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Codon optimized porcine circovirus type 2 Cap protein coding gene and application thereof

A technology for porcine circovirus and codon optimization, which is applied in the field of molecular biology and achieves the effects of low production cost, simple preparation method and high antibody level

Inactive Publication Date: 2013-12-18
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the production cost of the baculovirus expression system is high and the expression level is low
Some researchers also used the E. coli expression system to prepare Cap protein, but because the Cap protein gene contains a large number of rare codons, soluble expression cannot be achieved, and virus-like particles cannot be formed, that is, the prepared Cap protein cannot be folded correctly and has no good immunogenicity

Method used

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  • Codon optimized porcine circovirus type 2 Cap protein coding gene and application thereof
  • Codon optimized porcine circovirus type 2 Cap protein coding gene and application thereof
  • Codon optimized porcine circovirus type 2 Cap protein coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of recombinant bacteria containing porcine circovirus type 2 Cap protein gene

[0037] 1. Obtaining the Cap protein gene fragment

[0038] The DNA of PCV2 NJ strain was extracted by conventional methods. A pair of primers were designed according to the sequence of the Cap protein gene, and the DNA of the PCV2 NJ strain was used as a template to amplify the Cap protein gene and sequence it. The Cap protein gene sequence of PCV2 NJ strain has been submitted to GenBank with accession number JX678978.1. According to the codon preference of Escherichia coli, the Cap protein gene sequence of the PCV2 NJ strain was codon-optimized to obtain the codon-optimized porcine circovirus type 2 Cap protein-encoding gene, named mCap, and its sequence is shown in SEQ ID NO: 1 Show.

[0039] Restriction sites were added to the 5' end and 3' end of SEQ ID NO: 1, CATATG was added to the 5' end, and CTCGAG was added to the 3' end, which was synthesized by Shanghai Yi...

Embodiment 2

[0079] Example 2 Preparation of porcine circovirus type 2 virus-like particles

[0080] (1) Inoculate the recombinant bacterium pTXB-mCap / BL21 in LB liquid medium (1% tryptone, 0.5% yeast powder, 1% NaCl) containing ampicillin (100 μg / mL), and culture overnight at 37°C.

[0081] (2) Take 40 μl of the overnight culture and inoculate it into 4 ml of LB liquid medium containing ampicillin (100 μg / mL), shake and culture at 37°C and 220 rpm for about 2 hours to make the OD 600 Reach 0.5-0.6.

[0082] (3) Add IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 0.1mmol / L to the bacterial solution obtained in step (2) to induce expression, the induction temperature is 32°C, and induce The time is 6h, and the fermentation liquid is obtained.

[0083] (4) Put the fermented liquid obtained in step (3) at 4°C, 15000 g The cells were collected by centrifugation for 10 min under the conditions; the cells were washed twice with PBS buffer (0.05M, pH7.2) and then resus...

Embodiment 3

[0087] Example 3 Immune Test of Genetic Engineering Subunit Vaccine Using Porcine Circovirus Type 2 Virus-like Particles as Active Components

[0088] 1. Determination of the optimal immunization dose of subunit vaccine with porcine circovirus type 2 virus-like particles (abbreviated as porcine circovirus type 2 VLP) as the active ingredient

[0089] ①. Preparation of vaccine for the test According to the method in Example 2, porcine circovirus type 2 VLP was prepared, and its protein concentration was adjusted to 2 mg / ml. 2 mg / ml porcine circovirus type 2 VLP and 206 adjuvant were mixed and emulsified at a ratio of 1:1 (V / V) to obtain porcine circovirus type 2 VLP vaccine.

[0090] ②. Screening of test pigs Select 20 healthy piglets aged 2-3 weeks, use porcine circovirus type 2 ELISA antibody detection kit (Wuhan Keqian Biotechnology Co., Ltd.) to detect PCV2 antibody before the test, and use PCV2 PCR antigen detection reagent at the same time The box (Beijing Century...

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Abstract

The invention provides a codon optimized porcine circovirus type 2 Cap protein coding gene and application thereof, and belongs to the field of the molecular biology. The Cap protein coding gene is as shown in SEQ ID NO: 1. The invention also provides a recombinant vector containing the gene and recombinant bacteria containing the gene. A method for preparing porcine circovirus type 2 virus-like particles comprises the steps of inducing the recombinant bacteria to express a porcine circovirus type 2 Cap protein, lysing the recombinant bacteria, and taking the lysate for purification to obtain the porcine circovirus type 2 virus-like particles. The invention also provides a gene engineering subunit vaccine with the virus-like particles as an active ingredient. The codon optimized porcine circovirus type 2 Cap is capable of realizing abundant soluble expression. The virus-like particles (VLP) are assembled from the Cap proteins and have excellent immunogenicity. A vaccine prepared from the virus-like particles provided by the invention is capable of preventing porcine circovirus type 2 infection and has the advantages of good immunogenicity, high antibody level and effective protection after challenge.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a codon-optimized porcine circovirus type 2 Cap protein coding gene and application thereof. Background technique [0002] Porcine circovirus type 2 ( Porcine circovirus type 2 , PCV2) is the main pathogen that causes multisystemic wasting syndrome (PWMS) in weaned piglets. Porcine dermatitis nephritic syndrome (PDNS) is closely related to respiratory disease syndrome (PRDS). Since PMWS was first reported in Canada in 1991, the disease has spread to all parts of the world. It is very prevalent in my country's pig herds and has caused great economic losses to the pig industry all over the world. At the International Porcine Veterinary Congress (IPVS) in 2010, porcine circovirus type 2 infection became the disease with the highest attention, and it is one of the important infectious diseases recognized globally that endangers the pig industry. In recent years, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/34C12N15/70C12N1/21C12N7/04A61K39/12A61P31/20C12R1/93
Inventor 郑其升赵晓云陈瑾乔绪稳李鹏成侯立婷于晓明侯继波
Owner JIANGSU ACAD OF AGRI SCI
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