are shown. Figure (b) fusion inhibitory activity of CNV, LCVN, 10kPEG-LCVN, 20kPEG-LCVN, all experiments are statistically processed results after at least 3 independent experiments.

Fig. 19 is the CPE observation result of anti-HSV-1 activity of LCVN and its PEG modified product. in:

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-10kDa-L-CVN (3.125 μg/ml); G, mPEG-ALD-20kDa-L-CVN (3.125 μg/ml).

Detailed ways

Some preferred examples of the present invention are listed below to help further understanding of the present invention, but the embodiments of the present invention are not limited thereto.

The main materials involved in the embodiments of the present invention are as follows: host bacteria Escherichia coli BL21 (DE3) (purchased from Novagen), plasmid pET3c (purchased from Novagen), and pET3c-SUMO-CVN are preserved by our laboratory (the construction method has applied for a patent " The preparation method and application of recombinant cyanobacteria antiviral protein, application number: 200810198926.0", the construction of the plasmid can also adopt the known genetic engineering method, the basic idea is to first obtain the SUMO-CVN fusion sequence by the PCR method, and then connect it into the pET3c vector. Available plasmid pET3c-SUMO-CVN); SUMO protease (purchased from Haiji Biological Co., Ltd.); Taq enzyme, T4 DNA ligase, DNA molecular weight standard, and various restriction endonucleases were purchased from Dalian Bao Biological Company; protein molecular weight standard Products (purchased from Juyan Biotechnology Co., Ltd.), primers were purchased from Shanghai Sangon Biological Company; Ni ²⁺ Sepharose Fast Flow, SP Sepharose Fast Flow were purchased from GE Healthcare; MTT and WST were purchased from SIGMA, USA; herpes simplex virus Type 1 (HSV-1) F strain comes from Wuhan University Institute of Virology (CGMCCNo.0396); Vero cells (CCL-81 ^TM ), MOLT-4 cells (CRL-1582 ^TM ), MT-4 cells (CRL-1942 ^TM ), HIV-I/IIIB virus (CRL-1973 ^TM ), etc. were from the American Type Culture Collection (ATCC); mPEG-ALD (10K) and mPEG-ALD (20K) were purchased from Beijing Kaizheng Bioengineering 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, 20mmol/L imidazole), NTA-250buffer ( 20mmol/L Tris-HCl, pH 8.0, 0.15mol/LNaCl, 250mmol/L imidazole), enzyme digestion buffer (20mmol/L Tris-HCl, pH 8.0, 0.15mol/LNaCl), bufferA (20mmol/LNaAc-Ac, pH 4.0).

Example

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

The construction and synthesis of the SUMO-L-CVN gene is carried out in two steps. First, the L-CVN gene is synthesized by two PCRs. The first PCR uses the pET3c-SUMO-CVN plasmid as a template, and F1-CVN and R-CVN as upstream and downstream primers. The reaction system was 1 ng template, 1 μM upstream and downstream primers, 20 μl Taq PCR MasterMix, added water to 40 μl, the reaction mixture was denatured at 94 °C for 1 min, annealed to 55 °C, maintained for 1 min, extended at 72 °C for 1 min, and performed 29 cycles. The reaction product was subjected to 1% agarose gel electrophoresis, and the target fragment was recovered from the gel, which was used as a template for the next round of PCR. In the second PCR, the PCR product of the previous round was used as a template, and F2-CVN and R-CVN were used as upstream and downstream primer pairs (the F2-CVN primer contained a flexible polypeptide encoding 15 amino acid residues), and the full-length sequence of L-CVN was synthesized. . The full-length SUMO sequence was synthesized from pET3c-SUMO-CVN by PCR method. Perform PCR using the 26 bp overlapping complementary sequence between the end of the SUMO sequence and the front end of the L-CVN sequence: use the SUMO sequence and the L-CVN sequence as the extension template, F-SUMO and R-CVN as the upstream and downstream primers, and perform the reaction under conventional PCR conditions , the reaction product was subjected to 1% agarose gel electrophoresis and gel recovery to obtain the full-length sequence of SUMO-L-CVN.

