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Truncated PCV2-type capsid protein ORF2 (Open Reading Frame 2) virus-like particle and preparation method

A technology of capsid protein and protein, applied in the field of molecular biology, can solve the problems of low expression of ORF2 protein and loss of large-scale commercial application, etc., and achieve good protective effect, active protection, and simple process flow

Inactive Publication Date: 2014-09-03
SA BIOTECH (SUZHOU) PTE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many research results have shown that the N-terminal of ORF2 protein contains a nuclear localization signal consisting of 41 amino acids. Due to the codon bias of this signal peptide, the expression level of ORF2 protein in E.coli is very low and lost. The possibility of large-scale commercial application

Method used

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  • Truncated PCV2-type capsid protein ORF2 (Open Reading Frame 2) virus-like particle and preparation method
  • Truncated PCV2-type capsid protein ORF2 (Open Reading Frame 2) virus-like particle and preparation method
  • Truncated PCV2-type capsid protein ORF2 (Open Reading Frame 2) virus-like particle and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Protein expression of the N-terminal truncated PCV2 ORF2 gene with sequences 1-9

[0049] 1.1 Preparation of PCV2 ORF2 gene fragment used as template

[0050] The full-length PCV2 ORF2 gene optimized by Escherichia coli was synthesized by Shanghai Boya Biotechnology Co., Ltd. The synthesized gene fragment is 702bp in full length. The N-terminal truncated PCV2 ORF2 gene template of the present invention is prepared on the basis of the artificially synthesized PCV2 ORF2 gene fragment.

[0051] Construction of the non-fusion expression vector of the PCV2 ORF2 gene truncated at the 1.2N end

[0052] The PCV2 ORF2 full-length gene fragment synthesized in the previous step was used as a template for the PCR reaction. Use PCV2-N3F, PCV2-N6F, PCV2-N9F, PCV2-N12F, PCV2-N15F, PCV2-N18F, PCV2-N21F, PCV2-N24F and PCV2-N27F as forward primers, and PCV2-R as reverse primers , to amplify the N-terminal truncated PCV2 ORF2 gene. The 5' end of the forward primer introduc...

Embodiment 2

[0073] Example 2: The acquisition of N-terminal truncated PCV2 ORF2 protein with a purity of about 70%

[0074] Resuspend the bacteria at a ratio of 1 g of bacteria to 10 mL of lysate (20 mM Tris buffer pH 7.2, 300 mM NaCl), and crush the bacteria four times with a homogenizer at a pressure of 700 bar. The JA-14 rotor was centrifuged at 13500 rpm (28000 g) for 40 min, and the supernatant was collected and detected by 15% SDS-polyacrylamide gel electrophoresis. At this time, the purity of PCV2ORF2 in the supernatant was about 20%. Ammonium sulfate was used for fractionation and precipitation, and 30% saturated thiamine containing potassium chloride was used to precipitate part of the impurity protein, and then the target protein was precipitated with 50% thiamine containing potassium chloride. Resuspend the pellet with an equal volume of 10 mM phosphate buffer pH 7.0, 10 mM DTT, 300 mM NaCl, and stir for 30 min. JA-14 rotor (Beckman J25 high-speed centrifuge), 13500rpm (30000g...

Embodiment 3

[0075] Example 3: Chromatographic purification of N-terminal truncated PCV2 ORF2

[0076] Cation-exchange chromatography purification of N-terminally truncated PCV2 ORF2

[0077] Instrument system: AKTA purification and preparative liquid chromatography system produced by GE Healthcare former Amershan Pharmacia company.

[0078] Chromatography medium: Butyl Sepharose 4 Fast Flow.

[0079] Column volume: 5.5 cm x 20 cm.

[0080] Buffer: 20mM phosphate buffer pH8.0, 0.2M NaCl;

[0081] 20 mM phosphate buffer pH 8.0, 2M NaCl.

[0082] Flow rate: 25ml / min.

[0083] Detector wavelength: 280nm.

[0084] The sample is 3L of PCV2 ORF2 protein solution with a purity of about 70% N-terminal truncated.

[0085] The elution procedure was as follows: after breakthrough, 1500mM NaCl eluted the impurity protein, 200mM NaCl eluted the target protein, collected the 1000mM NaCl eluted product, and obtained a total of 900mL purified samples of N-terminal truncated PCV2 ORF2.

[0086] SP p...

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Abstract

The invention discloses a series of N-terminal truncated PCV2-type ORF2 (Open Reading Frame 2) genes, a virus-like particle of the ORF2 gene and a preparation method of the virus-like particle, a vaccine including the truncated virus-like particle and an application of the vaccine in the prevention of post-weaning multisystemic wasting syndrome infection. The virus-like particle assembled by truncated PCV-ORF2 genes disclosed by the invention has good immunogenicity and protection. Since no denaturing agent is used in the preparation and purification process disclosed by the invention, the activity of the target protein is protected to the greatest extent.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a porcine circovirus type 2 capsid protein gene, a carrier, a bacterial strain and an expression method thereof, a vaccine containing the virus-like particle and its role in preventing multisystemic wasting syndrome of weaned piglets (especially PCV2) infection. Background technique [0002] Porcine circovirus type 2 PCV2 belongs to the Circoviridae family and the genus Circovirus, and is a non-enveloped single-stranded circular DNA virus. This virus is widely prevalent in countries all over the world, and has caused great economic losses to the pig industry in the world. Type 2 porcine circovirus includes two subtypes: PCV2a and PCV2b. In recent years, PCV2b has the widest prevalence in China, accounting for the largest proportion, and PCV2b is more toxic. [0003] The PCV2ORF2 protein is the capsid protein of the virus with a molecular weight of 25kDa and is the mai...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N7/04C12N15/70C12N1/21C07K14/01C07K1/36C07K1/30C07K1/16A61K39/12A61P31/20
Inventor 袁于人莫小兵
Owner SA BIOTECH (SUZHOU) PTE LTD
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