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Porcine parvovirus VP2 protein

A parvovirus and protein technology, applied in the field of porcine parvovirus VP2 protein, can solve the problems of allergy, cumbersome antigen process, immunosuppressive side effects, etc., and achieve the effects of high antigen stability, convenient and accurate detection method, and strong specificity.

Inactive Publication Date: 2016-08-03
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some of these antigens can also cause allergies, immunosuppression and other side effects, which is the drawback of using the whole pathogen as a vaccine
Moreover, the production of inactivated vaccines currently used in China is cumbersome and the process of preparing antigens is cumbersome, and the attenuated vaccines also have the occurrence of vaccinations.

Method used

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  • Porcine parvovirus VP2 protein
  • Porcine parvovirus VP2 protein
  • Porcine parvovirus VP2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: Screening of porcine parvovirus (PPV) VP2 gene

[0016] In 2010, symptoms of porcine parvovirus disease appeared in many farms in Shandong Province, and the individual pigs with the disease had been injected with the existing parvovirus vaccine before, and it was speculated that the infected virus had mutated; Screening; the porcine parvovirus PPV1 was finally screened out.

[0017] In order to verify the antigenicity of the screened virus, five virus strains from different sources, including the screened PPV1 strain, were used as antigens to prepare vaccines. After immunizing SPF pigs, the screened virus liquid was used to challenge the virus. The results showed that Compared with other porcine parvovirus vaccines, the vaccine prepared by itself has a better immune effect (p<0.05), so it is confirmed that it has a genetic variation.

[0018] According to the antigenic characteristics and amino acid sequence of porcine parvovirus VP2 protein, a pair of pr...

Embodiment 2

[0020] Example 2: Preparation of recombinant porcine parvovirus VP2 protein

[0021] The method comprises the following steps: a. constructing an expression vector; b. constructing an expression strain; c. inducing, extracting and purifying the recombinant VP2 protein. The details are as follows: The positive cloned plasmid pMD18-T-VP2 and the expression vector pPICZα vector were digested with KpnI and NotI respectively, and after 1.2% agarose gel electrophoresis, they were recovered with a DNA gel recovery kit, and about 1.7 kb were obtained respectively. and 3.3kb fragments were oriented at 16°C to construct pPICZα-VP2 expression vector, after the correct identification by restriction enzyme digestion; after the plasmid was linearized, it was electrotransformed into Pichia pastoris competent cells to construct Pichia pastoris expression strain X33-VP2, The genome of the expressed strain X33-VP2 was extracted and identified correctly by PCR using primers primer1 and primer2. ...

Embodiment 3

[0022] Embodiment 3: the preparation of vaccine

[0023] The Pichia pastoris expression strain X33-VP2 strain producing porcine parvovirus VP2 protein was deposited in China Center for Type Culture Collection with the preservation number CCTCCM2016098.

[0024] 1. Preparation of bacterial solution for making seedlings Inoculate the X33-VP2 bacterial strain in YPD liquid medium containing bleomycin, and culture with shaking at 30°C for 16-18 hours. Then streak and inoculate in YPD solid medium with bleomycin, select 2 to 3 typical colonies and mix them in a small amount of YPD liquid medium, place them in a shaker at 30°C for 18 hours, and quantitatively pack them. After inspection, it is used as a first-class seed. Take the primary seeds and inoculate them in BMGY liquid medium, culture them with shaking at 30°C for 16-18 hours, and store them at 2-8°C after microscopic examination, as the secondary seeds.

[0025] 2. Preparation of protein for seedling production. Add BMGY ...

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Abstract

The invention aims at providing porcine parvovirus VP2 protein which is used for preparing a subunit vaccine so as to overcome deficiencies in the prior art .The amino acid sequence of the porcine parvovirus VP2 protein is SEQ ID NO:1 .The invention provides novel VP2 protein which lays a firm foundation for industrialized production of the porcine parvovirus subunit vaccine; the vaccine prepared through the protein is high in antigenic stability, high in purity, high in specificity and high in sensitivity, no other uncorrelated antibody is produced, an immune effect detection method is convenient and accurate, and production is very easy .Production is conducted without using animal bodies or embryoid bodies, tissue residues do not exist, and the safety is high.

Description

technical field [0001] The invention belongs to the technical field of vaccine antigen protein screening, in particular to a porcine parvovirus VP2 protein. Background technique [0002] Porcine parvovirus (Porcineparvovirus, PPV) is one of the important pathogens that cause reproductive disorders in sows, which can lead to abortion, premature birth, stillbirth, mummified fetuses, weak piglets, infertility of sows and mass death of newborn piglets. Since the 1980s in my country, PPV has been isolated from various places, and the survey found that the serological positive rate of the disease in some pig herds can reach more than 85%. PPV often presents as recessive infection, especially the phenomenon of low-dose persistent infection often occurs. In addition to causing reproductive disorders in sows, the virus can also cooperate with other pathogens to cause a variety of diseases, causing great economic losses to the pig industry. Porcine parvovirus has a single serotype a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/015C12N15/35A61K39/23A61P31/20
CPCC07K14/005A61K39/12A61K2039/5252A61K2039/54A61K2039/552C12N2750/14322
Inventor 刘新文孙健邹敏程增青李陆梅张志鸿徐保娟
Owner YEBIO BIOENG OF QINGDAO
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