CN103255151B - Coded sequence CVN (Cyanovirin-N) mutant with high expression quantity and high activity and application of coded sequence - Google Patents

Abstract

The invention discloses a coding sequence of a CVN mutant with high expression and strong activity and application thereof. The coding sequence is shown as SEQ ID NO.1. The coding sequence is used for CVN mutants, including the following steps: cloning the above-mentioned coding nucleotide sequence into an expression vector to obtain a recombinant vector; transforming the recombinant vector into a host cell for expression and purification to obtain a high expression amount and strong activity CVN mutants. The coding sequence significantly promotes the soluble expression of CVN in Escherichia coli, and the amount of soluble expression of the CVN mutant in Escherichia coli is significantly higher than that of the wild-type CVN. In addition, the in vitro anti-influenza virus activity of the CVN mutant obtained from the coding sequence is better than that of the wild-type CVN, indicating that the rational design and optimization of the protein-coding nucleotide sequence through the translation pause theory can not only increase the expression of CVN, but also The important thing is to significantly promote the soluble expression of CVN to obtain CVN protein with better biological activity.

Description

Coding sequence of CVN mutant with high expression and strong activity and its application

technical field

The invention belongs to the field of biomedicine, and specifically relates to a coding sequence of a CVN mutant with high expression and strong activity and application thereof.

Background technique

Cyanovirin-N (CVN) is an antiviral protein discovered in cyanobacteria by Boyd et al. in 1997. It can bind specifically and with high affinity to mannan oligosaccharides on the viral surface capsid protein gp120 , prevent the virus from binding to receptors on the surface of host cells, and prevent the spread of the virus between infected cells and normal cells. This feature makes it immune to virus mutation and is less likely to cause drug resistance. Low concentration of CVN protein (0.1-36.8nM) can effectively inhibit HIV infection of immune cells, and high concentration (45-400nM) has no direct toxic and side effects on normal cells.

In addition to HIV, CVN has antagonistic effects on influenza virus, herpes virus and parainfluenza virus, etc. It is a broad-spectrum, highly effective, and very valuable antiviral candidate drug that has attracted much attention. However, the large-scale preparation of CVN has been a bottleneck technology that limits its application. It is currently reported that CVN is expressed in the form of inclusion bodies in Escherichia coli, which is limited by various factors such as low expression, heterogeneous products, and incorrect modification (Mori, T., Gustafson, K.R., Recombinant Production of Cyanovirin-N, a Potent Human Immunodeficiency Virus-Inactivating Protein Derived from a Cultured Cyanobacterium. Protein Expression and Purification, 1998, 151-158); Some people also try to express CVN in transgenic plants, but no practical purification process has been established (Sexton, A., Drake, P.M., Mahmood, Transgenic plant production of Cyanovirin-N, an HIV microbicide. 2006, Vol. 20pp. 356-358).

The traditional biochemical theory "Anfinsen Principle" believes that the folding information of a protein is uniquely determined by its amino acid sequence in a certain environment (Taniuchi H., D.R. Davies, and C.B. Anfinsen, A comparison of the x-ray diffraction patterns of crystals of reconstituted nucleus -T and of nativestaphylococcal nuclease.J Biol Chem,1972.247(10):p.3362-4). But the inventor's research points out that this traditional theory is wrong. Although different DNA can be translated into the same amino acid, the speed of protein production (translation speed) is not constant, and it will be slower in some segments. This phenomenon is called translational pause (translational pause or translational attenuation) translation pause site It is highly related to protein folding. If the translation pause site is incorrect, the place that should be slow is fast, or the place that should be fast is slow, it will cause protein misfolding and aggregation, and functional soluble proteins cannot be obtained. That is to say, the protein spatial conformation is determined not only by the amino acid sequence, but also by the nucleotide sequence. (Zhang G., M.Hubalewska, and Z. Ignatova, Transient ribosomal attenuation coordinates protein synthesis and co-translational folding. Nat Struct Mol Biol, 2009.16(3): p.274-80.; Zhang G. and Z. Ignatova, Folding at the birth of the nascentchain: coordinating translation with co-translational folding. Curr Opin Struct Biol, 2011.21(1): p.25-31.). This theory is applicable to most proteins in the proteome (Zhang, G. and Z. Ignatova, Generic algorithm to predict the speed of translational elongation: implications for protein biogenesis. PLoS One, 2009.4 (4): p.e5036.; Fedyunin , I., et al., tRNA concentration fine tunes protein solubility. FEBS Lett, 2012.586(19):p.3336-40.).

