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Condon optimized African swine fever virus P54 gene, nucleic acid vaccine and application thereof

An African swine fever virus, codon optimization technology, applied in application, gene therapy, antiviral agent and other directions, can solve the problems of ineffective effect, poor safety of attenuated vaccine, chronic epidemic, etc., and achieve the effect of convenient operation

Inactive Publication Date: 2014-05-21
孙洁
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that the inactivated vaccine for African swine fever is not effective, and the attenuated vaccine is poor in safety and can easily lead to chronic epidemics. A new type of vaccine is urgently needed to overcome the current problems

Method used

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  • Condon optimized African swine fever virus P54 gene, nucleic acid vaccine and application thereof
  • Condon optimized African swine fever virus P54 gene, nucleic acid vaccine and application thereof
  • Condon optimized African swine fever virus P54 gene, nucleic acid vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: the construction of African swine fever virus P54 gene nucleic acid vaccine

[0056] 1.1 Design and synthesis of codon-optimized ASFV P54 gene

[0057] First, the EMBOSS software was used to analyze the ASFV P54 gene sequence to find out its codon usage bias and its codon usage bias with mammalian cells. According to the codon preference, the codon of the ASFV P54 gene is optimized, and the amino acid sequence of the protein encoded by the codon-optimized gene sequence remains unchanged. The sequence of the codon-optimized gene is shown as sequence 1 in the sequence listing. The codon-optimized ASFV P54 gene was synthesized by TAKARA Company and cloned into the pUC18 cloning vector to construct the recombinant plasmid pUC18-ASFV P54. The sequence of the synthesized gene was confirmed to be correct by sequencing.

[0058] 1.2 PCR amplification and sequence analysis of the codon-optimized ASFV P54 gene

[0059] First, according to the optimized ASFV ...

Embodiment 2

[0098] Example 2: Liposome-mediated transfection of BHK-21 cells and identification of expression products

[0099] The pcDNA3.3-ASFV P54 constructed in Example 1 was transfected into BHK-21 cells for transient expression. As a result, the P54 protein was expressed 72 hours after the transfection, and the indirect immunofluorescence was positive, while the empty plasmid pCDNA3.3 and the untransfected The BHK-21 cells of the plasmid group showed negative reaction. In vitro transfection experiments showed that all eukaryotic expression vectors containing P54 gene could be expressed in vitro, which provided a prerequisite for the next step of animal experiments.

[0100] 2.1LP-2000-mediated liposome transfection

[0101] Lipofectamine Lipofectamine TM 2000, purchased from Invitrogen Company, the steps are as follows:

[0102] 1) The day before transfection, cells were trypsinized and counted, and the cells were plated so that the density on the day of transfection was 90%. ...

Embodiment 3

[0115] Example 3: Immunogenicity Research of African Swine Fever Nucleic Acid Vaccine and Detection of Immune Response After Inoculating Animals

[0116] 3.1 Mass preparation and purification of African swine fever nucleic acid vaccine

[0117] A large amount of nucleic acid vaccines were prepared using a large plasmid extraction kit: Qiagen Plasmid Mega Kit, Qiagen, Inc.

[0118] Draw 5 μL of the correctly identified bacterial preservation solution and inoculate it into 300 mL of ampicillin-containing LB culture solution, grow overnight at 37°C and 200 rpm. Plasmid extraction was performed according to the instructions.

[0119] 3.2 Inoculated mice test

[0120] Grouping: Divide the mice into 4 groups with 5 mice in each group. The 1st to 3rd groups were injected with pcDNA3.3-ASFV P54, pcDNA3.3 empty plasmid, and normal saline respectively. The 4th group was healthy animal control and immunized three times , two weeks apart each time, intramuscular injection of tibialis a...

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Abstract

The invention relates to a condon optimized African swine fever virus P54 gene and a nucleic acid vaccine based on the gene, wherein the nucleic acid vaccine consists of an eukaryotic expression vector and the condon optimized African swine fever virus P54 gene. The nucleic acid vaccine is constructed through the following steps of optimizing the condon of P54protein, designing a specific primer, transferring sequence of the artificially synthesized African swine fever virus P54 gene into a cloning vector and recombining the purified P54 gene into the eukaryotic expression vector. The nucleic acid vaccine can be used for preventing the occurrence of African swine fever and is a powerful tool for solving the immune prevention and control of the African swine fever. Compared with the traditional vaccines, the nucleic acid vaccine has the advantages of small quantity, security, long-term effect, stability and convenience in operation when reaching same immune effect.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a codon-optimized African swine fever virus P54 gene, a DNA nucleic acid vaccine based on the gene, and an application of the gene in preparing a vaccine for preventing African swine fever. Background technique [0002] African swine fever (ASF) is a severe viral disease mainly infecting pigs caused by African swine fever virus (ASFV), which has spread since it was first discovered in Africa in 1909 It has spread to countries and regions throughout Africa, Europe, and South America, and many countries have suffered huge economic losses. The disease is listed as an important animal disease by the World Organization for Animal Health (OIE). Although my country has not found the African swine fever epidemic, many countries around my country have reported the prevalence of African swine fever, and my country has faced the serious threat of ASFV introduction...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/34C12N15/11A61K48/00A61K39/12A61P31/20C12R1/93
Inventor 孙洁杨俊兴廖立珊胡运发刘芸曾少灵刘建利林庆燕陶虹曹琛福张彩虹阮周曦唐金明吕建强花群义
Owner 孙洁
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