Application of Recombinant Eukaryotic Expression Plasmid Complex in Preparation of Equine Streptococcus Vaccine

A technique for expressing plasmids of streptococcus equine, which is applied in the field of biology and preparation of vaccines for preventing equine streptococcal disease, and can solve the safety hazards of attenuated vaccines, low immune effects of inactivated vaccines, and easy adverse reactions, etc. problem, to achieve the effects of good immune protection effect, good application and development and application prospects, and simple preparation method

Active Publication Date: 2018-04-27
XINJIANG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current use of Streptococcus adenoepides vaccines for equines is that the immune effect of inactivated vaccines is low and it is easy to cause adverse reactions, and the attenuated vaccines have potential safety hazards

Method used

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  • Application of Recombinant Eukaryotic Expression Plasmid Complex in Preparation of Equine Streptococcus Vaccine
  • Application of Recombinant Eukaryotic Expression Plasmid Complex in Preparation of Equine Streptococcus Vaccine
  • Application of Recombinant Eukaryotic Expression Plasmid Complex in Preparation of Equine Streptococcus Vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of recombinant eukaryotic expression plasmid complex

[0041] The recombinant eukaryotic expression plasmid complex is composed of recombinant eukaryotic expression plasmids PVAX1-S and PVAX1-E of S protein and E protein at a mass ratio of 1:1. The specific preparation method steps of the recombinant eukaryotic expression combination plasmid as follows:

[0042] (1) Amplify the S and E protein genes respectively, and use both genes Bam H I and xho Ⅰ endonuclease digestion and purification.

[0043] (2) The gene fragments after enzyme digestion and purification were connected to the Bam H I and xho In the eukaryotic expression vector after Ⅰ endonuclease digestion.

[0044] (3) The SeM gene fragments amplified by PCR were used respectively by the above steps xho I. Bam H Ⅰ After enzyme digestion, ligated into xho I. BamHI The eukaryotic expression vector PVAX-1 was ligated overnight at 16°C with T4 DNA ligase, and the ligated produ...

Embodiment 2

[0047] Example 2: Preparation of recombinant eukaryotic expression plasmid complex

[0048] The present invention provides in detail the specific preparation method steps of the above-mentioned recombinant eukaryotic expression plasmid complex as follows:

[0049] (1) Amplify the S protein gene and the E protein gene respectively, and use both genes Bam H I and xho Ⅰ Endonuclease digestion and purification, the amplified S protein gene and E protein gene are respectively shown in SEQ ID NO.1 and SEQ ID NO.2 in the attached gene sequence list.

[0050] (2) The gene fragments after enzyme digestion and purification were connected to the Bam H I and xho Ⅰ PVAX-1 in the eukaryotic expression vector after endonuclease digestion, the vector has the nucleotide sequence shown in SEQ ID No.3.

[0051] (3) The S gene fragments amplified by PCR were used respectively by the above steps xho Ⅰ. BamHI digested and then ligated into the warp, Bam H Ⅰ The eukaryotic expression vec...

Embodiment 3

[0055] Example 3: Construction of recombinant eukaryotic expression plasmid PVAX1-S

[0056] Acquisition of the gene encoding the S protein epitope of Streptococcus equi

[0057] Endemic strains of Streptococcus equine ( Streptococcus. equisubsp equi)XZ1 CGMCCNo.7830, the strain was isolated and obtained by the applicant in 2013, and was obtained by the "General Microbiology Center of China Microbiological Culture Collection Management Committee" on June 28, 2013, and its preservation number is CGMCC No. .7830. Pick a single colony of XZ1 strain on the sheep blood agar plate and inoculate it into Todd-Hewitt broth (THB) broth containing 5% calf serum (purchased from Hangzhou Sijiqing), culture it overnight at 37°C, and take 0.5mL of the bacterial liquid Centrifuge to extract chromosomal DNA, dissolve in pH 8.0 TE buffer, and freeze for future use.

[0058] The steps for extracting genomic DNA of Streptococcus equine XZ1 strain are as follows: respectively pick XZ1 colonies...

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Abstract

The invention discloses the application of the recombinant eukaryotic expression plasmid complex in the preparation of Streptococcus equine vaccine, by combining the recombinant eukaryotic plasmids PVAX1-S and PVAX1-E of S protein and E protein derived from Streptococcus equine with 1 The mass ratio of : 1 is combined to form a recombinant eukaryotic expression plasmid complex, which has good immunogenicity through immunization animal experiments, and the antibody level, cellular immunity level and immune protection effect of the recombinant plasmid complex after immunization Better than single antigen immunization alone. By selecting the recombinant eukaryotic expression plasmid complex of S protein and E protein of the prevailing strains in this area for immunization, it overcomes the disadvantage of poor immune protection effect produced by a single antigen, and has a good development and application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to the technical field of the application of a complex of recombinant eukaryotic expression plasmids derived from Streptococcus equi in the preparation of vaccines for preventing equine disease. Background technique [0002] Equine gland disease ( Stangles ) is an acute contagious disease of equine animals caused by Streptococcus adenipeptides, mainly in the neck and head of horses with purulent lymphangitis. The disease is commonly known as jet throat or groove knot, laryngeal bone swelling, and is an acute and febrile infectious disease of equine animals caused by Streptococcus equinides. It is characterized by acute, catarrhal, suppurative inflammation of the mucous membranes such as the nasopharynx and acute, suppurative inflammation of the subcollar lymph nodes. The disease has a high incidence in horse breeding areas and is distributed worldwide. It is pop...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/79C12N15/66A61K39/00A61P31/04
Inventor 苏艳苏玲玲张宝江邵俊高
Owner XINJIANG AGRI UNIV
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