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Preparation method and application of cyanovirin N

An anti-virus and protein technology, applied in the field of genetic engineering, can solve the problem of not getting good soluble expression

Inactive Publication Date: 2020-03-24
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] As early as the beginning of the discovery of CV-N, Boyd et al. used the method of artificial synthesis to connect the CV-N sequence and an 8-peptide leader sequence (Asp Tyr Lys Asp Asp Asp Asp Lys) to form a gene fragment, which was amplified by PCR Cloned on the pFLAG plasmid, constructed into a secretory expression vector, and successfully induced expression in E. Coli, expressed CV-N in the form of FLAG-CV-N fusion protein, but did not get good soluble expression [Boyd M R, Gustafson K R, Mcmahon J B, et al. 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 Chemother, 19797: 141 (19797), -1530.]

Method used

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  • Preparation method and application of cyanovirin N
  • Preparation method and application of cyanovirin N
  • Preparation method and application of cyanovirin N

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The construction of embodiment 1 plasmid pGEX-4T-1-cv-n plasmid

[0038] According to the characteristics of the pGEX-4T-1 vector sequence and cv-n sequence, select an appropriate restriction site, and design primers according to the general principles of primer design. Following primers are required primers of the present invention:

[0039]

[0040] Amplify the target sequence by PCR. The reaction system is 2 μl of PUC plasmid (about 1ng), 1 μl of cv-n R / cv-n F, 25 μl of PCRMIX, 21 μl of water, and 50 μl in total. Denaturation for 30s, annealing at 50°C for 30s, extension at 72°C for 45s, 30 cycles, and finally 5min at 72°C. The full length of the cv-n gene is 303bp, and the reaction product was identified by 1% agarose gel electrophoresis in line with the expected size ( figure 1 ).

[0041] Gel recovery obtained the full-length cv-n sequence, and the gel recovery product was connected to the T vector. The connection ratio was in accordance with the instruction...

Embodiment 2

[0043] Expression purification identification of embodiment 2 recombinant protein

[0044] 1. Screening and induction of expression strains

[0045] Pick the recombinants with correct sequencing, expand the culture, extract the plasmids and transform them into the expression host OrigamiB (DE3), screen the transformants with LB containing ampicillin and kana, pick a single colony until it contains 50 μg / ml ampicillin, 10 μg / ml In Kanna's 200ml LB liquid medium, expand the culture at 37°C and 220r / min. When the OD value reaches 0.6, take 1ml of the uninduced control, centrifuge to collect the bacterial pellet, add 1*SDS loading buffer, and boil it for later use. For the rest, IPTG was added to make the final concentration 0.1 mol / l. 30°C, 200r / min, induce expression for 3 hours.

[0046] 2. Purification of expression products

[0047] Collect the bacteria by centrifugation, add 3ml of bugbuster protein extraction reagent to resuspend 100ml of bacterial culture, and mix well...

Embodiment 3

[0051] Embodiment 3 chromatographic analysis of cyanobacteria antiviral protein N purity

[0052] The chromatographic column is Sepax SRT SEC-150A (7.8mm×300mm, 5μm), take an appropriate amount of CV-N, dissolve it in PBS to make the final concentration reach 1mg / ml, and use it as the test solution. Use 5mM ammonium acetate:methanol=100:1 solution as the mobile phase for isocratic elution, the flow rate is 0.6ml / min, and the column temperature is 30°C. Absorbance at 280 nm was detected with a DAD detector. Recorded and drawn by computer, by Figure 11 A single elution peak can be seen.

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Abstract

The invention relates to the field of gene engineering, in particular to separation and purification of recombinant cyanovirin N. The invention discloses a preparation method of cyanovirin N, and is characterized in that a strain capable of efficiently expressing soluble recombinant cyanovirin N is provided. A specific construction method comprises the following steps: firstly, based on an artificially synthesized cv-n gene sequence, obtaining a cv-n gene by utilizing PCR amplification technology; secondly, constructing an expression vector containing CV-N through a pGEX-4T-1 expression system, wherein the pGEX-4T-1 expression system is characterized in that a GST label is added in front of a target protein; finally, adopting IPTG for inducing expression in an expression host OrigamiB, andobtaining the soluble high-purity recombinant fusion protein GST-CV-N; and cutting the GST label by using thrombin to obtain the cyanovirin N with antiviral activity.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the separation and purification of recombinant cyanobacteria antiviral protein N. Background technique [0002] Cyanovirin-N (CV-N) is a water-soluble glycoprotein isolated from cyanobacteria, which can effectively inhibit the growth of various viruses. CV-N contains a unique sequence of 101 amino acids. BEWLEY et al. used high-resolution nuclear magnetic resonance technology to study the structure of dissolved CV-N, and found that most of it has a tertiary structure of β-sheet with elongated structure and 2 times pseudo-symmetry inside. [O'Keefe B R, Smee D F, Turpin J A, et al. Potent Anti-Influenza Activity of Cyanovirin-N and Interactions with ViralHemagglutinin[J]. Antimicrobial Agents & Chemotherapy, 2003, 47(8): 2518. Viruses are effectively resistant. CV-N interacts with mannose residues on the surface of many different viruses, including the envelope glycoprotein gp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/405C12N15/70C12N1/21A61K38/16A61P31/12C12R1/19
CPCC07K14/405C12N15/70A61P31/12A61K38/00
Inventor 欧瑜彭雯
Owner CHINA PHARM UNIV
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