Vaccine composition for improving immune protective rate of anti-Fasciola hepatica Cat L1 DNA, and preparation method thereof

A technology of DNA vaccine and Fasciola hepatica, which is applied in the field of vaccine combination and preparation to improve the immune protection rate of DNA vaccine against Fasciola hepatica, achieving high efficiency, easy operation and strong pertinence

Inactive Publication Date: 2013-07-17
SOUTHWEST UNIVERSITY
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Problems solved by technology

In the study of DNA vaccines, rats were immunized with Fasciola hepatica cathepsin L DNA vaccine and infected with metacercariae, although 70% of the immune protection could be obtained[32], but the immune route operation

Method used

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  • Vaccine composition for improving immune protective rate of anti-Fasciola hepatica Cat L1 DNA, and preparation method thereof
  • Vaccine composition for improving immune protective rate of anti-Fasciola hepatica Cat L1 DNA, and preparation method thereof
  • Vaccine composition for improving immune protective rate of anti-Fasciola hepatica Cat L1 DNA, and preparation method thereof

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preparation example Construction

[0043] 3. Preparation of Cat L1 polyclonal antibody

[0044] Five 6-7-week-old female BALB / c mice were injected intramuscularly with 40 μg / mouse of purified Cat L1 protein in the hind legs, and immunized for a total of 3 times, each time at an interval of 1 week, and blood was collected 1 week after the last immunization Separate the serum for later use. Antibody concentration was determined by Coomasie protein assay.

[0045] 4. Identification of target protein by Western blotting

[0046] The protein purified by the His-Tag fusion protein purification kit in method 2 was electrophoresed on SDS-PAGE (30V, 1h) and then transferred to a nylon membrane for Western blotting identification. Wash the membrane twice with 15ml 1×TBS, then add 5% non-fat milk powder TBS solution to block unbound protein sites. After washing twice with 1×TTBS, incubate the nylon membrane in polyclonal antibody for 1 hour, use the obtained Cat L1 polyclonal serum (diluted at 1:300), wash with 1×TTBS ...

specific Embodiment

[0061] The present invention is mainly divided into two major plates. One is to design and construct the required plasmids, and the other is to screen and evaluate vaccine combinations. The technology of the present invention will be described in detail below in conjunction with the accompanying drawings:

[0062] 1. Design and construct the required plasmid

[0063] First, in order to obtain the candidate vaccine gene FhCat L1, fresh worms obtained from bovine liver were used to extract mRNA (see QIAGEN mRNA kit for specific extraction methods). Then obtain the cDNA of FhCat L1 gene and the required ORF fragment with RT-PCR method, and connect it in the pMD-18 cloning vector, form pMD-Cat L1 plasmid, then carry out PCR sequencing identification (see figure 1 ), which is exactly in line with the expected results. IL4 and Flt3L were cloned directly from the peripheral blood of mice, and the method used was carried out according to the whole blood mRNA kit. Then, the two ge...

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Abstract

The invention relates to a vaccine composition for improving the immune protective rate of anti-Fasciola hepatica Cat L1 (FhCat L1) DNA. The vaccine composition is pVAX1-Cat L1+pIL4+pFlt3L. An FhCat L1 DNA plasmid is constructed and a host body Th2 immune response is utilized to well kill or inhibit worm infection, and Flt3L which can induce the maturity of dendritic cells (DCs) and increase the number and the frequency of the DCs and a cell factor IL4 which can induce the host immune response trending to Th2 are selected as a molecular adjuvant to improve the immunogenicity and the immune protective rate of FhCat L1 DNA after the immunization of BALB/c mice, so a new idea is provided for the research and development of an anti-Fasciola hepatica DNA vaccine.

Description

technical field [0001] The invention relates to a method for improving the protection rate of a DNA vaccine encoding Fasciola hepatica Cat L1 (FhCat L1) by improving Th2 immune response. Specifically, on the basis of constructing a DNA plasmid encoding Fasciola hepatica Cat L1 (FhCat L1), the Th2 immune response of the host body has a good killing or inhibiting effect on helminth infection, and the selection can induce dendritic cell (DC) maturation , Flt3L, which increases the number and frequency of DCs, and IL4, a cytokine that can induce host immune responses towards Th2, are used as molecular adjuvants of DNA vaccines to improve the immunogenicity and immune protection rate of FhCat L1 DNA vaccines after immunizing BALB / c mice, It provides a new idea for the research and development of DNA vaccine against Fasciola hepatica. Background technique [0002] Fasciola hepatica is a parasitic worm that can cause severe trematodesis in ruminants such as cattle and sheep as wel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/39A61K39/00A61P33/10C12N15/79C12N15/57
Inventor 罗洪林
Owner SOUTHWEST UNIVERSITY
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