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Bivalent DNA vaccine for Japanese blood fluke and preparation process thereof

A technology for DNA vaccines and schistosomiasis, applied in the field of nucleic acid vaccines against schistosomiasis, can solve the problems of increasing the unsafe factors of DNA vaccines and interfering with the immune regulation system

Inactive Publication Date: 2008-06-18
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, exogenous IL-12 has high homology with IL-12 produced in the human body, which will interfere with the body's own immune regulation system and increase the unsafe factors of DNA vaccines

Method used

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  • Bivalent DNA vaccine for Japanese blood fluke and preparation process thereof
  • Bivalent DNA vaccine for Japanese blood fluke and preparation process thereof
  • Bivalent DNA vaccine for Japanese blood fluke and preparation process thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The preparation of embodiment 1 monovalent DNA vaccine

[0041] 1. PCR primer design and synthesis

[0042] According to the Sj23 and SjFABP gene sequences, 2 pairs of primers were designed (primer length range: 15-30 nucleotides), and the 5′ end of each primer was respectively introduced with the following enzyme cutting sites (underlined), and the primers were provided by Shanghai Sangong Co. Synthesize instead:

[0043] Sj23(1) Upstream P1: CG G GAT CC A ATG GCG ACT TTG GGT ACT BamH I

[0044] Downstream P2: CGC GAA TTC TTA AAC ATT CTG ATA ATC GTG EcoR I

[0045] SjFABP(1) Upstream P1: CGT GGA TCC ATG TCT TCT TTC TTG BamH I

[0046] Downstream P2: GAC GAA TTC TGA CCA GTC TAT CAG T EcoR I

[0047] 2. Preparation of SjFABP and Sj23 target genes:

[0048] 1)PCR

[0049] The total reaction system is 50 μl, and the reaction system is as follows:

[0050]

[0051] For pVIVO2-Sj23, see "Chinese Journal of Medicine" 2005, 85: 193-197. Gan ...

Embodiment 2

[0095] The preparation of embodiment 2 bivalent DNA vaccine

[0096] 1. PCR primer design and synthesis

[0097] According to the Sj23 and SjFABP gene sequences, 2 pairs of primers were designed respectively, and the 5′ end of each primer was respectively introduced with the following enzyme cutting sites (underlined), and the primers were synthesized by Shanghai Sangong Company:

[0098] Sj23(2) Upstream P3: CG T CAT GA A ATG GCG ACT TTG GGT A BspH I

[0099] Downstream P4: CCG CCT AGG TTA AAC ATT CTG ATA ATC GTG AvR II

[0100] SjFABP(2) Upstream P3: GCA TCA TGA ATG TCT TCT TTC TTG G BspH I

[0101] Downstream P4: CGT CCT AGG TGA CCA GTC TAT CAG T AvRII

[0102] Primer length range: 10-35 nucleotides

[0103] 2. Preparation of SjFABP and Sj23 target genes:

[0104] 1)PCR

[0105] The total reaction system is 50 μl, and the reaction system is as follows:

[0106]

[0107] The cycle parameters of the PCR reaction are: denaturation at 95°C fo...

Embodiment 3

[0144] Embodiment 3: the effect evaluation experiment of the present invention

[0145] 1. Expression of plasmid DNA in vitro

[0146] 1). Preparation of plasmid DNA

[0147] Two plasmids, pVIVO2-mcs-SjFABP-Sj23 and pVIVO2-mcs were prepared with the plasmid mini-extraction kit from Omega Company, and the method was the same as before.

[0148] 2). Culture of HEK-293 cells before transfection

[0149] HEK-293 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO 2 . After the cell confluence reaches 95%, digest with 0.25% trypsin, centrifuge at 1500rpm for 5min, discard the supernatant, resuspend with a small amount of serum-free medium, and adjust the cell concentration to 1×10 6 / ml. Take 1ml of the cell suspension and add it to a cell culture dish (35mm in diameter), and set up a dish with a cover glass for each plasmid, and wait for the cells to grow on the plate. Cultivate for 24h.

[0150] 3). Transient transfection

[0151] Follow In...

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Abstract

The invention relates to a DNA vaccine of Japanese snail fever, wherein it comprises corn represent carrier and the target gene inserted into the multi colon points of corn represent carrier; the target gene is Sj23 and SjFABP; the corn represent carrier has two translate units; Sj23 and SjFABP are inserted into the multi colon point, to build said vaccine. The inventive vaccine has high safety and stable property, without refrigeration system.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a nucleic acid vaccine for zoonotic parasitic diseases, in particular to a nucleic acid vaccine for schistosomiasis. Background technique [0002] Schistosomiasis is a zoonotic parasitic disease that endangers human health and affects national economic development. According to WHO data in 1990, schistosomiasis is globally distributed. In 74 countries, about 600 million people are threatened, and 200 million people are affected to varying degrees. Infection, of which about 120 million are current patients and 20 million have serious complications. WHO / TDR listed schistosomiasis as one of the six major tropical diseases in the 1970s. my country is an endemic area of ​​schistosomiasis (schistosomiasis japonica), the most serious country among the 4 endemic countries (China, Philippines, Indonesia, Japan) of schistosomiasis, and also the 4 countries (Egypt, Sudan) with the most serious s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K39/00A61P33/12
CPCY02A50/30
Inventor 石佑恩胡媛
Owner HUAZHONG UNIV OF SCI & TECH