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Porcine circovirus type 2 subunit vaccine and preparation method thereof

A porcine circovirus and subunit vaccine technology, applied in the biological field, can solve the problems of difficult inactivated vaccines and attenuated vaccines, low value-added titers, etc., and achieve the effects of safe and reliable expression products, convenient preparation, and high biological activity.

Inactive Publication Date: 2010-11-17
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The proliferation titer of PCV2 on cells is very low, it is difficult to apply traditional inactivated vaccines and attenuated vaccines

Method used

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  • Porcine circovirus type 2 subunit vaccine and preparation method thereof
  • Porcine circovirus type 2 subunit vaccine and preparation method thereof
  • Porcine circovirus type 2 subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation method of porcine circovirus type 2 subunit vaccine

[0040] 1. Clone the porcine circovirus type 2 ORF2 gene and introduce the melittin signal peptide gene sequence, and transfer it into the pMD-18T vector to obtain a recombinant vector containing the porcine circovirus type 2 ORF2 gene:

[0041]First design primer Spe-ATG (5'-cgactagtatgacgtatccaaggaggcgt-3') and primer Hind-TAA (5'-gcaagctttatcattaagggttaagttggg-3'), use the genomic DNA of the PCV2-SH strain whose preservation number is CGMCC No.2389 as a template, and amplify The PCV2 ORF2 gene was amplified, and the amplified ORF2 gene was cloned into the pMD-18T vector to obtain the recombinant vector pT-ORF2. The size of the PCR product is about 730bp, see the attached results figure 1 . Among them, the first hole is DNA Marker2000; the second hole is PCV2 ORF2 amplification product; the third hole is PCR negative control. The PCR product was sequenced. According to the results of electro...

Embodiment 2

[0065] Efficacy detection of embodiment 2 PCV2 subunit vaccine:

[0066] Pigs from a pig farm in Luoyang were tested for major pathogens and related antibodies, and 10-14 day-old pigs negative for major pathogens such as porcine circovirus, swine fever virus, porcine blue ear disease virus, and porcine parvovirus were selected.

[0067] Qualified piglets were randomly divided into 9 groups, 10 in each group, and 7 groups were given subunit vaccines containing different amounts of recombinant ORF2 protein (comprising 0.2 μg / head, 1 μg / head, 2 μg / head, respectively) of recombinant ORF2 protein. head portion, 4 μg / head portion, 8 μg / head portion, 16 μg / head portion and 40 μg / head portion), one group was used as a challenge control, and the other group was used as a negative control. Among them, group 1 was given a subunit vaccine containing 0.2 μg / head of recombinant ORF2 protein; group 2 was given a subunit vaccine containing 1 μg / head of recombinant ORF2 protein; group 3 was gi...

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Abstract

The invention mainly aims to provide a porcine circovirus type 2 (PCV2) subunit vaccine and a preparation method thereof. Particularly, a baculovirus vector expression system is utilized to express a large amount of recombinant open reading frame type 2 (ORF2) protein in insect cells, so that the subunit vaccine with good immunity effect is developed. A bac-to-bac baculovirus expression system is adopted to perform whole gene amplification on the porcine circovirus type 2 ORF2 gene, and a melittin signal peptide nucleotide sequence is introduced into a terminal 5', so that the recombinant baculovirus of an open reading frame containing the melittin signal peptide nucleotide sequence and a PVC2ORF2 gene sequence is established, wherein the infected insect cell expresses the recombinant ORF2 protein with efficient and high PCV2 antigenicity; and thus the subunit vaccine containing the PCV2 recombinant ORF2 protein is prepared. The inoculation experiments of piglets show that the subunit vaccine has a good immunity protection effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a porcine circovirus type 2 subunit vaccine and a preparation method thereof. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) is a representative species of the genus Circovirus. It is a small, non-encapsulated, icosahedral symmetry, covalently closed, circular, single-stranded DNA virus with an average particle diameter of It is 17nm, which is the smallest animal virus found in veterinary medicine. According to the pathogenicity, antigenicity and nucleotide sequence of PCV, PCV can be divided into two types: porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2). PCV1 is not pathogenic to pigs and is a non-pathogenic PCV. PCV2 is pathogenic to pigs and can cause postweaning multisystemic wasting syndrome (Postweaning multisystemic wasting syndrome, PMWS), also known as PMWS-related PCV. Hamel et al. analyzed the PCV genome and found that bo...

Claims

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Application Information

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