Fusion protein, and canine toxoplasma subunit vaccine and vaccine composition thereof

A technology of fusion protein, Toxoplasma gondii, applied in the fields of immunology and biology

Inactive Publication Date: 2020-06-05
海木集团有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at current problems, the present invention provides a nucleotide sequence, carrier,...

Method used

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  • Fusion protein, and canine toxoplasma subunit vaccine and vaccine composition thereof
  • Fusion protein, and canine toxoplasma subunit vaccine and vaccine composition thereof
  • Fusion protein, and canine toxoplasma subunit vaccine and vaccine composition thereof

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: The construction of prokaryotic expression vector of Toxoplasma gondii recombinant protein, according to NCBI (http: / / www.ncbi.nlm.nih.gov) Toxoplasma gondii cyclophilin (cyclophilin, CyP) its accession number: KYF42711.1, altogether Contains 179 amino acids, using it as a reference, after analyzing with DNAstar software, remove the 2nd to 17th amino acids and the 173rd to 179th amino acids, and remove the random coil sequences at both ends that have little influence on the protein domain. It has little effect on the biological function of the protein and can also improve the stability of the protein. Replacing amino acids 116-125 with ENAGVRKAYM can effectively improve the soluble expression efficiency of the protein in Escherichia coli. The final amino acid sequence is Seq ID NO: 2, and a T cell is coupled behind the Toxoplasma gondii cyclophilin mutant Epitope, forming the fusion protein of toxoavidin mutant, its amino acid sequence is Seq ID NO:6. Acc...

Embodiment 2

[0040] Embodiment 2: Expression and purification of Toxoplasma gondii CyP fusion protein

[0041] (1) Soluble expression of Toxoplasma CyP fusion protein

[0042] The single colony of E.coli BL21 (DE3) transformed with the recombinant plasmid pET-28a-CyP was used as the engineering strain (BL21(DE3)-pET-28a-CyP) of the canine Toxoplasma gondii vaccine, and the pET-28a empty vector transformed The single colony of E.coli BL21(DE3) was the blank control strain (BL21(DE3)-pET-28a), and the above two strains were respectively inoculated in 50 μg / mL kanamycin containing LB liquid medium 100mL, cultured at 37°C, 180rpm for 6-8h, to make the bacteria OD 600 Between 0.6-1.0, add IPTG to make the final concentration 0.2mmol / L, continue to cultivate for 5h, collect the cells by centrifugation, suspend the cells with 10mL soluble protein lysate, add 100μL of 0.1M PMSF, freeze and thaw the cells repeatedly 2 After one time (-80°C, 1h / 37°C, 10min), 400w ultrasonic treatment for 30min, ce...

Embodiment 3

[0045] Embodiment 3: western blot identification

[0046] The fusion protein induced and expressed by BL21(DE3)-pET-CyP engineered bacteria was identified by western blot. SDS-PAGE electrophoresis. Membrane transfer: Cut out the PVDF membrane according to the size of the gel (slightly larger than the size of the gel), and immerse the gel together with the membrane transfer buffer to equilibrate for 10 minutes. According to filter paper / gel / PVFD membrane / filter paper, the gel side is placed on the negative side. Calculate the magnitude of the current based on the area of ​​the gel, turn on the power, and take out the PVDF membrane after 1 hour. Wash the PVDF membrane with TBST buffer, 5 minutes each time, shake slowly at room temperature, a total of three times. Transfer the PVDF membrane to blocking buffer and shake gently horizontally for 1 hour at room temperature. Add the PVDF membrane to the diluted Toxoplasma gondii antibody serum, shake slowly at room temperature for...

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Abstract

The invention provides a fusion protein and a nucleotide sequence thereof. The fusion protein comprises a toxoplasma cyclophilin protein subunit and a T cell epitope. The invention also provides a vaccine for preventing toxoplasmosis of dogs by using the fusion protein and a vaccine composition of the vaccine. The invention has the beneficial effects that the fusion protein has the characteristicsof high expression quantity and high immunogenicity; and the vaccine and the vaccine composition can effectively prevent canine toxoplasmosis and have high vaccine stability.

Description

technical field [0001] The present invention relates to the field of immunology and biology, specifically, the present invention relates to a kind of toxoplasma gondii genetic engineering subunit vaccine and preparation method and application thereof, mainly relate to a kind of nucleotide sequence for canine toxoplasma gondii vaccine, Fusion protein, vaccine and preparation method and application thereof. Background technique [0002] Toxoplasma gondii, also known as Toxoplasma gondii (T.gondii), belongs to the unicellular parasites of the kingdom Protozoa, the top door, the class Sporozoa, the order Eucoccidia, the family Sarcocystis, and the genus Toxoplasma. Nucleated cells that can infect almost all warm-blooded animals are strictly obligate intracellular parasites, and only the parasites that invade into the cells can proliferate. The discovery of Toxoplasma gondii has a history of more than 100 years. As early as 1900, scholar Laveran discovered a parasite suspected o...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/002A61P33/02
CPCA61K39/002A61P33/02C07K7/08C07K14/45C07K2319/00
Inventor 侯峰才学鹏曹利利刘业兵陈星远印春生赵耘李思明宫鹏涛丁鹤陈丽萍王典
Owner 海木集团有限公司
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