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Method for preparing recombined blue algae antiviral protein and application thereof

An anti-virus and protein technology, applied in the field of genetic engineering, can solve problems such as inactivity, complicated purification methods, and lack of amino acids in fusion expression, and achieve the effect of simplifying the purification route

Inactive Publication Date: 2010-05-12
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As early as the discovery of CVN, research on recombinant expression of CVN has already begun, and it has been successfully expressed in Escherichia coli, yeast and plant cells. However, these recombinant expression methods have low expression yield, easy formation of inclusion bodies and difficulty in renaturation. It is easy to form inactive dimer form, amino acid deletion occurs in fusion expression, and the purification method is complicated [Mori T, Gustafson KR, Pannell LK, et al. Recombinant production of cyanovirin-N, a potent humanimmunodefiiciency virus-inactivating protein derived from a cultured cyanobacterium[J]. Protein Expr Purif, 1998, 12(2): 151., Mori T, Barrientos LG, Han Z, et al. Functional homologs of cyanovirin-N amenable to mass production in prokaryotic and eukaryotic hosts[J]. Protein Expr Purif, 2002, 26(1): 42., Colleluori DM, Tien D, Kang F, et al.Expression, purification, and characterization of recombinant cyanovi-rin-N for vaginal anti-HIV microbicidedevelopment[J].Protein Expr Purif, 2005, 39(2): 229., Sexton A, Drake PM, Mahmood N, et al. Transgenic plant production of Cyanovirin-N, an HIV microbicide[J]. FASEB J, 2006, 20(2): 356. ]

Method used

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  • Method for preparing recombined blue algae antiviral protein and application thereof
  • Method for preparing recombined blue algae antiviral protein and application thereof
  • Method for preparing recombined blue algae antiviral protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction and sequence analysis of plasmid pET3c-SUMO-CVN

[0038] 1. Design of primer sequences

[0039] According to the codon preference of Escherichia coli, the original sequence of CVN was optimized, eleven primers were synthesized, and the complete sequence of SUMO-CVN was synthesized by multiple PCRs. The synthesis strategy was as follows: figure 1 shown.

[0040] The designed primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the sequences are shown in Table 1.

[0041] Table 1 Primers used to synthesize the full-length sequence of SUMO-CVN

[0042]

[0043] 2. Synthesize the full sequence of SUMO-CVN by PCR

[0044] The synthesis was carried out in two steps. First, the CVN gene was synthesized by multiple PCRs, and the first PCR primers were F1-CVN and R1-CVN. The reaction system is 1 μl of each primer, 8 μl sterile ddH2O, 10 μl Taq PCR MasterMix, the reaction mixture is denatured at 94°C for 1 min,...

Embodiment 2

[0047] Expression, purification and identification of embodiment 2 recombinant protein

[0048] 1. Screening and induction of expression strains

[0049] Pick the single clone that has been sequenced correctly, extract the plasmid and transform it into the expression host BL21(DE3), use a plate containing ampicillin to screen out positive transformants, pick the single clone and put it in 3ml of LB medium containing ampicillin, at 37°C, Cultivate to OD at 180rpm 600 = 0.6 to 1.0. Take 1ml as uninduced control, add IPTG to the rest to a final concentration of 1mmol / L, induce at 37°C for 4 hours, collect the bacteria by centrifugation, and resuspend the bacteria pellet in 100μl ddH 2 O, and added 1 μl lysozyme, incubated at room temperature for 30min. Centrifuge at 20,000×g for 15 minutes at 4°C, take the supernatant and the precipitate respectively, and conduct 12% SDS-PAGE analysis. Select a single clone with an expressed product for strain preservation.

[0050] 2. Analy...

Embodiment 3

[0066] Example 3 Recombinant CVN protein anti-herpes virus activity assay

[0067] Determination of cytotoxicity: MTT method [Xu Shuyun, Bian Rulian, Chen Xiu, editor-in-chief. Pharmacological Experimental Methodology [M]. 2nd Edition. Beijing: People's Medical Publishing House, 1992.] Determination. Add different concentrations of sample diluents to monolayer Vero cells, incubate with 5% CO2 for 48 hours, add 10 μl of 5 mg / ml MTT to each well, continue to incubate for 4 hours in 5% CO2, discard the supernatant, add 200 μl DMSO to each well, and place in the dark at room temperature After 30 minutes, shake the culture plate for about 10 minutes, use an enzyme label reader to measure the color (wavelength 570nm, reference wavelength 630nm), measure the absorbance and calculate the half cytotoxic concentration (50% cytotoxic concentration, TC50) of the sample.

[0068] Determination of anti-HSV-1 activity: 50 μl of samples with different dilutions and 100 TCID50 of HSV-1 virus s...

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Abstract

The invention provides an expression vector containing a recombined blue algae antiviral protein N nucleotide sequence, a host cell containing the expression vector and a method for preparing recombined blue algae antiviral protein on the basis to obtain the recombined blue algae antiviral protein and further proves that the expression vector and / or the host cell and / or the recombined blue algae antiviral protein can be applied to prepare an antiviral medicament. The method of the invention overcomes the disadvantages of low yield, an easily formed inclusion body and difficult purification and the like in the prior art. Peptide mass fingerprinting of the recombined CVN protein prepared by the method is totally consistent with theoretical peptide mass fingerprinting; and an antiviral experiment proves that the recombined protein has good antiviral activity.

Description

Technical field [0001] The present invention relates to the technical field of genetic engineering, specifically to a method for preparing a recombinant cyanobacterial antiviral protein, as well as the expression vector and host cells involved in this method. The present invention also relates to the expression vector and / or the host cell and / or the The application of the recombinant cyanobacterial antiviral protein obtained by the above method in the preparation of antiviral drugs. Background technique [0002] Cyanobacterial antiviral protein N (CVN) is a water-soluble glycoprotein isolated from cyanobacteria, also known as Cyanobacteria (Nostocetipsosporum). Its unique antiviral activity was originally reported in a study against human immunodeficiency virus ( Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development[J]. Antimicrob. Agents Ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/31C12N1/21C12P21/04C07K14/405A61K48/00A61K38/16A61P31/12C12R1/19
Inventor 熊盛高相雷王一飞
Owner JINAN UNIVERSITY
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