Contents of the invention

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide a coding sequence of a CVN mutant with high expression and strong activity.

Another object of the present invention is to provide a CVN mutant obtained through the above coding sequence.

Another object of the present invention is to provide the application of the CVN mutant with high expression and strong activity.

The object of the present invention is achieved through the following technical solutions: a coding sequence of a CVN mutant with high expression and strong activity is as follows:

CTTGGTAAATTCTCCCAGACCTGCTACAACTCCGCTATCCAGGGTTCTGTTCTGACCTCTACCTGCGAACGTACCAACGGTGGTTACAACACCTCCTCTATCGACCTGAACTCCGTTATCGAAAACGTTGACGGTTCTCTGAAATGGCAGCCGTCTAACTTCATCGAAACCTGCCGTAACACCCAGCTGGCTGGTTCCTCTGAACTGGCTGCTGAATGCAAAACCCGTGCTCAGCAGTTCGTTTCTACCAAAATCAACCTAGACGACCATATAGCTAACATAGACGGTACACTAAAATACGAA。

A CVN mutant with high expression and strong activity, prepared from the above coding sequence;

The CVN mutant with high expression and strong activity is prepared by the following method: cloning the above coding sequence into an expression vector to obtain a recombinant vector; transforming the recombinant vector into a host cell for expression, purification, and expression CVN mutant with high amount and strong activity;

The expression vector is preferably pET-28b;

The host cell is preferably Escherichia coli BL21 (DE3);

The application of the coding sequence of the CVN mutant with high expression and strong activity in the preparation of the CVN mutant comprises the following steps:

(1) Design the following primers:

F-sumo: 5'-GGAATTCCATATGGGCATGTCGGACTCAGAAGTC-3';

R-CVN: 5'-TTCCGCGGCCGCTATGGCCGACGTCGACTTCGTATTTCAGGGTACCGTC-3';

F1-mutant: 5'-GACGACCACATCGCTAACATAGACGGTACACTAAAATACGAATGATGAGTCGAC-3';

R1-mutant: 5'-GTCGACTCATCATTCGTATTTTAGTGTACCGTCTATGTTAGCGATGTGGTCGTC-3';

(2) The first PCR amplification: use the pET-3c-SUMO-CVN plasmid as a template, and use F-sumo/R-CVN as primers for PCR amplification. After purification, the obtained PCR product is digested with NdeI and SalI ; Ligate the PCR product after double digestion with the vector pET-28b that has been digested with NdeI and SalI; transform the ligated product into Escherichia coli DH5α competent cells, and screen to obtain the recombinant vector pET-28b-CVN;

(3) The second PCR amplification: using pET-28b-CVN as the PCR amplification template,

F1-mutant/R1-mutant was used as primers for PCR amplification, and the obtained PCR products were purified and digested with DpnI, and the digested products purified by the cycle pure Kit kit were directly transformed into Escherichia coli DH5α competent cells and screened to obtain the recombinant vector pET- 28b-CVN-M1;

(4) Induced expression: Transform the recombinant vector pET-28b-CVN-M1 into Escherichia coli BL21 (DE3) to obtain an expression strain; then use LB medium containing kanamycin to incubate the expression strain at 37°C, 180～ Cultivate under the condition of 200rpm, when OD ₆₀₀ =0.8, add IPTG, the final concentration of IPTG is 1mM, induce expression at 37°C for 4h, and collect the bacteria;

(5) Purification: Resuspend the bacterial cell pellet in NTA-10 buffer at a ratio of 1:10 by volume, crush the bacterial cell, centrifuge, and collect the supernatant; apply the supernatant to a Ni-NTA affinity chromatography column , first use NTA-10 buffer to wash back to the baseline, then use NTA-40 buffer to wash the impurity protein, and finally use NTA-200 buffer to elute to obtain the target protein CVN mutant; wherein, the composition of NTA-10 buffer is 20mmol/L Tris-HCl, pH8.0+0.15mol/L NaCl+10mmol/L imidazole, the composition of NTA-40 buffer is 20mmol/L Tris-HCl, pH8.0+0.15mol/L NaCl+40mmol/L imidazole , the composition of NTA-200 buffer is 20mmol/L Tris-HCl, pH8.0+0.15mol/L NaCl+200mmol/L imidazole;

