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A preparation method of virus vaccine expressing Plasmodium ovale ama1 protein

A technology of Plasmodium ovale and vaccine, which is applied in the fields of virology and genetic engineering, can solve the problem of no effective vaccine for Plasmodium ovale infection, etc., and achieve the effects of strong humoral immunity and cellular immune response, low price and short production cycle

Active Publication Date: 2021-03-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Aiming at the above-mentioned problems existing in the prior art, the present invention provides a method for preparing recombinant vesicular stomatitis virus vaccine using a novel virus vector, which solves the problem that there is no effective vaccine for P. provide ideas

Method used

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  • A preparation method of virus vaccine expressing Plasmodium ovale ama1 protein
  • A preparation method of virus vaccine expressing Plasmodium ovale ama1 protein
  • A preparation method of virus vaccine expressing Plasmodium ovale ama1 protein

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Experimental program
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Embodiment 1

[0038] Embodiment 1: Preparation of the recombinant vesicular stomatitis virus vaccine of AMA1 protein:

[0039] (1) Construction of pXN2-AMA1 recombinant plasmid: gene synthesis of Plasmodium ovale AMA1 gene (PocGH01_09039800), and insertion of the target gene AMA1 into the G and L restriction enzyme cutting sites of the vesicular stomatitis virus genome through the Xho1 and Nhe1 restriction sites Between points, construct the recombinant VSV genome plasmid VSV-XN2-AMA1, namely pXN2-AMA1;

[0040] (2) Inoculate 5x10 cells in a 10cm cell culture dish 6 BHK-21 cells were incubated with DMEM containing 10% FBS at 37°C, 5% CO 2 cultured in a cell culture incubator;

[0041] (3) After 24 hours, when the cells grow to 80%-90% confluence, the BHK-21 cell culture medium is discarded, and 10 6 The T7 poxvirus of PFU was diluted with 3ml DMEM serum-free medium to infect BHK-21 cells to provide the T7 RNA polymerase required for VSV recombinant virus packaging;

[0042] (4) After 2 ...

Embodiment 2

[0050] Embodiment 2: rVSV-AMA1 immune response comparison

[0051] experiment one)

[0052] Mice immunization: 6-8 week old male BALB / C mice were divided into rVSV-AMA1, AMA1pro and VSV control groups, with 6 mice in each group.

[0053] The recombinant vesicular stomatitis virus rVSV-AMA1 expressing Plasmodium ovale AMA1 obtained in Example 1 of the present invention was treated at week 0 with 10 6 Male BALB / C mice were immunized by intraperitoneal injection of PFU for 6-8 weeks as the rVSV-AMA1 group.

[0054] Purified AMA1 protein was mixed with Freund's adjuvant in equal proportions and injected intraperitoneally with 50 μg. The primary immunization at week 0 and the booster immunization at the third week were used as the AMA1pro group.

[0055] Empty vector virus VSV at week 0 with 10 6 PFU was injected intraperitoneally as a negative control group.

[0056] Experiment (2)

[0057] Detection of specific humoral immune response in mice: After five weeks of immunizatio...

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Abstract

The invention discloses a preparation method of a virus vaccine expressing Plasmodium ovale AMA1 protein, belonging to the technical fields of virology and genetic engineering. In the present invention, the gene expressing the apical membrane protein-1 of Plasmodium ovale is inserted into the genome vector of vesicular stomatitis virus VSV, and the backbone plasmid XN2-AMA1 of the recombinant virus vector is constructed, and the poxvirus containing T7 RNA polymerase is used to infect suckling hamster kidney cells (BHK-21), the plasmid XN2-AMA1 and the structural protein particles pN, pP, pL were co-transfected into BHK-21 cells, the recombinant virus was rescued in the cells, and the virus-like particles expressing AMA1 were packaged to form recombinant viruses rVSV-AMA1 is the vaccine of the present invention. The vaccine of the invention has the advantages of simple operation, high production titer, short immunization cycle, strong humoral and cellular immune responses, and great potential for clinical application.

Description

technical field [0001] The invention relates to the technical fields of virology and genetic engineering, in particular to a preparation method of a recombinant vesicular stomatitis virus vaccine expressing Plasmodium ovale AMA1 protein. Background technique [0002] Malaria is an ancient human disease, and it is still one of the most important infectious diseases in the world. It is prevalent in more than 90 countries around the world, and more than 300 to 500 million people are infected with malaria. Tens of thousands of people are infected, and children are the main group of people who die from infection. Malaria is a disease transmitted by Plasmodium through mosquito bite infection. There are four kinds of Plasmodium parasites in humans, namely, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Among them, Plasmodium falciparum is the most serious type of infection, and Plasmodium ovale causes mild infection symptoms compared with other...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/47C12N15/86C12N5/10A61K39/015A61P33/06
CPCA61K39/015A61K2039/522A61K2039/57A61K2039/575A61P33/06C07K14/445C12N15/86C12N2760/20243Y02A50/30
Inventor 程洋董春升石晓丹玄英花
Owner JIANGNAN UNIV
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