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Codon-optimized peste des petits ruminants virus F gene nucleic acid vaccine

A technology of codon optimization and Peste des Petits Ruminants, which is applied in the field of molecular biology and can solve the problem of inefficient expression of foreign genes

Active Publication Date: 2020-01-14
VETERINARY INST XINJINAG ACADEMY OF ANIMAL SCI CLINIC MEDICAL SCI RES CENT XINJIANG ACADEMY OF ANIMAL HUSBANDRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, nucleic acid vaccines also have many shortcomings, the most important of which is that the research and application objects of nucleic acid vaccines are mainly eukaryotes, and the vast majority of target genes come from prokaryotes such as viruses or bacteria. There are obvious differences in codon usage preferences, which leads to the inability of the foreign gene of the nucleic acid vaccine to be efficiently expressed in eukaryotic cells, and cannot effectively stimulate the body's immune system to respond

Method used

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  • Codon-optimized peste des petits ruminants virus F gene nucleic acid vaccine
  • Codon-optimized peste des petits ruminants virus F gene nucleic acid vaccine
  • Codon-optimized peste des petits ruminants virus F gene nucleic acid vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Design and synthesis of codon-unoptimized and optimized PPRV F gene sequences

[0030] 1. Synthesis of the codon-unoptimized PPRVF gene sequence

[0031] Taking the PPRV Nigeria / 75 / 1 strain as the reference sequence (GenBank: X74443), first select the F gene with a full length of 1641bp, which was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. and loaded into the vector pUC57 to form the recombinant plasmid pUC57-F , the synthesized sequence was confirmed to be correct by sequencing.

[0032] 2. Design and synthesis of codon-optimized PPRV F gene sequence without signal peptide

[0033] (1) Taking the PPRV Nigeria / 75 / 1 strain as the reference sequence (GenBank: X74443), first select the F gene, the full length is 1641bp, and the remaining 1584bp after removing the 5′ terminal signal peptide (57bp), and then use the software OptimumGeneTM to analyze its gene sequence , to find out its codon usage bias, and at the same time find out the codon sites...

Embodiment 2

[0038] Embodiment two: the construction of eukaryotic expression vector pcDNA3.1 (+)-F, pcDNA3.1 (+)-F-opt, pcDNA3.1 (+)-JEVSP-F-opt

[0039] 1. Obtaining the target fragment and vector

[0040] (1) Obtaining F fragment and large linear fragment of plasmid pcDNA3.1(+)

[0041] The recombinant vector pUC57-F containing the target sequence provided by Sangon Bioengineering (Shanghai) Co., Ltd. was digested with BamH I and Xba I to obtain the F fragment; the enzyme digestion reaction system was: 10×Tango Buffer 4 μL, plasmid pUC57 -F 10 μL, BamH I and Xba I 1.5 μL each, rehydrate to 40 μL, 37°C, 2h;

[0042] Digest vector plasmid pcDNA3.1(+) with BamH I and Xba I to obtain a large linear fragment of plasmid pcDNA3.1(+); enzyme digestion reaction system: 10×Tango Buffer 4μL, plasmid pcDNA3.1(+) 10μL , 1.5 μL each of BamH I and Xba I, add water to 40 μL, 37°C, 2h;

[0043] (2) Obtaining large linear fragments of F-opt and plasmid pcDNA3.1(+)

[0044] The recombinant vector pUC5...

Embodiment 3

[0060] Example 3: Identification of recombinant plasmids pcDNA3.1(+)-F, pcDNA3.1(+)-F-opt, pcDNA3.1(+)-JEVSP-F-opt

[0061] 1. Recombinant plasmids pcDNA3.1(+)-F, pcDNA3.1(+)-F-opt, pcDNA3.1(+)-JEVSP-F-opt transform Escherichia coli DH5α competent cells:

[0062] (1) Add 10 μL of the linker to the Ep tube containing 100 μL of DH5α competent cells, gently tap the tube wall several times, mix well, and ice-bath for 30 minutes. (2) Place the Ep tube in a water bath at 42°C for 90s;

[0063] (3) Slowly add 1 mL of LB medium to the Ep tube, shake at 37°C, 225 rpm, for 60 min;

[0064](4) Spread the bacterial solution on an LB plate containing ampicillin (0.1 g / L), and culture overnight at 37°C.

[0065] 2. Screen positive clones

[0066] Randomly pick a single colony, inoculate it into a culture test tube (LB medium containing 0.1 g / L ampicillin), shake at 225 rpm at 37°C, and culture overnight.

[0067] 3. A small amount of recombinant plasmids pcDNA3.1 (+)-F, pcDNA3.1 (+)-F-o...

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Abstract

The invention relates to the technical field of molecular biology, in particular to a codon-optimized peste des petits ruminants virus F gene nucleic acid vaccine. The codon optimization gene sequencegives consideration to the codon use preference of mammalian cells, and a sequence is shown as SEQ ID NO. 2. According to the peste des petits ruminants virus F gene nucleic acid vaccine, after signal peptide at the 5'end of an F gene is removed, the remaining sequence is optimized through codons, the 5 'end is connected with an encephalitis B virus signal peptide sequence, and finally the sequence is inserted into an eukaryotic expression vector. The nucleic acid vaccine effectively stimulates an immune system after immunizing a mouse, generates a neutralizing antibody and reflects good immunogenicity.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a codon-optimized F gene nucleic acid vaccine of Peste des petits ruminants. Background technique [0002] Peste-des-petits ruminants (PPR) is a severe and severe contagious disease caused by peste-des-petits ruminants virus (PPRV), characterized by fever, mouth and tongue Mucosal erosions, lacrimation, nasal discharge, diarrhea, and pneumonia. PPR is a Class A severe infectious disease that must be reported by the World Organization for Animal Health (OIE). Sheep and goats are more susceptible, and can also infect cattle, buffaloes, pigs and wild animals. PPRV infection can cause up to 100% morbidity and significant mortality in sheep and goats, seriously affecting food security and the survival of herders, especially in developing countries dependent on small ruminants. Since the first outbreak of PPR in Côte d'Ivoire in 1942, it has been prevalent in East Africa, ...

Claims

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Application Information

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IPC IPC(8): C12N15/45C12N15/85A61K39/155A61P31/14
CPCC07K14/005C12N15/85A61K39/12A61P31/14C12N2760/18422C12N2760/18434C12N2800/107C12N2800/22A61K2039/53A61K2039/552Y02A50/30
Inventor 夏俊汪萍苗书魁杜玮马文戈黄炯金映红沙吾尔江·阿不都力艾拉赵洁雅王文马晓艳席瑞陆桂丽魏玉荣米晓云沙依兰·卡依扎沈辰峰参都哈西·加吾丁徐猛木克日木·帕尔哈提
Owner VETERINARY INST XINJINAG ACADEMY OF ANIMAL SCI CLINIC MEDICAL SCI RES CENT XINJIANG ACADEMY OF ANIMAL HUSBANDRY SCI
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