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Recombinant DNA vector for efficiently preparing foot and mouth disease virus-like particles, application and vaccine

A foot-and-mouth disease virus and particle technology, used in recombinant DNA technology, veterinary vaccines, viruses/phages, etc., can solve the problems that animals cannot produce high-level neutralizing antibodies, cannot use immune prevention, and affect foot-and-mouth disease VLP. Large-scale production and purification, improved production and preparation, high safety effects

Active Publication Date: 2019-08-16
NOVARTIS BIOTECH WUHAN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, 3C protease can also destroy the protein of the host cell, causing toxicity to the host cell and seriously affecting the production of FMD VLP
Due to the insufficient purity and content of VLP, animals cannot produce high levels of neutralizing antibodies, and cannot be used for actual immune prevention

Method used

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  • Recombinant DNA vector for efficiently preparing foot and mouth disease virus-like particles, application and vaccine
  • Recombinant DNA vector for efficiently preparing foot and mouth disease virus-like particles, application and vaccine
  • Recombinant DNA vector for efficiently preparing foot and mouth disease virus-like particles, application and vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: implement site-directed mutagenesis to 3C protease by PCR method

[0059] The 3C protease of foot-and-mouth disease virus can effectively cut the P1 precursor protein of foot-and-mouth disease virus, making it into four viral coat proteins VP1-VP4 with structural functions, and the latter self-assembles into a hollow foot-and-mouth disease virus with twelve pentahedrons. shell. Therefore, the 3C protease is a key element for preparing the empty shell of the foot-and-mouth disease virus, that is, the foot-and-mouth disease virus-like particle VLP. However, the 3C protease can also destroy the protein of the host cell, causing toxicity to the host cell, thereby seriously affecting the production of FMD VLP. In order to solve this problem, this embodiment uses the PCR method to perform site-directed mutations at three sites on the 3C protease gene. The mutation sites are the 95th, 127th and 142nd amino acids of 3C protease. The specific operation is as fol...

Embodiment 2

[0084] Example 2: Artificial synthesis of a complete shRNA transcription cassette with restriction sites at both ends

[0085] In order to facilitate the construction of recombinant plasmids, restriction sites PmeI and AvrII were designed at both ends of the artificially synthesized shRNA transcription cassette sequence, corresponding to the restriction sites of the plasmid used. The components of the shRNA transcription cassette are connected in series, and the sequence is: human U6 promoter, shRNA nucleic acid sequence homologous to 3C protease, and a transcription termination signal composed of 5 Ts. Among them, the shRNA nucleic acid sequence homologous to 3C protease includes 19 sense nucleotides, 6 nucleotides constituting the hairpin loop and 19 antisense nucleotides corresponding to the sense nucleotides; the transcription termination signal is 5 An oligonucleotide composed of T (thymine). See SEQ ID No.3 for the nucleic acid sequence of human U6 promoter and 3C prote...

Embodiment 3

[0086] Embodiment 3: Construction of recombinant expression plasmid of foot-and-mouth disease virus-like particle protein

[0087] To construct the DNA recombination plasmid of FMD virus-like particle, the plasmid of insect cell-baculovirus expression system, the plasmid of mammalian cell expression system, the plasmid of bacterial expression system and the plasmid of yeast expression system can be used. In this example, the inventors took the plasmid of the insect cell-baculovirus expression system as an example, and elaborated a complete set of methods and steps for plasmid construction, protein expression, and virus-like particle formation. Recombinant plasmids of various expression systems obtained by those skilled in the art according to the methods and principles of the present invention, without making creative efforts, all belong to the scope of protection of the present invention.

[0088] There are as many as seven different serotypes of foot-and-mouth disease virus....

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Abstract

The invention relates to a recombinant DNA vector for efficiently preparing foot and mouth disease virus-like particles, the foot and mouth disease virus-like particles prepared by utilizing a recombinant expression plasmid vector containing the recombinant DNA vector to transfect a host cell, application thereof and a vaccine. Aiming at the problems of toxicity of 3C protease to the host cell andimpact of 3C protease on yield of foot and mouth disease VLPs, a 3CshRNA segment having disturbing effect on gene expression of 3C protease is designed, so that expression of 3C protease in the process of preparing the foot and mouth disease VLPs is reduced obviously; three sites are mutagenized on the basis of original foot and mouth disease virus 3C protease gene, so that action effect of 3C protease is regulated specifically. Through modifying the 3C protease gene, the toxicity of 3C protease to the host cell is lowered, and effective cutting performance of 3C protease to foot and mouth disease virus P1 precursor protein is improved, so that synthesis yield of the foot and mouth disease VLPs is increased greatly, and a foundation is laid for large-scale production and use of foot and mouth disease VLP vaccines.

Description

technical field [0001] The invention relates to the technical fields of bioengineering and virus vaccines, in particular to a recombinant DNA carrier for efficiently preparing foot-and-mouth disease virus-like particles, and foot-and-mouth disease virus-like particles prepared by transfecting host cells with recombinant expression plasmid vectors containing the recombinant DNA carrier , Application and vaccine of foot-and-mouth disease virus-like particles. Background technique [0002] Foot-and-mouth disease is an acute, febrile, and highly contagious infectious disease common to major domestic animals such as pigs, cattle, and sheep, and other domestic and wild cloven-hoofed animals. There are more than 70 susceptible animals, and it is also a zoonotic disease. The disease has epidemiological characteristics such as rapid prevalence, wide spread, acute onset, and great harm. The incidence rate in the epidemic area can reach 50%-100%, causing huge political and economic los...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/42C07K14/09A61K39/135A61P31/14G01N33/569
CPCC12N15/63C07K14/005A61K39/12A61P31/14G01N33/56983C12N2770/32122C12N2770/32123C12N2770/32134A61K2039/552A61K2039/5258G01N2333/09Y02A40/70
Inventor 许雁诺曼.吉利卡李改夏燕
Owner NOVARTIS BIOTECH WUHAN
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