The PCR conditions described in step (2) are preferably pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, extension at 68°C for 2 minutes, and 28 cycles; extension at 68°C for 5 minutes;

The PCR conditions described in step (3) are preferably pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, extension at 68°C for 12 minutes, and 18 cycles; extension at 68°C for 5 minutes;

The final concentration of kanamycin in the LB medium described in step (4) is preferably 50mg/L;

The centrifugation conditions described in step (5) are preferably 4°C, 25000g, 30min;

Application of the CVN mutant with high expression and strong activity in the preparation of antiviral drugs for prevention and/or treatment;

Said virus refers to influenza virus or HIV.

The innovation of the present invention lies in the rational optimization and design of the nucleotide sequence of CVN by using the theory of translation pause according to the relationship between protein structure domain and translation pause, and the introduction of translation pause site into the domain of CVN to construct CVN The mutants promoted the soluble expression of CVN in Escherichia coli, and finally purified the recombinant cyanobacteria antiviral protein; and the anti-influenza virus experiment proved that the prepared recombinant protein had good antiviral activity.

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

(1) The nucleotide sequence encoding the CVN mutant provided by the present invention improves the expression level of CVN in Escherichia coli; especially promotes the soluble expression of CVN in Escherichia coli, and the amount of soluble expression of CVN mutant in Escherichia coli Significantly higher than CVN.

(2) The in vitro anti-influenza virus activity of the CVN mutant provided by the present invention is better than that of CVN. It shows that the rational design and optimization of protein nucleotide sequence through translation pause theory can not only increase the expression of CVN, but more importantly, significantly promote the soluble expression of CVN, and obtain CVN protein with better biological activity.

Description of drawings

Figure 1 is a graph of the CVN-M1 translation pause; 1 is the blue line and 2 is the red line.

Figure 2 is the soluble expression analysis chart of CVN and its mutants; among them, picture (a) is an SDS-PAGE picture, and the arrow shows the target protein band of CVN; picture (b) is a Western blot picture, only CVN is detected; Lane M is the protein marker, the unit is KDa; lane 1 is the uninduced expression of CVN-BL21; lane 2 is the induced expression of CVN-BL21; lane 3 is the induced expression of CVN-M1-BL21.

Figure 3 is the SDS-PAGE image of CVN and its mutant proteins after purification. Lane M is the protein marker, the unit is KDa, and the arrow shows the target protein band of CVN.

Fig. 4 is a graph showing the activity results of CVN and its mutants against human influenza virus A/HK/8/68 (H3N2). The vertical axis is the percentage of virus inhibition. The higher the bar graph, the better the virus inhibition effect.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, 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) (Merck), plasmid pET-28b (Merck), pET3c-SUMO-CVN (constructed in this laboratory, and patent No. ZL200810198926.0, The title is "Preparation Method and Application of Recombinant Cyanobacterial Antiviral Protein" to disclose the detailed preparation process. At present, the simplest method is to directly synthesize the SUMO-CVN fusion sequence through the sequencing company, and connect it to the pET3c vector to obtain pET3c-SUMO-CVN); pfu mix enzyme, restriction endonuclease NdeI, SalI, DpnI, and prestained protein Marker were purchased from Fermentas Company, and primers were synthesized by Huada Gene Technology Co., Ltd.; Ni Sepharose ^TM 6Fast Flow was purchased from GE Company, and tetramethylazoazole Salt (MTT) was purchased from SIGMA, USA. All other reagents were of analytical grade. NTA-10 buffer (20mmol/L Tris-HCl, pH8.0+0.15mol/L NaCl+10mmol/L imidazole), NTA-40 buffer (20mmol/L Tris-HCl, pH8.0+0.15mol/L NaCl+40mmol/L imidazole), NTA-200 buffer (20mmol/L Tris-HCl, pH8.0+0.15mol/L NaCl+200mmol/L imidazole).

Example 1 CVN codon optimization and expression vector construction

Using the translation pause theory, optimize the nucleotide sequence encoding CVN protein, rationally design the translation pause site, and carry out synonymous mutations on the nucleotide sequence of CVN by mutation PCR technology.

