Fusion protein as well as toxoplasma subunit vaccine and vaccine composition thereof

A fusion protein, Toxoplasma gondii technology, applied in the fields of immunology and biology, can solve the problems of reversion of virulence, time-consuming purification, high price, etc., and achieve the effect of high expression

Active Publication Date: 2020-05-12
JILIN UNIV FIRST HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whole insect vaccines include inactivated vaccines and live attenuated vaccines, but inactivated vaccines lack immune protection for mice, so they have no practical application value; Toxoplasma gondii tachyzoites are weakened after being treated with ultraviolet rays, radiation and chemical reagents. Can induce a strong immune response, such as Ts-4, T-263 and S48, but attenuated live vaccines have risks such as insufficient attenuation and virulence reversion, so attenuated vaccines cannot be widely used; subunit vaccines are derived from insects Specific components are extracted from body lysates or excretion-secretion antigens as vaccines, which have good immunogenicity, but purification is time-consuming, laborious, and expensive; genetic engineering subunit vaccines are toxoplasma antigen genes in high-efficiency expression vectors expression, resulting in a large amount of purified single antigen

Method used

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  • Fusion protein as well as toxoplasma subunit vaccine and vaccine composition thereof
  • Fusion protein as well as toxoplasma subunit vaccine and vaccine composition thereof
  • Fusion protein as well as toxoplasma subunit vaccine and vaccine composition thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Construction of the prokaryotic expression vector of Toxoplasma gondii recombinant protein

[0042] According to the open reading frame of Toxoplasma gondii P30 gene sequence and the physical map of prokaryotic expression vector pET28a, primers were designed and restriction sites BamH I and Hind III were introduced.

[0043] Upstream: GGATCCatgtcggtttcgctgcacca

[0044] Downstream: AAGCTTtggctcctttggaaag

[0045] In Escherichia coli, under normal circumstances, protein expression with a relatively large molecular weight generally exists in the form of inclusion bodies, and the codons of the natural sequence are not conducive to expression in Escherichia coli, and codon optimization is required to facilitate the preference of the Escherichia coli expression system sex. At the same time, in the research process of the present invention, we found that the main antigenic epitope of the P30 gene is mainly located in the first 480 nucleotides (that is, compare...

Embodiment 2

[0046] Example 2: Expression and purification of expressed protein

[0047] (1) Extraction and solubility verification of expressed protein

[0048] A single colony of E. coli BL21 (DE3) transformed with the recombinant plasmid pET-28a-P30 was used as the engineering strain of Toxoplasma gondii vaccine (BL21 (DE3)-pET-P30), and E. A single colony of coli BL21(DE3) was used as a blank control strain (BL21(DE3)-pET-28a). The above two strains were inoculated in 8 mL LB liquid medium respectively, and incubated overnight at 37°C with shaking. The next day, the seed solution was transferred to 400mL LB liquid medium for propagation at 37°C. When the OD600 of the bacterial solution was monitored to 0.6-0.7, the final concentration of IPTG was added to 0.1Mm / L, and the expression was induced at 18°C ​​for 16 hours. Collect the bacteria by centrifugation, suspend the bacteria with 10mL soluble protein lysate, add 100μL 0.1M PMSF, freeze and thaw 3 times (-80℃, 1h / 37℃, 10min), after ...

Embodiment 3

[0051] Embodiment 3: western blot identification

[0052] Using Toxoplasma gondii-positive serum as the primary antibody, horseradish peroxidase-labeled rabbit anti-dog IgG as the secondary antibody, and DAB as the substrate chromogenic reagent, the fusion of BL21(DE3)-pET-P30 engineered bacteria induced expression Proteins were identified by western blot. BL21(DE3)-pET-28a served as a negative control. as a result of Figure 4 , Figure 4 Middle: M: pre-stained with Marker III; 1, BL21(DE3)-pET-P30; 2, BL21(DE3)-pET-28a. .

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Abstract

The invention provides fusion protein and a nucleotide sequence thereof. The fusion protein comprises a toxoplasma P30 protein subunit and a T cell antigenic epitope. The invention further provides avaccine using the fusion protein to prevent abortion of pregnant sows due to toxoplasmosis and a vaccine composition thereof. The fusion protein has the beneficial effects of the characteristics of high expression quantity and high immunogenicity. The vaccine and the vaccine composition can effectively prevent abortion of the pregnant sows due to the toxoplasmosis and are high in vaccine stability.

Description

technical field [0001] The present invention relates to the fields of immunology and biology. Specifically, the present invention relates to a genetically engineered subunit vaccine of Toxoplasma gondii, a preparation method and its application, and mainly relates to a nucleotide sequence, a fusion vaccine for Toxoplasma gondii vaccine Proteins, vaccines and preparation methods and their applications. Background technique [0002] Toxoplasmosis, also known as toxoplasmosis or toxoplasmosis, is a worldwide distribution of zoonotic parasitic diseases caused by Toxoplasma gondii (Toxoplasma gondii). It is widely spread among livestock and wild animals, and seriously threatens the public health of humans and animals. Toxoplasma can infect more than 200 species of vertebrates. According to foreign reports, the average per capita infection rate is 25% to 50%. It is estimated that at least 500 million people in the world are infected with Toxoplasma gondii. According to statisti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/002A61P33/02
CPCA61K39/002A61K2039/53A61K2039/552A61P33/02C07K14/45C07K2319/40
Inventor 杨阳涂正坤安倍莹高修竹李海军郭魏莹李天阳李天祺
Owner JILIN UNIV FIRST HOSPITAL
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