One coding gene of rotavirus VP6 protein and its application

A technology of rotavirus and coding gene, which is applied to the coding gene of rotavirus VP6 protein and its application field, which can solve the problems of unsuitable for commercial production of oral vaccine, reduced content of antigenic protein, no effective treatment and prevention means for diarrhea, etc. , to achieve the effects of low cost, easy preparation, and reduced production cost

Inactive Publication Date: 2006-05-17
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] So far, a variety of dicotyledonous and monocotyledonous plants have been used to produce vaccines, and the reported plant materials expressing antigenic proteins are mainly concentrated in tobacco and potato, mainly because the genetic transformation of these two plants is easy to operate, However, tobacco contains harmful ingredients such as nicotine, and potatoes are not suitable for raw food, and the content of antigenic protein will be reduced by 50% after being cooked, so these two plants are not suitable for commercial production of oral vaccines
[0008] For the diarrhea caused by rotavirus infection in humans and animals, there is no effective treatment and prevention means, and there is an urgent need to develop a safe and efficient vaccine in this field

Method used

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  • One coding gene of rotavirus VP6 protein and its application
  • One coding gene of rotavirus VP6 protein and its application
  • One coding gene of rotavirus VP6 protein and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Example 1. Human group A rotavirus VP6 gene-sVP6 gene and sVP6-SEKDEL gene synthesized using dicotyledonous plant preferred codons

[0061] Using DNAMAN software to analyze the coding region sequence of the human group A rotavirus VP6 gene (GenBank AF260931), analyze its A+T percentage content, restriction endonuclease sites, translated amino acid sequence, and codon usage preference . Compare the difference between the sequence of the VP6 gene coding region and the codon preference of dicotyledonous plants, and without changing a single amino acid sequence, modify the sequence of the VP6 coding region to make its codon preference close to that of dicotyledonous plants, and try to avoid using Rare codons NCG and NTA.

[0062] In addition, to ensure the production of mature translatable mRNA, the following sequence was modified

[0063] 1) Transform the six polyadenylic acid (Poly(A)) signal sequences in the sequence of the coding region of the VP6 gene, so as to preve...

Embodiment 2

[0067] Embodiment 2, preparation rotavirus oral vaccine

[0068] 1. Construction of plant transformation vector expressing sVP6 gene

[0069] Utilize restriction endonuclease NcoI and SacI to digest pUsVP6 plasmid, reclaim the sVP6-SEKDEL gene fragment that size is 1.2kb, and insert it into the NcoI / SacI position of vector pRTL2 (J.Virol., 1990,64:1590-1597) point, the recombinant plasmid pRsVP6 containing the sVP6-SEKDEL gene was obtained. Then use HindIII and Sac I double enzymes to cut pRsVP6, and reclaim the cauliflower mosaic virus (CaMV) 35S promoter+tobacco etch virus (TEV) 5' untranslated region (5'-UTR) containing "double enhancer" with a size of 2.4kb ) + Kozak sequence + sVP6-SEKDEL” fragment, which was ligated with the pBI121 plasmid that had been digested with the same double restriction enzymes, to obtain the binary expression vector pBsVP6. The vector construction diagram is shown in Figure 2A.

[0070]The T-DNA region of pBsVP6 plasmid contains two gene expre...

Embodiment 3

[0089] Example 3. Oral immunization of mother mice with transgenic alfalfa expressing rotavirus VP6 protein can protect offspring mice

[0090] In this study, CpG ODN whose backbone was modified by phosphorothioation was used as an immune adjuvant. Its sequence is TCCATGACGTT CCTGACGTT (CpG ODN 1826), which was synthesized by Shanghai Sangon Bioengineering Company and used at a dose of 10 μg / time. After asexual reproduction of transgenic alfalfa with high expression of sVP6 protein, a large amount of material can be obtained for immunization of mice within three months. After 30% and 60% (NH 4 ) 2 SO 4 The transgenic alfalfa stem total soluble protein (TSP) obtained by fractional precipitation was redissolved in 0.1M PBS, and TSP was dialyzed with normal saline for 24 hours and then used for oral immunization of mice.

[0091] Thirty BALB / c female mice aged 6-8 weeks were divided into 3 groups, 10 in each group, and were immunized by intragastric administration once a week ...

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Abstract

The present invention discloses one coding gene of rotavirus VP6 protein and its application, and aims at providing one coding gene of rotavirus VP6 protein with high expression amount in dicotyledonous plant, especially clover, method of producing oral rotavirus vaccine with the coding gene and the obtained oral vaccine. The rotavirus VP6 protein coding gene has one of the following nucleotide sequences: 1. DNA sequence of SEQ ID No. 1 in the sequence list; 2. DNA sequence of SEQ ID No. 2 in the sequence list; 3. the nucleotide sequence capable of hybridizing in high strict condition with the DNA sequence limited by SEQ ID No. 1 or SEQ ID No. 2; and 4. DNA sequence possessing over 80 % homology with the SEQ ID No. 1 or SEQ ID No. 2 limited DNA sequence and high expression amount in dicotyledonous plant. The oral rotavirus vaccine is used in preventing various rotavirus infection caused acute diarrhea of infant and young animal.

Description

technical field [0001] The invention relates to a coding gene of rotavirus VP6 protein and its application, in particular to a method for producing a rotavirus oral vaccine by using the coding gene and the obtained rotavirus oral vaccine. Background technique [0002] Rotavirus infection is ubiquitous in humans, mammals, and birds, and is the main pathogen of viral diarrhea. Rotaviruses are divided into seven groups (A-G), among which group A rotaviruses are the main cause of diarrhea in infants and young animals, and are widely prevalent in the world. Regardless of developed or developing countries, more than 90% of infants are infected with rotavirus before the age of 3. In developing countries, about 18 million children are hospitalized each year due to rotavirus infection, and about 1 million of them die, causing huge economic losses (Dodet et al.1997), ranking second among infant deaths in my country . In addition, rotavirus is also one of the important causes of deat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/46A61K39/15A61P1/12C07K14/14C12N15/82
Inventor 王涛董江丽
Owner CHINA AGRI UNIV
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