The full-length sequences of plasmids pET3C and 6His-SUMO-LCVN were digested with Nde I and BamHI respectively, the digested products were electrophoresed on 1% agarose gel, the digested products were recovered, T ₄ DNA ligase, and the ligated products were transformed into Escherichia coli JM109 competent cells were spread on LB plates containing ampicillin, cultured overnight at 37°C, plasmids were extracted, amplified by PCR and identified by Nde I and BamHI double enzyme digestion, and the positive plasmids were sent to Yingjun Company for sequencing.

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

2. Shake flask culture, protein purification and purity determination of LCVN engineering bacteria

The correctly sequenced plasmid was transformed into Escherichia coli BL21(DE3) to obtain engineering bacteria BL21[pET3c-6His-SUMO-LCVN]. A single clone was selected for culture and induced expression, and the bacterial protein was collected for SDS-PAGE electrophoresis analysis. The results showed that BL21[pET3c-6His-SUMO-LCVN] positive clones could express a fusion protein with a size of about 28kDa after being induced, and the uninduced control group had no visible expression at the corresponding position, and the fusion protein was found after ultrasonic crushing Located in the supernatant, it is soluble expression, and the soluble protein of interest accounts for 28.3±3.4% of the total protein of the supernatant through gel densitometric scanning ( FIG. 3 ).

Select high-expression strains and inoculate them into 1L LB medium containing ampicillin (100mg/L), culture at 37°C and 180rpm until OD600=0.6-1.0, then cool down to 20°C and add IPTG to a final concentration of 0.5mM to induce expression for 24h. Collect the cells by centrifugation at 4°C, 6000×g, 10 min, freeze and thaw once, then resuspend the cell pellet in NTA-10 buffer at a ratio of 1:10, and ultrasonically break (working time 5s, intermittent time 5s, 99 times, repeat 3 2 times), 4°C, 25000×g, 30min centrifugation to collect the supernatant.

The supernatant was loaded onto a Ni-NTA affinity chromatography column with a column bed volume of 20ml, the flow rate was 0.6ml/min, and the NTA-0 buffer was washed back to the baseline. off-target protein. The purified target protein 6His-SUMO-LCVN was deimidazoled by Sephadex G-25 molecular sieve, and then subjected to SUMO protease digestion to remove the SUMO fusion protein.

Adjust the concentration of 6His-SUMO-LCVN to 1mg/ml, add 1U SUMO protease/mg fusion protein, and digest at 30°C for 1h. Since the 6His-SUMO tag and SUMO protease both contain 6×His tags, the digested samples were loaded on the Ni-NTA affinity layer column for purification again to remove SUMO with 6His tags, undigested 6His-SUMO-LCVN and SUMO protease, to obtain non-fused target protein LCVN, desalted through G-25 molecular sieve column and freeze-dried to obtain LCVN products for subsequent physical and chemical property determination, activity determination or PEG modification.

Figure 3 is the protein expression profile of engineering bacteria BL21[pET3c-6His-SUMO-LCVN] induced by IPTG. It can be seen that after induction, a significantly thickened protein band (lane 3) appears at the molecular weight of 28kD, which is similar to that of 6His-SUMO- The theoretical molecular weight of LCVN was consistent, and after sonication, the target protein was located in the supernatant of the crushed cells, and the content accounted for 40% of the soluble proteins of the cells (lane 2). Figure 4 is the SDS-PAGE analysis of the LCVN purification process, where * indicates the 6His-SUMO-LCVN fusion protein, and ** indicates the LCVN protein. Fig. 5 is the reverse high performance liquid chromatography (RP-HPLC) purity analysis result of LCVN product, plant type is C-18 reverse column, detector wavelength is 280nm, mobile phase A: contain 0.1% trifluoroacetic acid (TFA) Ultrapure water, mobile phase B: acetocyanide, gradient elution with 40%-60% B, the elution peak of LCVN appears between 4-6min retention time.