The coding nucleotide sequence of CVN protein is as follows:

CTTGGTAAATTCTCCCAGACCTGCTACAACTCCGCTATCCAGGGTTCTGTTCTGACCTCTACCTGCGAACGTACCAACGGTGGTTACAACACCTCCTCTATCGACCTGAACTCCGTTATCGAAAACGTTGACGGTTCTCTGAAATGGCAGCCGTCTAACTTCATCGAAACCTGCCGTAACACCCAGCTGGCTGGTTCCTCTGAACTGGCTGCTGAATGCAAAACCCGTGCTCAGCAGTTCGTTTCTACCAAAATCAACCTGGACGACCACATCGCTAACATAGACGGTACACTAAAATACGAA。

(1) Design the nucleotide sequence encoding the CVN mutant through the theory of translation pause, without changing the amino acid sequence of CVN, the codons (ATC, ACC, CTG) that translate faster in the last 20 codons of CVN Replaced with slower translation codons (ATA, ACA, CTA) to create translation pause sites (calculated by RiboTempo software, http://bioinformatics.jnu.edu.cn/software/ribotempo), the mutants were processed by RiboTempo The translation pause curves calculated by the software are shown in Figure 1. The red line of the translation pause curve forms a translation pause site in the predetermined region (the last 20 codons) (the red line is lower than the blue line), which meets the requirements. The designed nucleotide sequence encoding the CVN mutant is as follows:

The coding nucleotide sequence of mutant 1 (CVN-M1):

CTTGGTAAATTCTCCCAGACCTGCTACAACTCCGCTATCCAGGGTTCTGTTCTGACCTCTACCTGCGAACGTACCAACGGTGGTTACAACACCTCCTCTATCGACCTGAACTCCGTTATCGAAAACGTTGACGGTTCTCTGAAATGGCAGCCGTCTAACTTCATCGAAACCTGCCGTAACACCCAGCTGGCTGGTTCCTCTGAACTGGCTGCTGAATGCAAAACCCGTGCTCAGCAGTTCGTTTCTACCAAAATCAACCTAGACGACCATATAGCTAACATAGACGGTACACTAAAATACGAA。

For the coding nucleotide sequence of mutant 1 (CVN-M1), the mutant PCR primers designed are as follows:

F1-mutant: 5'-GACGACCACATCGCTAACATAGACGGTACACTAAAATACGAATGATGAGTCGAC-3';

R1- _mu t _an t: 5'-GTCGACTCATCATTCGTATTTTAGTGTACCGTCTATGTTAGCGATGTGGTCGTC-3'.

(2) Construct the expression plasmid of pET-28b-CVN

Use pET-3c-SUMO-CVN plasmid as PCR amplification template, and F-sumo/R-CVN as primers for PCR amplification. The amplification system is: 2 μL of each primer (10 μmol/L), 25 μL of pfu mix, 1 μL of template, and Bacterial ultrapure water 20 μL; amplification conditions: the reaction mixture was pre-denatured at 94°C for 3 minutes; denatured at 94°C for 1 minute, annealed to 55°C for 1 minute, extended at 68°C for 2 minutes, and performed 28 cycles; extended at 68°C for 5 minutes.

F-sumo: 5'-GGAATTCCATATGGGCATGTCGGACTCAGAAGTC-3'

R-CVN: 5'-TTCCGCGGCCGCTATGGCCGACGTCGACTTCGTATTTCAGGGTACCGTC-3'.

The obtained PCR product was recovered using a DNA purification kit (OMEGA), and the purified PCR product and plasmid pET-28b were digested with NdeI and SalI respectively (10×Fastdigest buffer, 37°C water bath for 30min), and the digested product was processed Mass volume ratio 1% agarose gel electrophoresis, recover the digested product, use T4 DNA ligase (reaction system prepared according to the standard system recommended by the manual), connect at 16°C overnight, connect the two digested products, and construct a recombinant plasmid pET-28b-CVN. The recombinant plasmid was transformed into Escherichia coli DH5α competent cells (Merck), spread on LB plates containing 50 μg/mL kanamycin, and cultured at 37°C overnight, and the positive clones obtained by screening were sent to Huada Gene Technology Co., Ltd. for sequencing, and obtained The CVN clone pET-28b-CVN conforming to the expected design.

