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Preparation of self-assembled virus-like particle by using escherichia coli to express feline parvovirus VP2 protein

A feline parvovirus and parvovirus technology, applied to viruses, viral peptides, viruses/bacteriophages, etc., can solve problems such as research work and application obstacles, and low production costs

Inactive Publication Date: 2021-10-01
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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  • Preparation of self-assembled virus-like particle by using escherichia coli to express feline parvovirus VP2 protein
  • Preparation of self-assembled virus-like particle by using escherichia coli to express feline parvovirus VP2 protein
  • Preparation of self-assembled virus-like particle by using escherichia coli to express feline parvovirus VP2 protein

Examples

Experimental program
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Effect test

Embodiment 1

[0027] The invention discloses a prokaryotic method for expressing feline parvovirus VP2 protein. In the method, the VP2 protein is co-expressed with a chaperone protein, and a recombinant expression protein with good solubility and high activity can be obtained. Its specific steps are as follows:

[0028] 1.1 Construction of recombinant vector pET30a-VP2

[0029] 1.1.1 Artificial modification and synthesis of FPV main immunogenic gene VP2

[0030] Referring to the FPV gene sequence, the sequence was optimized according to the codon preference of Escherichia coli, and the artificially synthesized complete feline parvovirus VP2 gene was inserted into the vector pUC to construct the plasmid pUC-VP2.

[0031] 1.1.2 Construction of pET30a-VP2 vector

[0032] 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, and then the double-digested products of vector pET30a ...

Embodiment 2

[0044] Preparation of feline parvovirus virus-like particles

[0045]2.1 Purification of VP2 protein and determination of VLP

[0046] 2.1.1 Purification of recombinant VP2 protein

[0047] Add saturated ammonium sulfate solution to the supernatant of ultrasonic cracking obtained in Example 1, so that the final concentrations of ammonium sulfate are 15%, 20%, 25%, 30%, 35%, 40%, and 45% saturation respectively. Stir at 4°C for 1 h, centrifuge at 4000 rpm for 30 min, take the supernatant and resuspend the pellet with lysate (50 mM Tris, 150 mM NaCl, pH 8.0). After processing each supernatant and precipitation sample, SDS-PAGE detection, the results are as follows Image 6 , VP2 protein can remove a large number of foreign proteins after passing through 25% ammonium sulfate.

[0048] Add 25% ammonium sulfate precipitated protein to an equal volume of reconstituted solution (0.25M (NH4)2SO4, 20mM Tris, 2mMNaCl) to redissolve, use the reconstituted solution to wash the hydropho...

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Abstract

The invention belongs to the technical field of vaccine preparation, relates to a novel feline parvovirus vaccine, and discloses a coding gene of a feline parvovirus VP2 protein. According to the invention, an FPV VP2 protein sequence is coded and optimized through a genetic engineering technology, a recombinant plasmid is constructed, a recombinant protein is expressed by using escherichia coli, and the FPV VP2 protein is co-expressed with four molecular chaperones respectively; and a target protein is purified by different purification methods to obtain a virus-like particle which is similar to natural viruses in size and has good hemagglutination. A new thought and a new basis are provided for novel feline parvovirus vaccines.

Description

technical field [0001] The invention belongs to the field of VLP vaccines. The invention relates to the recombination expression of FPV main capsid protein VP2 by using Escherichia coli system, and self-assembly into virus-like particles. When the exogenous gene is highly expressed in E. coli, the intracellular protein concentration is too high, and there is not enough time for folding to form non-crystalline and amorphous protein aggregates. The present invention adopts co-expression with four molecular chaperones pG-KJE7, pG-KJE8, pGro7 and pTf16 to increase the proportion of soluble protein. Proteins purified by different methods can form virus-like particles with uniform particles, good shape and similar in size to natural viruses under the specific environment of the present invention. It provides a basis for the study of the VLP vaccine of feline parvovirus, and further solves the problems faced by pet cats and felines. Background technique [0002] Feline parvoviru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/35C07K14/015C12N15/70C12N1/21C12R1/19
CPCC07K14/005C12N15/70C12N2750/14022C12N2750/14051C12N2750/14023
Inventor 赵林烨王博夏霖亚吴丛梅殷玉和
Owner CHANGCHUN UNIV OF TECH
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