3. Pilot-scale fermentation and protein preparation of LCVN engineering bacteria

The engineering strain BL21[pET3c-6His-SUMO-LCVN] was selected for culture and induced expression in a test tube, and a high-expression strain was selected for pilot fermentation. Cultivate primary and secondary seeds in Erlenmeyer flasks respectively, cultivate secondary seeds to a suitable concentration, and inoculate 15L of fermentation medium with 10% inoculum. Automatically control the temperature at 37°C, adjust the speed and ventilation in time to maintain a suitable dissolved oxygen level, and adjust the pH to 7.0-7.2 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 0.5mM to induce expression for 20h. 100ml/min, 9000g, continuous flow centrifugation to collect bacteria, 15L of fermentation medium can harvest about 550g of wet bacteria, and the collected bacteria are stored in a -20°C refrigerator.

The cell pellet was suspended in NTA-0 buffer at a ratio of 1:10, ultrasonically disrupted (working time 5 s, intermittent time 5 s, 99 times, repeated 3 times), and the supernatant was collected by centrifugation at 4°C, 25000×g, 30 min. The Ni-NTA filler with a column bed volume of 20ml is loaded with a sample volume of 250ml supernatant, the flow rate is 1ml/min, NTA-0buffer washes back to the baseline; the flow rate is 1ml/min, NTA-20buffer washes impurities, and NTA-250buffer elutes the target protein . The purified target protein 6His-SUMO-LCVN was deimidazoled by Sephadex G-25 molecular sieve, and then subjected to SUMO protease digestion to remove the SUMO fusion protein.

Adjust the concentration of 6His-SUMO-LCVN to 1mg/ml, add 1U SUMO protease/mg fusion protein, and digest at 30°C for 1h. Since the 6His-SUMO tag and SUMO protease both contain 6×His tags, the digested samples were loaded on the Ni-NTA affinity layer column for purification again to remove SUMO with 6His tags, undigested 6His-SUMO-LCVN and SUMO protease is used to obtain non-fused target protein LCVN, and the buffer is exchanged through a G-25 molecular sieve column, and then the LCVN protein is concentrated with an ultrafiltration tube with a cutoff of 3KD for subsequent physical and chemical property determination, activity determination or PEG modification.

4. PEG modification of LCVN protein

PEG modification of pharmaceutical proteins is an effective method to improve various druggable properties such as pharmacokinetic properties, stability and immunogenicity. The sites available for PEG modification of proteins include side-chain amino groups, N-terminal amino groups, side-chain carboxyl groups, C-terminal carboxyl groups, and side-chain sulfhydryl groups. The existing carboxyl modification technology is prone to non-specific cross-linking reactions, so amino modification is more commonly used and the technology development is more mature. The research results of Zappe et al. showed that the modification of the side chain amino group or the N-terminal amino group of wild-type CVN would destroy the activity of the protein. Therefore, the authors first performed site-directed mutation on CVN to obtain the Q62C mutant, and then modified the Cys The active protein was obtained by modifying the side chain sulfhydryl group. Since 2 pairs of disulfide bonds are formed between the existing 4 Cys residues in the natural CVN, and in solution, the disulfide bonds of the protein are in a state of dynamic isomerism and equilibrium, so it is difficult to avoid the introduction of the Q62C mutation. Cys does not interfere with the correct matching of disulfide bonds, or non-62-position Cys modification, the author's final experimental results also proved that the activities of the CVN Q62C mutant and its modified products were lower than those of wild-type CVN.

Since a hydrophilic and flexible 15-peptide sequence is introduced into the N-terminus of the LCV prepared by the present invention, it is possible to try to perform site-specific amino modification of the N-terminus. Our experimental results prove that after LCVN is modified by the N-terminal site-directed amino group, not only active modified products can be obtained, but also the antiviral activity of the modified products is further enhanced.

There are many methods for PEG modification of amino groups, and there are many alternative modifiers. The present invention selects mPEG-ALD (10K) and mPEG-ALD (20K) as modifiers, from substrate: modifier ratio, modification pH, modification reaction The optimal PEG modification conditions of LCVN were screened in three aspects including time.