(3) Construction of recombinant vectors expressing CVN mutants

Obtaining the target gene: pET-28b-CVN was used as PCR amplification template, and F1-mutant/R1-mutant was used as primers for PCR amplification. The amplification system was: 2 μL of each primer, 25 μL of pfu mix, 1 μL of template, sterilized ultrapure 19 μL of water; amplification conditions: the reaction mixture was pre-denatured at 94°C for 3 minutes; denatured at 94°C for 1 minute, annealed at 55°C for 1 minute, extended at 68°C for 12 minutes, and performed 18 cycles; extended at 68°C for 5 minutes.

The obtained PCR product was recovered using a DNA purification kit (OMEGA), and the purified PCR product was digested with DpnI, and the digested product purified by the cycle pure Kit kit (OMEGA) was directly transformed into Escherichia coli DH5α competent cells (Merck) , spread LB plates containing 50 μg/mL kanamycin, and send the screened positive clones to Huada Gene Technology Co., Ltd. for sequencing, and obtain clones containing the nucleotide sequence encoding CVN mutant 1 that meet the expected design. pET-28b-CVN-M1.

Example 2

(1) Preparation of expression strains:

① Preparation of Escherichia coli BL21 (DE3) competent cells: For the preparation process, refer to the third edition of "Molecular Cloning Experiment Guide"; [US] J. Sambrook, translated by Huang Peitang.

②Transform the expression vectors pET-28b-CVN and pET-28b-CVN-M1 into Escherichia coli BL21 (DE3) competent cells: see the third edition of "Molecular Cloning Experiment Guide" for details of the transformation process; [US] J. Sha Written by Mbruk, translated by Huang Peitang.

(2) Induced expression and solubility analysis of CVN and its mutants

① Inoculate the expression strains CVN-BL21 and CVN-M1-BL21 obtained in step (1) into 20 mL of LB medium containing 50 μg/mL kanamycin, culture at 37 °C and 180 rpm, when OD ₆₀₀ =0.8 , add IPTG, the final concentration is 1mM, after inducing expression at 37°C for 4 hours, measure the OD ₆₀₀ respectively to ensure that the volume × OD ₆₀₀ of CVN and its mutants are the same, and collect the cells by centrifugation at 5000g and 4°C for 10 minutes . The bacteria were resuspended with 20mmol/L Tris-HCl (pH8.0, 0.15mol/L NaCl) buffer, the cells were homogeneously broken under high pressure (1000bar), centrifuged at 18000g, 4°C for 30min, and the supernatant and sediment were reserved for subsequent SDS-PAGE electrophoresis (5% stacking gel, 12% separating gel) and Western blot analysis.

BCA kit (Beiyuntian Biotechnology Co., Ltd.) was used to determine the total protein content in the centrifuged supernatant of crushed CVN and its mutants, to ensure the same amount of protein loading, western blot (for the experimental steps, see "Refined Molecular Biology Experiment Guide" Fifth Edition; (US) Osper editor-in-chief, translated by Jin Youxin) Analysis of CVN protein content in supernatant. The results showed (Fig. 2), the amount of soluble expression of CVN mutant was significantly higher than that of CVN.

(3) Shake flask fermentation and purification of CVN and its mutants

The expression strains CVN-BL21 and CVN-M1-BL21 obtained in step (1) were respectively inoculated into 1 L LB medium with a kanamycin content of 50 μg/mL, and shake flask fermentation and induction were carried out according to the aforementioned expression conditions. Collect the cells by centrifugation at 4°C, 6000×g, and 10 min, then resuspend the cell pellet in NTA-10 buffer at a volume ratio of 1:10, and break the cells homogeneously under high pressure (1000 bar). Collect the supernatant by centrifugation at 4°C, 25000×g, 30min.

Load the supernatant to a Ni-NTA affinity chromatography column with a column bed volume of 20mL at a flow rate of 0.6mL/min, wash back to baseline with NTA-10 buffer, and wash impurities at a flow rate of 1mL/min with NTA-40 buffer , the target protein was eluted with NTA-200 buffer; the purified CVN protein was concentrated by Sephadex G-25 molecular sieve deimidazole and 3KDa ultrafiltration tube, and the purity of the purified CVN protein was identified by SDS-PAGE electrophoresis.

