Coding nucleotide sequence of cupular virus capsid protein with codon optimization and use thereof

A nucleotide sequence and codon optimization technology, applied in applications, viral peptides, biochemical equipment and methods, etc., can solve problems such as unsatisfactory yields, limited applications, and production limitations

Inactive Publication Date: 2007-10-10
王健伟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the production of calicivirus VLP is limited by the amount of protein expression, and the yield is not ideal, which limits its application [see: JiangX, et al.Diagnosis of Human Caliciviruses by Use of EnzymeImmunoassays.J.Infect.Dis.2000; 181 (Suppl 2): S349-59; Venkataram Prasad BV. et al. Structural Studies of Recombinant Norwalk Capsids. J. Infect. Dis. 2000; 181 (Suppl 2): ​​S317-21; Kitamoto N, et al. Cross-Reactivity among Several Recombinant Calicivirus Virus-LikeParticles (VLPs) with Monoclonal Antibodies Obtained from MiceImmunized Orally with One Type of VLP. J. Clin. Microbiol. 2002, 40, 2459-2465; Bertolotti-Ciarlet A, et al. The 3End of Norwalk Virus mRNA Contains Determinants That Regulate the Expression and Stability of the Viral Capsid Protein VP1: a Novel Function for the VP2 Protein. J. Virol. 2003, 77, 11603-11615.]

Method used

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  • Coding nucleotide sequence of cupular virus capsid protein with codon optimization and use thereof
  • Coding nucleotide sequence of cupular virus capsid protein with codon optimization and use thereof
  • Coding nucleotide sequence of cupular virus capsid protein with codon optimization and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Optimization of the codon of the Norovirus capsid protein coding gene and its expression in insect cells

[0042] The content of this example is to optimize the codon design of the Noro virus capsid protein coding gene, artificially synthesize the optimized gene and use the baculovirus as a carrier to express the codon-optimized Noro virus GGII1, GGII3, GGII4 and GGII7-type capsid protein genes, and their expression levels were compared with wild-type Noro virus capsid protein genes to evaluate the effect of codon optimization. Specific steps are as follows:

[0043] 1. Screening of Calicivirus Positive Diarrhea Stool Specimens

[0044] (1) Specimen processing

[0045] The diarrhea stool specimen was made into a 20% suspension with normal saline, after repeated freezing and thawing twice, centrifuged at 8000rpm for 10min, the supernatant was taken, and immediately used for viral RNA extraction or stored at -80°C for later use.

[0046] (2) Extraction of vir...

Embodiment 2

[0090] Example 2 Code optimization Noro virus capsid protein coding gene expression in mammalian cells is improved

[0091] In order to verify whether the Noro virus capsid protein gene optimized according to the preferred codons used by insect cells can also increase its expression in mammalian cells, in this example, we used adenovirus as a vector to express the codon-optimized Noro virus Virus GGII1, GGII3, GGII4, GGII7 and wild-type GGII3 capsid protein genes were detected in human embryonic kidney epithelial cells 293 and their differences in expression levels. Specific steps are as follows:

[0092] 1. Construction of recombinant adenoviral vector

[0093] (1) Cloning the codon-optimized capsid protein gene into the shuttle vector pShuttle-CMV

[0094] Digest the shuttle plasmid pShuttle-CMV (Stratagen, USA) with restriction endonucleases Bgl II and XhoI: pShuttle-CMV 10 μl, BamH I and XhoI 1 μl each, 10× buffer 2 μl, H 2 O 6 μl. Incubate at 37°C for 2 hours, and rec...

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Abstract

The invention is about encoding nucleotide sequence, which is about condon's optimization and calicivirus capsid albumen, and includes the recombinant vector and host cell of this sequence. This invention also relates to the optimization by calicivirus capsid albumen gene condon, and the method to improve the expression of calicivirus capsid albumen.

Description

technical field [0001] The invention belongs to the field of genetic engineering. Specifically, the present invention relates to a nucleotide sequence encoding a codon-optimized calicivirus capsid protein, a recombinant vector and a host cell containing the sequence. The invention also relates to a method for improving the expression of the calicivirus capsid protein by optimizing the codon of the calicivirus capsid protein gene. Background technique [0002] Human caliciviruses are important pathogens causing acute nonbacterial gastroenteritis, mainly causing infections in school-age children, adults and their close contacts in the home or community (see Fields BN, et al. Fields Virology, 2nd ed, pp671-693.). In 1972, Kapikian et al. used immunoelectron microscopy to find virus particles with a diameter of 27nm in stool samples of diarrhea patients in the Norwalk area of ​​the United States, and named them Norwalk virus (see Kapikian AZ, et al.Visualization by immune elec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/33C12N15/63C12N15/86C12N15/79C12P21/02C07K14/005
Inventor 王健伟郭丽周红莉洪涛
Owner 王健伟
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