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

In a similar way, the optimal modification pH and feed ratio of mPEG-ALD(20K) modified LCVN were studied. After mPEG-ALD (20K) modifies LCVN, the apparent molecular weight of the single modification product on SDS-PAGE is about 50KD. The experimental results show that: at pH5.0, the molar ratio of LCVN to mPEG-ALD (20K) is 1:1 Under the condition, the rate of single modification is the highest (Fig. 18).

After determining the optimal modified pH and feed ratio, mPEG-ALD (10K) and mPEG-ALD (20K) modified LCVN were sampled at 1h, 3h, 5h, 7h, 9h, 12h and 24h of the reaction, Tricine-SDS- PAGE electrophoresis to identify the best reaction time. The results of SDS-PAGE analysis showed that the reaction time had no significant effect on the reaction of mPEG-ALD(10K) and mPEG-ALD(20K) modified LCVN. Considering the time cost, the optimal modification reaction time was room temperature shaking reaction for 2h (Fig. 10 and Figure 11).

5. Separation and purification of PEG-modified products of LCVN

After LCVN was modified by PEG-ALD, the reaction mixture mainly contained unmodified LCVN, the remaining PEG, LCVN with multiple PEG modifications, and LCVN with single PEG modification. Use AKTA prime plus separation and purification system, SP sepharose chromatography column to separate and purify the mono-modified mPEG-ALD-LCVN, the mobile phase is 20mM Na-Ac buffer (buffer A), and use 5 times the column volume of buffer A before loading Equilibrate the column, collect the breakthrough peak after loading the sample, and then use buffer A containing different concentrations of NaCl for elution, and collect each elution peak. The flow rate is 1 ml/min, and the detection wavelength is 280 nm. The collected samples were detected by SDS-PAGE gel electrophoresis.

The mixture of mPEG-ALD (10K) modified LCVN, after purified by SP sepharose cation chromatography, the modifier mPEG-ALD and the multi-modification product were not combined with SP sepharose and were penetrated, and the elution product of buffer A containing 80mM NaCl was a single modification mPEG-ALD _(10K) -LCVN; the buffer A elution product containing 400mM NaCl is unmodified LCVN. Figure 12 is the elution curve of mPEG-ALD _(10K) -LCVN separation and purification, peak 1 is the modifier and multiple modification products, peak 2 is the single modification product, and peak 3 is the unmodified substrate. Figure 13 is the SDS-PAGE analysis of each elution component, swimming lane M is the protein molecular weight standard; swimming lane A is the loading sample, unmodified substrate, single modified product and multiple modified products can be seen; swimming lane 1 is the breakthrough peak; swimming lane 2 It is a single modification product, that is, the target protein mPEG-ALD _(10K) -LCVN; lane 3 is an unmodified product.

mPEG-ALD (20K) modified LCVN mixture, after purified by SP sepharose cation chromatography, the modifier mPEG-ALD and multi-modified products also exist in the loading breakthrough peak, the buffer A containing 70mM NaCl eluted as a single Modified mPEG-ALD _(20K) -LCVN; buffer A containing 400mM NaCl elutes unmodified LCVN.

Figure 14 is the elution curve of mPEG-ALD _(20K) -LCVN separation and purification, peak 1 is the modifier and multiple modification products, peak 2 is the single modification product, and peak 3 is the unmodified substrate. Figure 15 is the SDS-PAGE analysis of each elution fraction, swimming lane M is the protein molecular weight standard; swimming lane A is the loading sample, showing unmodified substrate, single modified product and multiple modified products; swimming lane 1 is the breakthrough peak; swimming lane 2 It is a single modification product, that is, the target protein mPEG-ALD _(10K) -LCVN; lane 3 is the elution fraction of 400mMNaCl, unmodified product can be seen, but also contains

There are some single-modification products.