According to the relationship between the CVN protein domain and the translation pause, the CVN mutant with translation pause introduced into the CVN domain can be detected by Western blot from the broken centrifuged supernatant of the bacteria after induction of expression at 37°C protein (Fig. 2b). Due to the extremely low amount of soluble expression of unoptimized native sequence CVN, the amount of CVN protein obtained after purification is extremely low, so that it cannot be detected by Coomassie brilliant blue staining; while the optimized CVN mutant (identified as CVN-M1) Although there is no amino acid sequence change, a large amount of CVN protein can be purified (Figure 3). This shows that it is feasible to promote soluble expression of CVN by rationally introducing translation pause sites in the CVN domain to reduce the translation rate of CVN.

Example 3

Study on Anti-Influenza Virus A/HK/8/68(H3N2) Activity of CVN

Dog kidney epithelial cells MDCK cells (ATCC, USA) were digested with 0.25% mass-volume trypsin solution, and added to 96-well cell culture plates at 2.5×10 ⁵ cells/well (MEM medium, containing 10% volume percentage of small After the cells grow into a monolayer, discard the growth solution, and use the maintenance solution (MEM medium, not containing calf serum) to separate the drug CVN protein (preserved in the laboratory, according to the publication number CN101638435, named "one A kind of cyanobacterial virus protein N mutant, its modified derivatives and application "prepared by the disclosed steps) and the CVN mutant protein purified in Example 2 were diluted into 6 serial concentrations (100, 50, 25, 12.5, 6.25, 3.125 μM), set 3 replicate wells for each dilution, set normal cell control group, positive drug (ribavirin) control group, culture in 37°C, 5% CO ₂ incubator for 48h, observe the changes of cells every day, The OD value of cells in each well was measured by MTT method, and the half toxic concentration TC50 was calculated.

Dilute the drug into different concentrations within the non-toxic concentration range (TC50>50 μM), take 50 μL of each drug dilution, human influenza strain A/HK/8/68 (H3N2) (ATCC company, Influenza A virus (H3N2), ATCC VR-1679 ^TM )) 50 μL virus dilution solution (100TCID50) was mixed and directly added to the monolayer MDCK cells, with 3 replicate wells for each concentration, and a positive drug group, a virus control group, and a negative control group were set. Place at 37°C, 5% CO ₂ , cultivate for 48 hours, add 10 μL of MTT solution with a concentration of 5 mg/mL, continue to cultivate for 4 hours, carefully discard the supernatant, add 100 μL DMSO to each well, and place on a shaker in the dark for 15 minutes. The instrument (Bio-Rad550) reads the OD value, the measurement wavelength is 490nm, and the reference wavelength is 630nm.

Calculate the inhibition rate % at each drug concentration = (OD drug group - OD virus control group) / (OD cell control group - OD virus control group).

The results of anti-influenza virus H3N2 activity of CVN and its mutants (Figure 4) show that: CVN and its mutants have significant inhibitory effect on human influenza strain A/HK/8/68 (H3N2), and CVN mutants have significant inhibitory effect on human influenza virus The inhibitory effect of strain A/HK/8/68(H3N2) was better than that of CVN, which indicated that the relationship between protein domains and translation pauses was used to rationally introduce translation pauses in protein domains, and the theory of translation pauses was used to control the core of CVN. The redesign and optimization of the nucleotide sequence can not only promote the soluble expression of CVN, but also obtain CVN protein with better activity.

In summary, the CVN mutant constructed by the present invention was successfully expressed in Escherichia coli BL21 (DE3), and the amount of soluble expression was significantly higher than that of CVN; it overcomes the disadvantages of easy formation of inclusion bodies and difficult purification of CVN. The anti-human influenza A/HK/8/68 (H3N2) activity results of CVN and its mutants showed that the activity of CVN mutants was better than that of CVN.

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.

Claims (8)

1. an encoding sequence for expression amount height and active strong CVN mutant, is characterized in that: as shown in SEQ ID NO.1.

2. the CVN mutant that expression amount is high and activity is strong, is characterized in that: by encoding sequence claimed in claim 1, prepared.

3. the CVN mutant that expression amount according to claim 2 is high and activity is strong, is characterized in that preparing by the following method: encoding sequence claimed in claim 1 is cloned on expression vector, obtains recombinant vectors; Recombinant vectors is transformed in host cell and is expressed, and purifying, obtains the CVN mutant that expression amount is high and activity is strong.

