Genetic engineering subunit mixed vaccine as well as preparation method and application thereof

A genetic engineering and mixed vaccine technology, applied in the field of veterinary vaccine research, can solve the problems of incomplete cleavage, strong virulence, and decreased immunogenicity of the vaccine, and achieve the effect of high expression product yield and good antigenicity.

Active Publication Date: 2016-06-15
SOUTH CHINA AGRI UNIV
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  • Abstract
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Problems solved by technology

As the virulence of the attenuated vaccine decreases, its immunogenicity also decreases, and sometimes the virulen

Method used

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  • Genetic engineering subunit mixed vaccine as well as preparation method and application thereof
  • Genetic engineering subunit mixed vaccine as well as preparation method and application thereof
  • Genetic engineering subunit mixed vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The recombinant protein preparation of embodiment 1, APPApxIA, ApxⅡA and OMPD

[0063] 1. Main material

[0064] 1.1 The strain APP is a product of the China Veterinary Drug Administration, and the number is ActinobacilluspleuropneumoniaeCVCC259.

[0065] 1.2 Plasmids, vectors and competent cells

[0066] Escherichia coli (E.coli) BL21(DE3) and Escherichia coli (E.coli) DH5α competent cells were purchased from Takara Company, and expression vectors PET-32a(+) and PGEX-4T-1 were purchased from Novagen Company.

[0067] 2. Method

[0068] 2.1 Use the PCR method to expand the sequences in APPApxⅠA, ApxⅡA and OMPD genes SEQIDNO:1, SEQIDNO:2, and SEQIDNO:3, and the amplified lengths are 753bp, 843bp, and 2382bp respectively. The results are as follows figure 1 shown. The upstream and downstream primers F1, F2, F3, F4, F5, and F6 used were the aforementioned primers, all of which contained BamHI and XhoI restriction sites. PCR reaction system: 2×KODFXBuffer 25 μL, 2mMdN...

Embodiment 2

[0116] Example 2 Analysis of Immune Efficacy of Porcine Infectious Actinobacillus Pleuropneumoniae ApxⅠA, ApxⅡA and OMPD Genetic Engineering Subunit Mixed Vaccine

[0117] 1. Main material

[0118] 1.1 Animals: 4-6 weeks old female Balb / c mice, purchased from Guangzhou Southern Medical University Experimental Animal Technology Co., Ltd.

[0119] 1.2 Vaccine: It is the purified protein obtained in Example 1 and a mixture of three kinds of proteins in equal mass. The protein is emulsified with Freund's complete adjuvant and Freund's incomplete adjuvant, both of which are purchased from SIGMA.

[0120] 2. Method

[0121] 2.1 Take 60 Balb / c mice aged about 4 to 6 weeks and divide them into 6 groups at random, rApxIA, rApxIIA, rOMPD and rApxIA+rApxIIA+rOMPD proteins are each in one group, and normal saline is used as the blank control group and pigs infected with The trivalent inactivated vaccine of acute pleuropneumonia (purchased from Wuhan Keqian Company) was used as a positiv...

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Abstract

The invention discloses a genetic engineering subunit mixed vaccine as well as a preparation method and an application thereof and belongs to the field of research of veterinary vaccines. Three recombinant proteins rApxIA, rApxIIA and rOMPD are expressed abundantly with a genetic engineering method and have better antigenicity. The rOMPD and rApxIA antibody levels of the mixed vaccine are higher than those during separate immunization of three proteins, the antibody level of rApxIIA and the antibody level of the mixed vaccine are remarkably higher than that of a control group, and the result proves that humoral immunity reaction induced by the recombined subunit mixed vaccine and rApxIA and rOMPD antibodies play an important role in cross immunization protection of mice. After inoculation, the genetic engineering subunit mixed vaccine is safe and harmless to an immune animal, is a novel vaccine with wide prospect, provides material reserve and technical support for control of porcine contagious pleuropneumonia in china and has great significance.

Description

technical field [0001] The invention belongs to the field of veterinary vaccine research, and relates to a porcine infectious actinobacillus pleuropneumoniae genetically engineered subunit mixed vaccine and its preparation method and application. Genetic engineering subunit mixed vaccine and its preparation method and application. Background technique [0002] Porcine contagious pleuropneumonia (PCP), caused by Actinobacillus pleuropneumoniae (APP), is a highly contagious disease characterized by pulmonary hemorrhage, necrosis and fibrinous exudation. There are 15 serotypes of APP serotypes, and the serotypes circulating in each country are also different. In my country, in 1987, the Harbin Veterinary Research Institute discovered clinical cases for the first time, and proved the existence and prevalence of PCP in my country by isolating the pathogen and conducting serotype and serological experiments on the isolated pathogen. In my country, serotypes 1, 3 and 7 are the mo...

Claims

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Application Information

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IPC IPC(8): A61K39/02A61P11/00A61P31/04
CPCA61K39/02A61K2039/70A61K2300/00
Inventor 陈金顶王利军赵明秋刘翠翠孙秀吴云燕朱俊灵吴祖雄
Owner SOUTH CHINA AGRI UNIV
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