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Vaccine for simultaneously preventing neospora and toxoplasma infection, preparation method and application thereof

A technology of Neospora and Toxoplasma gondii, which is applied in the field of parasitic disease vaccine genetic engineering, can solve the problem that the recombinant adenovirus vector vaccine for simultaneous prevention of Neospora and Toxoplasma gondii, the low protection rate, the inability to use neosporosis, etc. problem, to achieve the effect of good immune protection, high transgenic efficiency and high safety

Inactive Publication Date: 2019-01-29
YANBIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Most of the research on Neospora vaccines adopts traditional methods, such as using the surface antigen protein gene of Neospora to prepare recombinant protein subunit vaccines, or using eukaryotic expression vectors to prepare nucleic acid vaccines, etc., and the prepared subunit vaccines or nucleic acid vaccines exist The problems of poor immune effect and low protection rate cannot be used for the effective prevention and control of neosporosis. Although the research on Toxoplasma gondii vaccine has made some progress, the immune effect of the vaccine is not satisfactory.
Moreover, there is no simultaneous prevention of Neospora and Toxoplasma vaccine at home and abroad, and there is no relevant report on the simultaneous prevention of Neospora and Toxoplasma recombinant adenovirus vector vaccine

Method used

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  • Vaccine for simultaneously preventing neospora and toxoplasma infection, preparation method and application thereof
  • Vaccine for simultaneously preventing neospora and toxoplasma infection, preparation method and application thereof
  • Vaccine for simultaneously preventing neospora and toxoplasma infection, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

no. 1 example

[0050] In the vaccine of the present invention for simultaneously preventing the infection of Neospora and Toxoplasma, the amino acid sequence of the vaccine antigen is:

[0051] MSKIKASTELGKCAEFAYKTTAMDKSNQATQYRYPFVYDSKRRLCYILSVSMQRMEGSKYCSTNDDPVNLTWYCFEPQKSPTANHNLIFGSAYVGKDPDAFLTKCPNQALKGYRFGHWTNGRCHDYTELADSWIEAVDSKAQCWVKTFTNDEVASDKGHSCALGDQSHGDQPRGDSHGDHSGFDQPHWWNDKG

[0052] MSKIKASTDLGRCAEFAFKTVAMDKNNKATKYRYPFVYDSKKRLCHILYVSMQLMEGKKYCSVKGEPPDLTWYCFKPRKSVTENHHLIYGSAYVGENPDAFISKCPNQALRGYRFGVWKKGRCLDYTELTDTVIERVESKAQCWVKTFENDGVAQPHDTGLVDSGVDTGLVIDQVDTGLVIDQCWDTG

[0053] The nucleotide sequence encoding the above vaccine antigen is:

[0054] CCGCTCGAGGCCACCATGAGCAAAATCAAGGCGAGTACAGAGCTTGGGAAGTGCGCCGAATTCGCCTACAAGACGACTGCTATGGATAAAAGCAATCAGGCGACTCAGTACCGCTATCCATTTGTGTATGACTCTAAGCGGCGGCTGTGTTACATCCTTTCCGTATCGATGCAGCGAATGGAGGGCAGTAAGTACTGTTCTACCAACGATGACCCGGTTAATCTCACATGGTATTGCTTCGAGCCCCAAAAGAGTCCTACGGCGAATCATAATCTCATCTTCGGATCGGCCTACGTTGGAAAAGACCCAGATGCGTTCCTCACTAAATGCCCAAACCAAGCTCTTAAG...

no. 1 example

[0070] Animal immunity test of the recombinant adenovirus vector vaccine prepared in the first embodiment of the present invention

