Preparation method of viral vaccine expressing plasmodium ovale AMA1 protein

A technology of Plasmodium ovale, XN2-AMA1, which is applied in the fields of virology and genetic engineering, can solve the problem of no effective vaccine for Plasmodium ovale infection, etc., achieves strong humoral immunity and cellular immune response, simple preparation and low price.

CN109022456AActive Publication Date: 2018-12-18JIANGNAN UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2018-12-18

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Abstract

Belonging to the technical field of virology and genetic engineering, the invention discloses a preparation method of a viral vaccine expressing plasmodium ovale AMA1 protein. According to the invention, a gene expressing plasmodium ovale apical membrane protein-1 is inserted into a vesicular stomatitis virus VSV genome vector to construct the recombinant virus vector skeleton plasmid XN2-AMA1, apoxvirus containing T7RNA polymerase is employed to infect the baby hamster kidney cell (BHK-21), then the plasmid XN2-AMA1 and the structural protein plasmids pN, pP, pL are utilized for cotransfection of BHK-21 cells to realize rescue of recombinant virus in cells, virus-like particles expressing AMA1 are packaged, and the recombinant virus rVSV-AMA1, i.e. the vaccine can be formed. The vaccineprovided by the invention has the advantages of simple operation, high production titer and short immune cycle, can induce strong humoral and cellular immune response, and has the great potential of clinical application.
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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...

Examples

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...