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Artificial codon optimized pseudorabies virus gE protein and application thereof

A pseudorabies virus, codon optimization technology, applied in the direction of virus, virus peptide, virus/bacteriophage, etc., can solve problems such as death, reproductive barriers of sows, dyspnea of ​​adult pigs, etc., and achieve the effect of high-efficiency expression

Active Publication Date: 2019-12-13
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In practice, the clinical manifestations of pigs infected with PRV are diverse. Newborn piglets show neurological disorders and death, adult pigs show dyspnea, and sows are characterized by reproductive disorders, causing serious economic losses to the pig industry.
In recent years, with the widespread application of PRV-deficient vaccines, the prevention and control of this disease has achieved remarkable results, but with the occurrence of mixed infections in pig farms, the incidence of pseudorabies has increased year by year

Method used

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  • Artificial codon optimized pseudorabies virus gE protein and application thereof
  • Artificial codon optimized pseudorabies virus gE protein and application thereof
  • Artificial codon optimized pseudorabies virus gE protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0030] Example 2 Constructing recombinant pseudorabia virus-like gE protein expression plasmid pET19b-like-gE

[0031] The artificially synthesized pseudorabies virus gE-like protein gene was inserted into the pET19b vector through NdeI and BamHI digestion PCR amplification products, and transformed into Escherichia coli JM109, and the bacterial solution was spread on LB agar plates (containing ampicillin 100 μg / ml, chloride Mycin 34μg / ml), after culturing overnight at 37°C, the clones were picked, and the recombinant plasmid was extracted for double-enzyme digestion identification. The results showed that the recombinant plasmid pET19b-like-gE of the pseudorabies virus-like gE protein was successfully constructed, and sequenced Identification (the recombinant plasmid was sequenced and identified by Shanghai Handsome Biological Co., Ltd.), and the vector NTI 10 software was used to deduce the amino acid sequence. The result was consistent with the originally designed pseudorabi...

Embodiment 3

[0032] Example 3 Expression and Purification of Recombinant Pseudorabies Virus-like gE Protein

[0033] After the recombinant plasmid pET19b-like-gE was transformed into Escherichia coli BL21 Star(DE3)pLysS, it was spread on LB agar plates (containing ampicillin 100 μg / ml, chloramphenicol 34 μg / ml), and cultured overnight in a 37°C incubator. Colony into 800ml LB culture medium (containing ampicillin 50μg / ml, chloramphenicol 34μg / ml), shake at 220rmp at 37°C, cultivate until OD600 is about 0.5, add 1mM IPTG, induce for 3 hours at 37°C, and incubate at 4°C Centrifuge at 8000rpm for 10 minutes, discard the supernatant, and collect the bacterial pellet. After the bacteria were lysed, the supernatant was purified by His-binding resin (Novagen, Madison, Wis.), and its purity was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Detected by 12.5% ​​SDS-PAGE, the bacterium liquid induced by IPTG appears target protein expression band (such as Figure...

Embodiment 4

[0034] Example 4 Western blotting detection of recombinant pseudorabies virus-like gE protein

[0035]Take 6 μg of recombinant pseudorabies virus gE protein, add an equal volume of sample buffer and 1 / 10 volume of β-mercaptoethanol, mix well, denature at 95°C for 5 minutes, centrifuge at 14,000rpm for 10 minutes, and use the supernatant for SDS-PAGE loading. After the electrophoresis, transfer the protein to the PVDF membrane. After the electrotransfer, wash the membrane with PBST for 30 minutes, add PBS containing 5% skimmed milk, and block at room temperature for 1 hour in a wet box; discard the blocking solution, and add mouse anti- His antibody (diluted 1:250), incubated overnight at 4°C; washed membrane with PBST, 30 minutes; added HRP-Goat Anti-mouse IgG antibody (diluted 1:250), incubated at room temperature for 1 hour; washed membrane with PBST, 30 minutes, with Konica Immunostaining HRP-100 developed the color, and the reaction was terminated after 20 minutes. The res...

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Abstract

The invention belongs to the technical field of biology, in particular to an artificial codon-optimized pseudorabies virus gE protein as well as a coding gene and an application thereof. According tothe invention, the gE-like protein gene which can be recognized by a pseudorabies virus gE antibody is optimized and synthesized through an artificial codon, an expression vector is constructed by utilizing an artificially synthesized nucleotide sequence, and the specific recognition capability of the pseudorabies virus gE protein and the pseudorabies virus gE antibody is analyzed through westernblotting, ELISA and sequencing. The pseudorabies virus gE protein obtained by the invention can be used for preparing a differential diagnosis kit for porcine pseudorabies virus infected wild strains.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a pseudorabies virus-like gE protein, in particular to an artificial codon-optimized pseudorabies virus-like gE protein, its encoding gene and its application. The obtained pseudorabies virus gE-like protein can be used to prepare a differential diagnosis kit for pigs infected with wild strains of pseudorabies virus. Background technique [0002] Pseudorabies (Pseudorabies, PR) is an infectious disease notified by the World Organization for Animal Health (OIE), and it is also one of the pig diseases listed as priority prevention and treatment in the "National Medium and Long-term Animal Disease Prevention and Control Plan (2012-2020)". Pseudorabies virus (Pseudo rabievims, PRV) is an acute infectious disease caused by a variety of domestic and wild animals such as pigs, cattle, sheep, dogs, rabbits, mice, mink, and foxes. In practice, the clinical manifestations of pigs infected with ...

Claims

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Application Information

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IPC IPC(8): C07K14/03C12N15/38C12N15/70C12N1/21G01N33/569C12R1/19
CPCC07K14/005C12N15/70G01N33/56994C12N2710/16722C12N2800/22G01N2333/032
Inventor 付永锋周晶雨程训佳
Owner FUDAN UNIV
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