[0071] With BALB / c mice as experimental animals, 60 BALB / c mice were randomly divided into 3 groups, each with 20 mice, namely Ad5-Nc / Tg AMA1 vaccine group, pVAX1-Nc / TgAMA1 plasmid group and PBS control The group received intramuscular injection every 2 weeks for a total of 3 injections. Among them, pVAX1-Nc / TgAMA1 refers to the subcloning of Nc / TgAMA1 gene into pVAX1 eukaryotic expression vector. pVAX1-Nc / TgAMA1 was constructed and stored by the Laboratory of Preventive Veterinary Medicine of Yanbian University. The immune effect of the vaccine was compared. Ad5-Nc / TgAMA1 vaccine group one immunization 6×10 9 TCID 50 , 1.2×10 for the second and third immunity respectively 10 TCID 50 The pVAX1-Nc / TgAMA1 plasmid group was vaccinated with 0.1mg, 0.2mg for the second and third immunizations; PBS control group was vaccinated with 0.1mL for the first im...

no. 2 example

[0073] Neospora / Toxoplasma attack experiment

[0074] After three immunizations for 2 weeks, the 20 BABL / c mice in each group were divided into two groups, 10 in each group, and each in the abdominal cavity attacked the Neospora tachyzoites 10 6 Each of the two groups attacked the Toxoplasma RH strain tachyzoites 10 3 First, carry out the immune protection test of the vaccine, and observe the clinical symptoms and statistical survival rate of the mice. After 21 days of challenge experiment, BABL / c mice vaccinated with Ad5-Nc / TgAMA1 recombinant adenovirus vaccine had an 80% survival rate against Neospora tachyzoites, while the pVAX1-Nc / TgAMA1 plasmid group and PBS control group were respectively Survival rates are 50% and 20%; BABL / c mice vaccinated with Ad5-Nc / TgAMA1 recombinant adenovirus vaccine have 60% survival rates against Toxoplasma gondii, while the pVAX1-Nc / TgAMA1 plasmid group and PBS control group are respectively The survival rate is 20% and 0%.

[0075] The experiment...

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Abstract

The invention provides a vaccine capable of simultaneously preventing neospora caninum infection and toxoplasma infection, a nucleotide sequence used for encoding a vaccine atigen, a preparation method of the vaccine and application of the vaccine. The amino acid sequence of the vaccine atigen is shown as SEQ. ID. NO. 2 and SEQ. ID. NO. 4. The preparation method of the vaccine comprises the following specific steps of according to open reading frames of a neospora caninum AMAI (Apical Membrane Antigen) gene sequence and a toxoplasma AMAI gene sequence, designing a universal primer of neospora caninum and toxoplasma cross antigen AMAI genes, building pMD18-T-Nc / TgAMAI recombined cloning plasmid, recombinant adenovirus shuttle plasmid pCR259-Nc / TgAMAI and expression plasmid H294-Nc / TgAMAI, packaging Ad5-Nc / TgAMAI recombinant adenovirus by a liposome mediated transfection QBI-HEK293 cell, amplifying and purifying to prepare an Ad5-Nc / TgAMAI recombinant adenovirus vector vaccine.

Description

Technical field [0001] The present invention relates to the technical field of parasitic disease vaccine genetic engineering, and provides a vaccine for simultaneously preventing the infection of Neospora and Toxoplasma and its preparation method and application. Background technique [0002] Neosporidiosis and toxoplasmosis are the main parasitic diseases that seriously endanger the healthy development of aquaculture and human physical and mental health. There are no effective drugs and vaccines for prevention and control. [0003] Neosporosis is a kind of protozoal disease caused by the parasitization of Neospora caninum (Neospora caninum) in the cells of various mammals such as dogs, cattle, sheep, etc. The disease mainly causes miscarriage, stillbirth, or dyskinesia of newborns. And neurological diseases. Dogs are the final host of Neospora. Many mammals can be used as intermediate hosts. Its parasitic sites are the central nervous system, brain, liver, muscle and other intern...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/002A61K39/015A61K47/46C07K14/45C07K14/44C12N15/30C12N15/861A61P33/02
Inventor 贾立军刘明明张蕾李积旭张守发玄学南
Owner YANBIAN UNIV
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