Synthetic peptide vaccine for O-type foot and mouth disease of swine and preparation method thereof

A technique for synthesizing peptide vaccines and foot-and-mouth disease, applied in the field of genetic engineering, can solve problems such as high cost and complex production process, and achieve the effects of low production cost, simple separation and purification, and high expression efficiency

Inactive Publication Date: 2012-07-18
GENIFARM LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The object of the present invention is to provide a cheap, safe and efficient synthetic peptide vaccine for porcine O-ty...

Method used

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  • Synthetic peptide vaccine for O-type foot and mouth disease of swine and preparation method thereof
  • Synthetic peptide vaccine for O-type foot and mouth disease of swine and preparation method thereof
  • Synthetic peptide vaccine for O-type foot and mouth disease of swine and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] (1) Construction of recombinant expression vector: According to the partial tropism of the Pichia pastoris gene, the amino acid sequence of the synthetic peptide SP, and the multiple cloning site of the expression vector pGAPZαA, the SP gene was designed and synthesized, connected to the plasmid Puc57 vector, and double-enzyme Cut the Puc57 recombinant vector and the expression vector pGAPZαA, and then connect and transform to construct the recombinant expression vector ( figure 1 ). Another two primers were designed for detection, the primers are as follows: p1: SEQ ID NO: 4, p2: SEQ ID NO: 5, and the enzyme cutting sites are EcoRI and NotI.

[0026] (2) Construction of genetically engineered bacteria: Transform the host bacteria with the recombinant expression vector constructed above, and the host bacteria is Pichia pastoris X33.

[0027] (3) Expression of peptides synthesized by recombinant yeast: Use a sterilized toothpick to pick out a single colony with Zeocin ...

Embodiment 2

[0030] Preparation of pig O-type foot-and-mouth disease synthetic peptide vaccine: SDS-PAGE and Western-Blot analysis of the supernatant expressed in Figure 3~4 ), and then mixed penicillin and streptomycin with the separated and purified protein, and adjusted the pH value of the mixture to the physiological value, that is, pH7. agent.

Embodiment 3

[0032] Determination of expression level of swine type O foot-and-mouth disease synthetic peptides: Bradford protein concentration assay kit was used for the determination of SP expression supernatant protein concentration. Since there is a small amount of bacterial protein in the expression supernatant, the measured concentration is the total protein concentration. The specific steps are as follows:

[0033]1. Completely dissolve protein standard BSA (5 mg / mL), take 10 μL and dilute to 100 μL, so that the final concentration is 0.5 mg / mL. Standards were diluted with PBS.

[0034] 2. Add 0, 1, 2, 4, 8, 12, 16, 20 μL of the diluted standard to the standard wells of the 96-well plate, and add PBS diluent to make up to 20 μL.

[0035] 3. Add 10 μL of the supernatant of synthetic peptides expressed at 48h, 72h, and 96h to the sample wells of the 96-well plate, and add PBS diluent to 20 μL.

[0036] 4. Add 200 μL of G250 staining solution to each well and let stand at room tempera...

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Abstract

The invention discloses synthetic peptide vaccine for O-type foot and mouth disease of swine and the preparation method thereof. In the invention, synthetic peptide genes are expressed by selecting an eukaryotic expression system, then an expression product is emulsified into vaccine, since the application of genetic engineering technology, the synthetic peptide for O-type foot and mouth disease of swine can be efficiently and stably expressed, the production cost is low, the amplification is easy, and the toxic side effect and worries about biosafety can be avoided. Therefore, the synthetic peptide vaccine for the O-type foot and mouth disease of swine is suitable for large scale industrial production, and is classified as novel synthetic peptide vaccine for O-type foot and mouth disease of swine, is used for clinically preventing, controlling and curing foot and mouth disease.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a synthetic peptide vaccine for porcine O-type foot-and-mouth disease and a preparation method thereof. Background technique [0002] Foot-and-Mouth Disease (FMD) is an acute thermal and highly contagious infectious disease caused by Foot and mouth virus (FMDV) in pigs, cattle, sheep and other artiodactyls, which seriously endangers the productivity and The quality and safety of livestock products are listed as Class A important infectious diseases by OIE. At present, the developed countries mainly adopt the slaughter policy for the prevention and control of the disease, and the developing countries adopt the isolation and immunization policy, among which the immunization before the epidemic is an effective means to specifically protect livestock from infection. my country currently controls the prevalence of the disease by inoculating some new vaccines such as inactivated vac...

Claims

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Application Information

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IPC IPC(8): A61K39/135C12N15/42A61P31/14
Inventor 刘德辉何俊
Owner GENIFARM LAB INC
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