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Encoding nucleotide sequence of codons optimizing rotavirus protein, recombinant and uses thereof

A nucleotide sequence and codon optimization technology, applied in the fields of application, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of translation process blockage, increased difficulty of gene synthesis, and influence on gene stability, etc.

Inactive Publication Date: 2008-08-20
王健伟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, if there is no mobility in codon usage, it is difficult to avoid the generation of some repetitive elements and secondary structures in the gene and post-transcriptional mRNA, which inhibit the passage of ribosomes and lead to a block in the translation process
In addition, the presence of repetitive elements may increase the difficulty of gene synthesis
The presence of too many repetitive elements can also affect the stability of the gene in the host

Method used

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  • Encoding nucleotide sequence of codons optimizing rotavirus protein, recombinant and uses thereof
  • Encoding nucleotide sequence of codons optimizing rotavirus protein, recombinant and uses thereof
  • Encoding nucleotide sequence of codons optimizing rotavirus protein, recombinant and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Codon optimization of rotavirus gene and improvement of expression in mammalian cells

[0079] The content of this example is to optimize the codon design of the rotavirus gene, artificially synthesize the optimized gene and use adenovirus as a carrier to express the codon-optimized G1, G2, G3 type VP7 protein genes and VP6 protein gene, and its expression level was compared with that of the wild-type protein gene to evaluate the effect of codon optimization. Specific steps are as follows:

[0080] 1. Optimization of four rotavirus protein gene codons

[0081] Artificially designed according to human preferred codons (see: http: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=Homo+sapiens+[gbpri]), commissioned by Shanghai Sangon Bioengineering Co., Ltd. The optimized gene of coat protein VP7 and the optimize gene of inner coat protein VP6 of currently popular rotavirus G1, G2, G3 types in my country were synthesized, and their sequences are respectivel...

Embodiment 2

[0097] Example 2 Enhancing the Expression of Rotavirus Genes Optimized in Code in Insect Cells

[0098] In order to prove that the expression of the rotavirus structural protein gene optimized according to the preferred codons of human cells can also be increased in insect cells, in this example, we used baculovirus as a vector to express the codon-optimized rotavirus G1VP7(opt), G2VP7(opt), G3VP7(opt), VP6(opt) and wild-type G3VP7 protein genes were detected in insect cell Sf9 for gene expression and the differences in their expression levels. Specific steps are as follows:

[0099] 1. Construction of Baculovirus Expression Vector

[0100] Digest the baculovirus expression vector pFastBac 1 (hereinafter referred to as pFB1, Invitrogen, USA) with EcoR I and Sal I (TAKARA Company), the system is: pFB110 μg each 2 μl of EcoR I and Sal I, 5 μl of 10× buffer, H 2 O 23 μl. Incubate at 37°C for 3 hours, recover carrier fragments with the agarose gel recovery kit from Tianwei Time...

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Abstract

The invention pertains to the field of genetic engineering. More particularly, the invention relates to a coding nucleotide sequence of a codon optimized rotavirus VP6 or VP7 protein, a recombinant vector and a host cell which contain the sequence. The invention also relates to a method for optimizing and improving the amount of protein expression by the rotavirus structural protein gene codon and a usage on the immunology.

Description

technical field [0001] The invention belongs to the field of genetic engineering. Specifically, the present invention relates to a codon-optimized coding nucleotide sequence of rotavirus VP6 or VP7 protein, a recombinant vector containing the sequence and a host cell. The invention also relates to a method for improving protein expression by optimizing the codon of the rotavirus structural protein gene and its application in immunology. Background technique [0002] Rotavirus [0003] Group A rotavirus (Rotavirus, RV) is the most important cause of severe diarrhea in infants and young children worldwide (seeing Parashar, U.D., Bresee, J.S., Gentsch, J.R. & Glass, R.I.Rotavirus.Emerg.Infect.Dis.1998, 4 :561-570). In developing countries, approximately 600,000 to 870,000 children die each year from rotavirus infection. In view of the serious harm of rotavirus and no effective treatment, the World Health Organization has listed the development of rotavirus vaccine as one of...

Claims

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Application Information

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IPC IPC(8): C12N15/46C12N15/63C12N15/85C12P21/02A61K48/00C12N15/861C12N15/866
Inventor 王健伟王敏束弋洪涛
Owner 王健伟
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