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Preparation and use of truncated Cap protein of porcine circovirus type 2

A porcine circovirus and truncated type technology, applied in the field of genetic engineering, can solve the problems of restricted expression, high price, low expression of PCV2Cap protein, etc., and achieve the effect of solving the problem of high cost

Inactive Publication Date: 2011-01-19
PU LIKE BIO ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the expression of PCV2 Cap protein in Escherichia coli, there are also related literature reports at home and abroad, such as: Liu et al. have carried out the expression of the whole gene of PCV2 Cap protein in Escherichia coli (Protein Exp.Purif.21 (2001b), 115-120) , Zhou et al expressed the truncated Cap protein in Escherichia coli (Journal of Biotechnology 118 (2005) 201-211), but in general, the expression of PCV2 Cap protein was very low, respectively 1mg / L and 6.142mg / L, and requires protein renaturation and the addition of expensive IPTG, both of which limit the expression of PCV2 Cap protein in E. coli

Method used

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  • Preparation and use of truncated Cap protein of porcine circovirus type 2
  • Preparation and use of truncated Cap protein of porcine circovirus type 2
  • Preparation and use of truncated Cap protein of porcine circovirus type 2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: the preparation method of porcine circovirus type 2 truncated Cap protein vaccine

[0043] 1. Cloning the porcine circovirus type 2 truncated Cap protein gene into an expression vector to obtain a recombinant expression vector.

[0044] ① Design of primers

[0045] Using the PCV2-SH strain nucleic acid sequence as a reference (GenBank: AY686763), use Oligo6.0 primer design software to design porcine circovirus type 2 truncated Cap gene-specific primers, upstream primers: restriction site NcoI 5′-CATGCCATGGTGAATGGCATCTTC -3'; downstream primer: restriction site HindIII 5'-CCCAAGCTTTTAAGGGTTAAGT-3', the length of the target gene for PCR amplification is 585bp.

[0046] ② Extraction of PCV2 virus nucleic acid

[0047] 1ml DNAzol Reagent (Invitrogen ) into 0.1ml of the PCV2-SH virus strain sample with the preservation number CGMCCNo.2389, shake the centrifuge tube upside down; let stand at room temperature for 5min; centrifuge at 4°C (10,000r / min, 10min), ...

Embodiment 2

[0068] Example 2: Efficacy and biological testing of porcine circovirus type 2 truncated Cap protein vaccine

[0069] The oil-emulsion vaccine prepared in Example 1 was tested for finished vaccines in accordance with the requirements of the veterinary biological products regulations, mainly for character testing, safety testing, efficacy testing, and determination of formaldehyde and thimerosal residues for the finished vaccines. The results showed that the sampling samples had a uniform appearance and no mold growth; the safety inspection and efficacy inspection all met the requirements; the residues of formaldehyde and thimerosal also met the requirements. Use qualified products to carry out clinical trials - piglet challenge tests.

[0070] ①Safety test

[0071] Five 25-day-old piglets (provided by a pig farm in Jiangsu) were inoculated intramuscularly with 2 times the dose of PCV2Cap vaccine (4ml / head), and there was no abnormal reaction after 28 days of clinical observat...

Embodiment 3

[0074] Example 3 Identification of immunogenicity of porcine circovirus type 2 truncated Cap protein and its application in the preparation of detection reagents for PCV2

[0075] ①Western-blot test

[0076] The purified porcine circovirus type 2 truncated Cap protein was identified by Western-blot. The molecular weight of the truncated PCV2 Cap protein is about 22.5Ku, and it has a positive result with porcine circovirus type 2 positive seroreaction; it has a negative result with negative seroreaction; Positive sera of porcine pseudorabies and porcine parvovirus have no reaction, the results are shown in Figure 5 . Among them, 1-6 were positive for porcine circovirus type 2 truncated Cap protein and swine fever, porcine reproductive and respiratory syndrome, porcine pneumonia, porcine pseudorabies, porcine parvovirus disease positive serum and porcine circovirus type 2 negative Serum reaction result; 7 is positive serological reaction result of porcine circovirus type 2 t...

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Abstract

The invention relates to a vaccine of the truncated Cap protein of porcine circoviruses type 2 and a preparation method thereof. The preparation method comprises: firstly, cloning the Cap protein gene, in which 41 amino acid residues in an N terminal is removed, of the porcine circoviruses type 2 into a plasmid pKK-233-2 to obtain a recombinant expression plasmid pKK-233-2-Cap; and transforming Escherichia coli DH5 alpha by the recombinant expression plasmid to obtain recombinant genetic engineering bacteria DH5 alpha / pKK-233-2-Cap. The addition of IPTG or lactose for induction is avoided, the recombinant genetic engineering bacteria express the truncated Cap protein constitutively, the truncated Cap protein is separated and purified, and an adjuvant is added to obtain the vaccine of the truncated Cap protein of porcine circoviruses type 2. The Cap protein can also be used for preparing reagent for diagnosing related diseases caused by the porcine circoviruses type 2.

Description

technical field [0001] The invention relates to a genetically engineered vaccine of porcine circovirus type 2 and its preparation method, and uses truncated Cap protein as an ELISA detection antigen to identify porcine circovirus type 2 infection, especially porcine circovirus type 2 truncated type A Cap protein vaccine belongs to the field of genetic engineering. Background technique [0002] Porcine circovirus (PCV) is the smallest known animal virus. According to the pathogenicity, antigenicity and nucleotide sequence of PCV, it is divided into two serotypes, PCV1 (porcine circovirus type 1) and PCV2 (porcine circovirus type 2). PCV1 widely exists in pigs and pig-derived cell lines, and it has been proved by animal infection experiments that PCV1 has no pathogenicity. PCV2 is associated with a variety of diseases that occur in pigs, such as multisystemic wasting syndrome in weaned piglets, porcine dermatitis nephrotic syndrome, porcine respiratory disease syndrome, and ...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N15/70C12N1/21C07K14/01A61K39/12A61P31/20G01N33/569C12R1/19
Inventor 张许科孙进忠乔荣岑
Owner PU LIKE BIO ENG
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