Recombinant CL7-CVN protein, and preparation method and applications thereof

A CL7-CVN and protein technology, which is applied in the field of recombinant CL7-CVN protein and its preparation, can solve the problems of difficult protein expression, amino acid deletion in fusion expression, and low expression yield, and achieve good antiviral effect, high purification efficiency, and high heat The effect of stability

Active Publication Date: 2020-03-20
HUBEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The molecular weight of CVN protein is 11kDa, and there are two disulfide bonds in the molecule, which makes it difficult to express the protein in E. coli
As early as the discovery of CVN, research on recombinant expression of CVN has already begun, and it has been successfully expressed in E. co

Method used

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  • Recombinant CL7-CVN protein, and preparation method and applications thereof
  • Recombinant CL7-CVN protein, and preparation method and applications thereof
  • Recombinant CL7-CVN protein, and preparation method and applications thereof

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Experimental program
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Effect test

Embodiment 1

[0037] The construction of embodiment 1 recombinant CL7-CVN protein prokaryotic expression vector

[0038] 1. The first PCR amplification: the mutant CL7 gene was obtained by site-directed mutation (the nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence of the mutant protein is shown in SEQ ID NO: 4);

[0039] Using the pET23a-CL7 plasmid as a template, F1 shown in SEQ ID NO: 6 and R1 shown in SEQ ID NO: 7 were used as primers to simultaneously introduce linker, His and terminal homologous sequences for PCR amplification to obtain CL7- linker-His linear fragment, see image 3 Middle lane 3; according to the CVN gene sequence announced by the gene bank, carry out codon optimization design and synthesis of the CVN gene, and use the constructed pET28a-CVN vector as a template, with F2 as shown in SEQ ID NO:8 and as SEQ ID NO: R2 shown in 9 is a primer, and 3C and terminal homologous sequences are introduced at the same time, and PCR amplification is perfor...

Embodiment 2

[0049] Example 2 Induced expression of recombinant CL7-CVN protein and optimization of expression conditions

[0050] The recombinant plasmid pET28a-CL7-linker-His-3C-CVN constructed in Example 1, the empty plasmid pET28a, and the control plasmid pET28a-CVN were respectively transformed into Escherichia coli Rosetta (DE3) to obtain expression strains. The expression strain was inoculated in 6ml of liquid LB medium containing kanamycin, and cultured on a shaker at 37°C at 220rpm. When the OD600 value was 0.6, the inducer IPTG was added, and the induction culture was continued on a shaker at 37°C at 220rpm. The recombinant protein expression was detected by SDS-PAGE electrophoresis method, the results are shown in Image 6 , the induced pET28a-CL7-linker-His-3C-CVN (amino acid sequence shown in SEQ ID NO: 1) has an obvious target band at about 28kDa, which is consistent with the expected size, and the expressed protein is soluble; The CL7-labeled control pET28a-CVN showed the t...

Embodiment 3

[0055] The stability study of embodiment 3 recombinant CL7-CVN protein

[0056] Inoculate pET28a-CL7-linker-His-3C-CVN expression strains into 200ml liquid LB medium containing kanamycin, culture at 37°C until OD600 value is 0.6, add final concentration 1mM / LIPTG, and incubate at 18°C Collect the bacteria after 20 hours of induction on a shaking table; resuspend the bacteria with 20ml of lysate, break the bacteria by ultrasonic, and collect the supernatant by centrifugation; take 500ul of the supernatant in EP tubes, and place them in water bath for 15min and 30min at 90°C and 100°C respectively , 45min, 60min, 75min, 90min, 105min, 120min, the supernatant was collected by centrifugation, protein samples were prepared, and the stability of the recombinant protein was detected by SDS-PAGE electrophoresis. see results Figure 10(A, B), after 90℃, 100℃ high temperature treatment for 2 hours, most of the miscellaneous proteins were denatured, but the recombinant CL7-CVN protein w...

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Abstract

The invention discloses a recombinant CL7-CVN protein, and a preparation method and applications thereof. The preparation method includes cloning a coding sequence on an expression vector to obtain arecombinant expression vector; and converting the recombinant expression vector into host cells to perform expression and purification so as to obtain the recombinant CL7-CVN protein. Through a synthesized CVN gene and gene assembly method, the fusion expression vector can be built, and the highly soluble expression of the vector in escherichia coli can be realized; and according to the heat resistance of the recombinant CL7-CVN protein and through heat treatment and 3C protease digestion, the recombinant CL7-CVN protein and CVN protein can be rapidly obtained through one-step affinity chromatography, and purity can reach 99%. The provided preparation method can make the recombinant protein expression significantly enhanced, make activity more stable and make a purification method simpler;and both the prepared recombinant CL7-CVN protein and CVN protein have antiviral activity.

Description

technical field [0001] The invention belongs to the technical field of recombinant gene protein medicine, and specifically relates to a recombinant CL7-CVN protein and its preparation method and application. Background technique [0002] Cyanovirin-N (CVN), a water-soluble glycoprotein isolated from cyanobacteria, whose unique antiviral activity was initially identified in a natural drug screening program against human immunodeficiency virus (HIV) Later studies showed that it has a wide range of anti-enveloped virus effects, can inhibit the infection of host cells by influenza virus, Ebola virus, herpes virus and hepatitis C virus, and has stable physical and chemical properties, can resist Treatment of denaturants, detergents and organic solvents. CVN has a high affinity with mannan oligosaccharides on the surface glycoprotein of the virus, which can prevent the combination of the host cell surface receptors and the virus, and prevent the spread of the virus. This feature ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C07K14/195C12P21/06C07K1/22A61K38/16A61P31/22A61P31/18C12R1/19
CPCA61K38/00A61P31/18A61P31/22C07K14/195C07K2319/20C07K2319/21C12N15/70C12N2800/22C12P21/06
Inventor 余晓岚王斌王飞马立新杨智刘敏高丹陈冠军
Owner HUBEI UNIV
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