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Raccoon dog parvovirus VP2 gene, expression vector, recombinant bacterium, method for preparing VP2 protein and assembly method

A technology of parvovirus and expression vector, which is applied in the fields of raccoon parvovirus VP2 gene, preparation of VP2 protein, recombinant bacteria, and expression vector. It can solve the problems of research work and application obstacles, low production cost, etc., achieve good immune activity, and improve correctness. The effect of improving the folding rate and solubility

Pending Publication Date: 2020-02-28
CHANGCHUN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In contrast, the prokaryotic expression system has high protein expression, low production cost, and simple production operation. However, in actual operation, most proteins form inclusion bodies due to incorrect folding, which brings obstacles to subsequent research work and application.

Method used

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  • Raccoon dog parvovirus VP2 gene, expression vector, recombinant bacterium, method for preparing VP2 protein and assembly method
  • Raccoon dog parvovirus VP2 gene, expression vector, recombinant bacterium, method for preparing VP2 protein and assembly method
  • Raccoon dog parvovirus VP2 gene, expression vector, recombinant bacterium, method for preparing VP2 protein and assembly method

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Experimental program
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Effect test

Embodiment 1

[0064] A method for prokaryotic expression of raccoon parvovirus VP2 protein. In the method, VP2 protein and chaperone protein are co-expressed to obtain recombinant expression protein with good solubility and high activity. The specific steps are as follows:

[0065] 1.1 Construction of recombinant vector pET30a-VP2

[0066] 1.1.1 Artificial modification and synthesis of main immunogenic gene VP2 of RDPV

[0067] Referring to the RDPV gene sequence, the sequence was optimized according to the codon preference of Escherichia coli, and the artificially synthesized complete raccoon parvovirus VP2 gene was inserted into the vector pUC to construct the plasmid pUC-VP2.

[0068] 1.1.2 Construction of pET30a-VP2 vector

[0069] Plasmid pUC-VP2 and vector pET30a were double-digested with restriction endonucleases NdeI and HindIII, respectively, and the reaction conditions were 37°C for 2 hours. The enzyme digestion system was shown in Table 1; then the double-enzymes of vector pET3...

Embodiment 2

[0082] Preparation of raccoon parvovirus virus-like particles

[0083] 2.1 Purification of VP2 protein and determination of VLP

[0084] 2.1.1 Purification of recombinant VP2 protein

[0085] Add saturated ammonium sulfate solution to the supernatant of ultrasonic cracking obtained in Example 1, so that the final concentrations of ammonium sulfate are 10%, 15%, 20%, 25%, and 30% saturation respectively. Stir at 4°C for 1 h, centrifuge at 10,000 rpm for 30 min, take the supernatant and resuspend the pellet with lysate solution (50 mM Tris, 150 mM NaCl, pH 8.0). After processing each supernatant and precipitation sample, SDS-PAGE detection, the results are as follows Figure 6-7 , VP2 protein can remove a lot of miscellaneous proteins after passing through 30% ammonium sulfate.

[0086] Add 60%, 50%, 40% and 30% sucrose solutions in sequence to a 38.5mL sucrose density gradient centrifuge tube, 8mL for each gradient; add 6.5mL of the sample after 30% ammonium sulfate precipit...

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Abstract

The invention provides raccoon dog parvovirus VP2 gene, an expression vector, a recombinant bacterium, a method for preparing VP2 protein and an assembly method, and belongs to the technical field ofvaccine preparation. The raccoon dog parvovirus VP2 gene has a nucleotide sequence shown as SEQ ID No. 1. According to the invention, the raccoon dog parvovirus VP2 gene and a molecular chaperone arejointly transformed into ER2566 cells, and the molecular chaperone improves the correct folding rate of VP2 protein. The raccoon dog parvovirus VP2 gene provided by the invention can form high-qualityraccoon dog parvovirus-like particles. A raccoon dog parvovirus-like particle vaccine provided by the invention has a hemagglutination titer of 216 after being purified, and has a hemagglutinating property similar to that of a natural virus.

Description

technical field [0001] The invention belongs to the technical field of vaccine preparation, and in particular relates to a raccoon parvovirus VP2 gene, an expression vector, a recombinant bacterium, a method for preparing VP2 protein and an assembly method. Background technique [0002] Raccoon dogparvovirus (RDPV) can cause raccoon parvovirus enteritis, which is an acute and highly contagious infectious disease with high morbidity and mortality. It is one of the important infectious diseases that endanger the breeding and development of raccoon dogs. one. At present, raccoon parvovirus enteritis widely exists in various countries in the world. Raccoon raccoon dogs of different ages and breeds are susceptible, but especially 50-60-day-old young raccoon dogs and young raccoon dogs are the most susceptible. The incidence rate of young raccoon dogs is over 70%, and the mortality rate is as high as 90%. The incidence rate of adult raccoon dogs is It is about 30%, and the morta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/35C12N15/70C12N1/21C07K14/015C12R1/19
CPCC07K14/005C12N15/70C12N2750/14022C12N2750/14023
Inventor 殷玉和雷欢孙博赫玉芳罗国良李希辰吴丛梅
Owner CHANGCHUN UNIV OF TECH
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