4. the CVN mutant that expression amount according to claim 3 is high and activity is strong, is characterized in that: described expression vector is pET-28b; Described host cell is intestinal bacteria (Escherichia coli) BL21 (DE3).

5. the application of the encoding sequence of expression amount height claimed in claim 1 and active strong CVN mutant in preparation CVN mutant, is characterized in that comprising following steps:

(1) design following primer:

F-sumo：5’-GGAATTCCATATGGGCATGTCGGACTCAGAAGTC-3’；

R-CVN：5’-TTCCGCGGCCGCTATGGCCGACGTCGACTTCGTATTTCAGGGTACCGTC-3’；

F1-mutant：5’-GACGACCACATCGCTAACATAGACGGTACACTAAAATACGAATGATGAGTCGAC-3’；

R1-mutant：5’-GTCGACTCATCATTCGTATTTTAGTGTACCGTCTATGTTAGCGATGTGGTCGTC-3’；

(2) pcr amplification for the first time: take pET-3c-SUMO-CVN plasmid as template, take F-sumo/R-CVN as primer carries out pcr amplification, use NdeI and SalI double digestion after the PCR product purification obtaining; Carrier pET-28b by the PCR product after double digestion with process NdeI and SalI double digestion, connects; Connect product and transform intestinal bacteria (Escherichia coli) DH5 α competent cell, screening, obtains recombinant vectors pET-28b-CVN;

(3) pcr amplification for the second time: take pET-28b-CVN as pcr amplification template, F1-mutant/R1-mutant is that primer carries out pcr amplification, the PCR product purification obtaining is cut through DpnI enzyme, the enzyme of cycle pure Kit test kit purifying is cut product and is directly transformed bacillus coli DH 5 alpha competent cell, screening, obtains recombinant vectors pET-28b-CVN-M1;

(4) abduction delivering: recombinant vectors pET-28b-CVN-M1 is proceeded to e. coli bl21 (DE3), obtain expression strain; Then use the LB substratum that contains kantlex to cultivate under 37 ℃, the condition of 180～200rpm this expression strain, at OD ₆₀₀=0.8 o'clock, add IPTG, the final concentration of IPTG is 1mM, after 37 ℃ of abduction delivering 4h, collects thalline;

(5) purifying: by bacterial sediment by volume 1:10 ratio Eddy diffusion after NTA-10 damping fluid, thalline is carried out to fragmentation, centrifugal, collect supernatant; In supernatant loading Ni-NTA affinity column, first use NTA-10 damping fluid to wash back baseline, then wash foreign protein with NTA-40 damping fluid, finally use NTA-200 buffer solution elution, obtain target protein CVN mutant; Wherein, NTA-10 damping fluid consist of 20mmol/LTris-HCl, pH8.0+0.15mol/L NaCl+10mmol/L imidazoles, NTA-40 damping fluid consist of 20mmol/L Tris-HCl, pH8.0+0.15mol/L NaCl+40mmol/L imidazoles, NTA-200 damping fluid consist of 20mmol/L Tris-HCl, pH8.0+0.15mol/L NaCl+200mmol/L imidazoles.

6. the application of the encoding sequence of expression amount height according to claim 5 and active strong CVN mutant in preparation CVN mutant, is characterized in that: the PCR condition described in step (2) is 94 ℃ of denaturation 3min; 94 sex change 1min, be annealed to 55 ℃ and keep 1min, 68 ℃ to extend 2min, carry out 28 circulations; 68 ℃ are extended 5min;

PCR condition described in step (3) is 94 ℃ of denaturation 3min; 94 sex change 1min, be annealed to 55 ℃ and keep 1min, 68 ℃ to extend 12min, carry out 18 circulations; 68 ℃ are extended 5min;

The final concentration of the kantlex described in step (4) is 50mg/L;

Centrifugal condition described in step (5) is 4 ℃, 25000g, 30min.

7. the high and active strong CVN mutant of expression amount claimed in claim 2 prevents and/or treats the application in antiviral in preparation.

8. the high and active strong CVN mutant of expression amount according to claim 7 prevents and/or treats the application in antiviral in preparation, it is characterized in that: described virus is influenza virus or virus of AIDS.