Application Example 1 WST method is used to measure the cytotoxicity of LCVN to T cells

CVN, LCVN and PEG-modified products were serially diluted 5 times with RPMI-1640 culture medium, added to 96-well cell culture plate, 50 μl per well, the dilution range of CVN and LCVN was 10 μg/ml to 0.04 μg/ml, PEG-modified LCVN The dilution range is 50 μg/ml to 0.19 μg/ml. Adjust the concentration of MT-4 cells to 1×10 ⁵ /ml, 100 μl per well, mix well, and culture in a cell incubator at 37°C, 5% CO ₂ , and set a cell control and a positive drug control (63nM azidothymidine, AZT), each sample was determined in parallel with 3 replicate holes. After 4 days, add 10 μl of WST-1 (water-soluble tetrazolium, 5mmol/L) solution to each well of the culture plate, continue to cultivate for 4 hours, read the absorbance (A) on the plate reader, the wavelength is 450/650nm, and count the cells Survival rate (Relative percentage, RP, %), and calculate 50% toxic concentration (CC ₅₀ ) according to RP.

Cell survival rate (Relative percentage, %)=A value of drug treatment group/A value of cell control group×100%.

Figure 16 shows the cell survival rate after MT-4 was treated with four kinds of proteins including CVN, LCVN and their modified products at different concentrations. N.C. in the figure refers to the untreated cell control, and the density of the cell control is 100%. AZT is the positive control drug, 63nM azidothymidine. The calculated 50% toxic concentration is shown in Table 2.

Table 2 50% toxicity concentration of LCVN and its PEG modification product

The anti-human immunodeficiency virus activity of application embodiment 2WST method is measured LCVN

CVN, LCVN and PEG-modified products were serially diluted 5-fold with RPMI-1640 culture medium, and added to 96-well cell culture plates, 50 μl per well. Adjust the concentration of MT-4 cells to 1×10 ⁵ /ml, 100 μl per well, mix well, then add 50 μl of HIV-1/IIIB virus suspension, the titer is 100TCID50. They were cultured in a 37°C, 5% _CO2 cell incubator, and a cell control, a virus control, and a positive drug control (azidethymidine, AZT) were set at the same time, and each sample was measured in triplicate in multiple wells. After 4 days, add 10 μl of WST-1 (water-soluble tetrazolium, 5 mmol/L) solution to each well of the culture plate, continue to incubate for 4 hours, and read the absorbance (A) on a plate reader at a wavelength of 450/650 nm.

Cell survival rate (Relative percentage, %)=A value of drug treatment group/A value of cell control group×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%

Calculate the 50% inhibitory concentration (IC50) by Reed-Muench method, and calculate the selection index (TI) according to the 50% toxic concentration (CC50) of Example ×, TI=CC50/IC50.

It can be seen from Table 3 that for the positive drug AZT, CVN and LCVN have stronger inhibitory activity on HIV-1/IIIB proliferation.

Table 3CVN and LCVN are to the inhibitory activity of HIV-1/IIIB proliferation

The anti-human immunodeficiency virus activity of LCVN and its PEG-modified product was determined by fusion inhibition method in Example 3

Existing studies have shown that the transmission of HIV between T cells is mainly mediated by gp120 expressed on the surface of infected cells, which binds to receptors on uninfected cells to form fusion cells. CVN can specifically bind gp120 to inhibit the fusion between infected cells and normal cells and block the spread of the virus. Therefore, we applied 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].

Adjust the density of MOLT-4 cells and MOLT-4/IIIB cells grown to the logarithmic phase to 1×10 ⁶ /ml, take 250 μl of cell suspension, mix in equal volumes, and add them to a 24-well cell culture plate (the total number of cells is 5×10 ⁵ /500μl/well); CVN, LCVN and their PEG-modified products were serially diluted 4 times in RPMI-1640 medium containing fetal bovine serum and antibiotics, and 3 concentrations (452nM, 113nM , 28nM), an equal volume was added to the cell suspension, and various control groups were set at the same time, all samples were duplicated in parallel, and cultured at 37° C. and 5% CO ₂ for 24 hours. After 24 hours, the cell suspension was taken, stained with trypan blue, filled into the cell counting tank, and the number of surviving MOLT-4 cells was counted under a microscope. Because MOLT-4/IIIB cells can continuously produce HIV-I/IIIB virus particles, and form large multinucleated cells (syncytia) to infect normal host cells by fusion with normal MOLT-4 cells, during cell counting, fusion Cells cannot enter the counting pool. Therefore, counting the number of normal-sized MOLT-4 or MOLT-4/IIIB cells in the cell counting pool can calculate the number of cells forming syncytia, and compare the number of cells in the co-cultured group with that in the non-co-cultured group. The number of MOLT-4 cells in the group, calculate the fusion index (FI, fusionindex):

