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Preparation of PCV2 ORF2 capsid protein virus-like particles derived from escherichia coli

A capsid protein, Escherichia coli technology, applied in the field of molecular biology, can solve the problems of low expression and loss of large-scale commercial application

Inactive Publication Date: 2013-06-26
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 contains a nuclear localization signal composed of 41 amino acids. Due to the codon bias of this signal peptide, the ORF2 protein is in the E. coli The expression level in is very low, has lost the possibility of large-scale commercial application, and the present invention therefore comes

Method used

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  • Preparation of PCV2 ORF2 capsid protein virus-like particles derived from escherichia coli
  • Preparation of PCV2 ORF2 capsid protein virus-like particles derived from escherichia coli
  • Preparation of PCV2 ORF2 capsid protein virus-like particles derived from escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Expression of PCV2 protein with sequence 1

[0046] Preparation of PCV2 ORF2 gene fragment used as template

[0047] The full-length PCV2 ORF2 gene optimized by Escherichia coli was synthesized by Shanghai Boya Biotechnology Co., Ltd. The synthesized gene fragment is 702 bp in full length. The PCV2 ORF2 protein template of the present invention is prepared on the basis of the artificially synthesized PCV2 ORF2 gene fragment.

[0048] Construction of gene non-fusion expression vector

[0049] The PCV2 ORF2 full-length gene fragment synthesized in the previous step was used as a template for the PCR reaction. Using PCV2-F: 5'- GGA TTC CCC GTG CCC GTG AGC AAG -3' as the forward primer, the 5' end introduces a restriction endonuclease BamH I site and a protective base, and the BamH I site sequence is GGA TCC, ATG is the start codon in the E. coli system; PCV2-R: 5'-CTC GAG CA CCT CTT CAC CTT CTT C -3' is the reverse primer, and its 5' end introduces...

Embodiment 2

[0068] Example 2: The acquisition of PCV2 ORF2 protein with a purity of about 70%

[0069] 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 4 times with a homogenizer at a pressure of 700 bar. The JA-14 rotor was centrifuged at 13500 rpm (28000g) for 40 min, and the supernatant was collected and detected by 15% SDS-polyacrylamide gel electrophoresis. At this time, the purity of PCV2 ORF2 in the supernatant was about 20%. Ammonium sulfate was used for fractional separation and precipitation. Initially, 30% saturated thiamine was used to precipitate part of the impurity protein, and then 50% thiamine was used to precipitate the target protein. Resuspend the pellet with an equal volume of 10mM phosphate buffer pH7.0, 10mM DTT, 300mM NaCl, and stir for 30min. JA-14 rotor (Beckman J25 high-speed centrifuge), 13500rpm (30000g), centrifuged for 20min, centrifuged to get the supernatant, filter ...

Embodiment 3

[0070] Example 3: Chromatographic purification of PCV2 ORF2

[0071] Purification of PCV2 ORF2 by Cation Exchange Chromatography

[0072] Instrument system: AKTA purification and preparative liquid chromatography system produced by GE Healthcare former Amershan Pharmacia company.

[0073] Chromatography medium: Butyl Sepharose 4 Fast Flow.

[0074] Column volume: 5.5 cm x 20 cm.

[0075] Buffer: 20mM phosphate buffer pH8.0, 0.2M NaCl;

[0076] 20mM phosphate buffer pH 8.0, 2M NaCl.

[0077] Flow rate: 25ml / min.

[0078] Detector wavelength: 280nm.

[0079] The sample is 3L of PCV2 ORF2 protein solution with a purity of about 70%.

[0080] 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 PCV2 ORF2 purified sample.

[0081] Purification of SP

[0082] Instrument system: AKTA purification ...

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Abstract

The invention relates to porcine circovirus PCV2 ORF2 gene optimized by using escherichia coli expression codon and a preparation method of PCV2 ORF2 capsid protein virus-like particles derived from the escherichia coli.

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 PCV is a non-enveloped single-stranded circular DNA virus belonging to the Circoviridae family and the genus Circovirus. This virus is widely prevalent in countries all over the world. The virus not only causes slow growth of piglets, but also infringes on the immune system of pigs, leading to a decline in the immune function of pigs, causing immunosuppression of the body, and causing great economic losses to the world's pig industry. . [0003] The genome is very small. According to the difference in genetic composition and antigenicity, PCV can be divided into...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/34C07K14/01C12N15/63C12N15/70C12N1/21C12N7/04A61K39/12A61P31/20C12R1/93
Inventor 袁于人莫小兵
Owner SA BIOTECH (SUZHOU) PTE LTD
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