Method of preparing porcine parvovirus virus-like particle subunit vaccine by using Escherichia coli expression system and application of method

A parvovirus and expression method technology, applied in the field of bioengineering, can solve the problems of research work and application barriers, low production costs, etc., achieve good immunogenicity, improve the correct folding rate, and improve the solubility

Inactive Publication Date: 2016-11-23
HENAN ACAD OF AGRI SCI +1
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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 operations. However, in actual operation, most proteins form inclusion bodies due to incorrect folding, which brings obstacles to subsequent research work and applications.

Method used

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  • Method of preparing porcine parvovirus virus-like particle subunit vaccine by using Escherichia coli expression system and application of method
  • Method of preparing porcine parvovirus virus-like particle subunit vaccine by using Escherichia coli expression system and application of method
  • Method of preparing porcine parvovirus virus-like particle subunit vaccine by using Escherichia coli expression system and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The invention discloses a prokaryotic method for expressing porcine parvovirus VP2 protein. In the method, PPV VP2 protein and chaperone protein are co-expressed to obtain a recombinant expression protein with good solubility and high activity. The specific steps are as follows:

[0047] 1.1 Construction of recombinant vector pET28a-VP2

[0048] 1.1.1 Artificial modification and synthesis of PPV main immunogenic gene VP2

[0049] Referring to the VP2 gene sequence of PPV China strain (GenBank: AY583318), the sequence was optimized according to the codon preference of Escherichia coli, and the VP2 gene was artificially synthesized, and its nucleotide sequence is shown in SEQ ID NO.1.

[0050] 1.1.2 Construction of pET28a-VP2 vector

[0051] The primer sequences used to amplify the VP2 protein-encoding gene are as follows:

[0052] F: 5'- GGATCC ATGTCGGAAAATGTGGAACA-3' ( Bam HI)

[0053] R: 5'- AAGCTT ATACAGTTTCCGTGGAATGA-3' ( Hind III)

[0054] Among them, t...

Embodiment 2

[0064] Example 2 Preparation of porcine parvovirus virus-like particles

[0065] 2.1 Purification of VP2 protein and determination of VLP

[0066] 2.1.1 Purification of recombinant VP2 protein

[0067] The supernatant of ultrasonic lysis was purified by Ni-NTA chromatography column (Merck). The specific process was: after Ni-NTA was equilibrated with buffer A, the sample was slowly flowed through the chromatography column, and then at least 10 times the column volume Buffer B (50mmol / L PB, 250mmol / L NaCl, 30mmol / L imidazole, pH7.0) was passed through the column to wash the impurities, and finally buffer C (50mmol / L PB, 250mmol / L NaCl, 300mmol / L imidazole , pH7.0) to elute the target protein, collect the eluted fractions, and use SDS-PAGE and Western-blot detection, the results are as follows Figure 4 , the purity of VP2 protein can reach more than 95% after purification by affinity chromatography, and it has a good reaction with His monoclonal antibody.

[0068] 2.1.2 Prot...

Embodiment 3

[0074] Example 3 PPV virus-like particle immunogenicity detection

[0075] 3.1 Determination of VP2 protein concentration and determination of endotoxin content

[0076] The concentration of the purified VP2 protein was determined with the BCA protein content assay kit (Pierce Biotechnology), and the specific steps refer to its instructions. The measured concentration of VP2 protein is 0.57mg / mL, and the expression level is relatively high, which can meet the requirements of industrial production.

[0077] Endotoxin content assay kit (ToxinSensor TM Endotoxin Detection System, GenScript) was used to determine the endotoxin content of the purified protein. For specific steps, refer to its instructions. The measured endotoxin content of the VP2 protein was less than 0.3mU / mg.

[0078] 3.2 Animal Immunization

[0079] Referring to the method in "Pharmacopoeia of the People's Republic of China" (2005 edition), the VLP obtained in Example 2 was prepared into a vaccine.

[0080...

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Abstract

The invention discloses an encoding gene of porcine parvovirus VP2 protein, a method of prokaryotically expressing VP2 protein virus-like particles, and application of the method in vaccine preparation. Sequences are optimized, VP2 gene is artificially synthesized, the synthesized gene is inserted into pET28a vector, the gene and chaperone protein plasmids are co-transferred to BL21(DE3) host bacteria, the VP2 protein and chaperone protein are co-expressed to promote correct folding of the VP2 protein. Experiments prove that recombinant bacteria expressed VP2 protein can be self-assembled in vitro and has good immunogenicity; by immunizing mice and guinea pigs with the virus-like particle subunit vaccine prepared with the VP2 protein expressed herein, it is possible to induce the production of a high level of hemagglutination inhibition antibodies and neutralizing antibodies, and the vaccine can prevent guinea pigs from being affected by strong porcine parvovirus. The recombinant bacteria according to the invention can be utilized to efficiently prepare porcine parvovirus virus-like particles, the production cost is low, operation is simple, and biosafety is better.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a method for soluble expression of porcine parvovirus VP2 protein, an encoding gene, a preparation method of porcine parvovirus virus-like particles, and the application of the virus-like particles as a subunit vaccine. Background technique [0002] Porcine parvovirus (PPV) is one of the main causes of reproductive disorders in pigs. Its main characteristics are abortion, stillbirth, mummification of primiparous sows and death of newborn piglets. Since Mayr and Mahnel discovered and confirmed the existence and pathogenicity of PPV in 1966, scholars at home and abroad have conducted extensive and in-depth research on it. In recent years, PPV infection has been expanding and rising, which has caused huge economic losses to the global pig industry. In my country, Pan Xuezhu first isolated PPV in 1983. Since then, several strains of PPV have been isolated successively in va...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/35C07K14/015C12N15/70C12N1/21C12N7/04A61K39/23A61P31/20C12R1/19
CPCC07K14/005A61K39/12A61K2039/5258C12N7/00C12N15/70C12N2750/14322C12N2750/14323C12N2750/14334C12N2800/101C12N2800/22
Inventor 张改平刘运超王爱萍陈玉梅邢广旭姬鹏超刘文英王娟冯景刘东民邓瑞广
Owner HENAN ACAD OF AGRI SCI
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