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

Comparing the fusion index of co-cultured cells with administration and non-administration co-culture cells, calculate the fusion inhibition rate (FIR, fusioninhibition rate):

FIR (%)=[1-(FI _T /FI _C )]×100, wherein F _T is the fusion index of the administered sample, and _FIC is the fusion index of the untreated co-cultured cells.

Table 4 LCVN and its PEG-modified product inhibit the fusion of cells (%)

As can be seen from Table 4, the fusion inhibitory activity of LCVN to HIV-1/IIIB is significantly higher than that of CVN no matter in any dose group; Unmodified LCVN.

Figure 18 (a) is the (1) MOLT-4 cells, (2) MOLT-4/IIIB cells, (3) cells after co-cultivation, (4) after co-cultivation of sham treatment after 24 h of culture observed under a phase contrast microscope cells, (5-8) co-cultured cells treated with CVN/LCVN/10kPEG-LCVN/20kPEG-LCVN at a concentration of 113nM, the black arrows indicate fused giant cells, and there are no fused cells in the non-co-cultured group , typical fusion cells appeared in the co-culture group, but almost no fusion cells were seen in the administration group.

Fig. 18(b) shows the fusion inhibitory activity of CNV, LCVN, 10kPEG-LCVN, and 20kPEG-LCVN. All experiments were statistically processed results after at least three 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 activity of LCVN PEG-modified products gradually increases with the increase of molecular weight , but in the low-dose group, the activity of PEG-modified products gradually decreased with the increase of molecular weight.

The anti-herpes simplex virus (HSV-1) activity of application embodiment 4MTT assay LCVN

Herpes simplex virus type I (HSV-I) is a DNA virus that causes widespread infection in the population, and humans are its only host. About 90% of healthy adults are infected with HSV-1. HSV-I can cause multiple diseases such as cold sores, herpetic keratoconjunctivitis, and neonatal encephalitis. Since HSV-I can infect latently in the ganglion, symptoms tend to recur. In this experiment, MTT method was used to measure the toxicity of recombinant LCVN and its modified products, and wild-type CVN to Vero cells; CPE method was used to observe the activity of drugs on cells.

To the monolayer of Vero cells, 50 μl each of the test drug and 100 TCID50 of the HSV-1 virus solution of different dilutions were added, and a normal cell control and a virus control group were set at the same time. Incubate in 5% CO ₂ for 48 hours, add 10 μl of 5 mg/ml MTT to each well, continue to incubate in 5% CO ₂ for 4 hours, discard the supernatant, add 200 μl DMSO to each well, place in the dark at room temperature for 30 minutes, shake the culture plate for about 10 minutes, and enzyme label The reader is colorimetric (wavelength 570nm, reference wavelength 630nm), absorbance is measured and the 50% cytotoxic concentration (50% cytotoxic concentration, CC ₅₀ ) of the sample is calculated.

Figure 19 is the morphology of 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 drug on the cells. The results showed that both LCVN and its modified products had good anti-HSV-1 activity. Under the conditions of close mass concentration and lower molar concentration, LCVN and its PEG modified products showed antiviral activity close to that of the positive control drug ACV. . The results of toxicity assay showed that the toxicity of PEG-modified LCVN to Vero cells was also significantly reduced.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Jinan University

<120> A mutant of cyanobacterial virus protein N, 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 connecting peptide (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)

Amino acid sequence of <223> 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> nucleotide sequence encoding 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 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 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 encoding His-SUMO-LCVN in pET3c-6His-SUMO-LCVN vector

<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 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 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> Primer F1-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> Primer F2-CVN

<220>

<221>misc_feature

<222>(1)..(49)

<223> Primer 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> Primer 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.

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