schistosomiasis japonica arborization carrier-DNA vaccine and preparation method thereof

A DNA vaccine and schistosomiasis technology, applied in recombinant DNA technology, DNA / RNA fragments, pharmaceutical formulations, etc., can solve the problems of short retention and expression, difficulty in treatment and prevention, etc.

Inactive Publication Date: 2010-04-07
JIANGSU INST OF PARASITIC DISEASES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because its protection rate has not yet reached the level of protection higher than 40% stipulated by WHO, how to improve the protection of Schistosoma japonicum vaccine has become an urgent problem to be solved for current schistosomiasis vaccines, and it is also the research of DNA vaccines against Schistosomiasis today. hotspot
[0004] The delivery process of DNA into cells is a key factor affecting the level of DNA transfection, naked DNA is inefficient for eukaryotic cells at both in vitro and in vivo transfection levels, and DNA entering cells is vulnerable to intracellular nucleases Enzyme cleavage effects, so that the retention and expression time is very short, it is difficult to meet the needs of treatment and prevention

Method used

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  • schistosomiasis japonica arborization carrier-DNA vaccine and preparation method thereof
  • schistosomiasis japonica arborization carrier-DNA vaccine and preparation method thereof
  • schistosomiasis japonica arborization carrier-DNA vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Characteristic Analysis of Dendritic Vector-DNA Vaccine

[0048] 1. Electrophoretic retardation experiment: Determine the optimal charge ratio R for complete complexing of plasmid DNA and PAMAM-Lys dendrimer by agarose gel electrophoresis + / - . The results showed that PAMAM-Lys had the strongest ability to bind DNA when the charge ratio was 4.

[0049] 2. Morphological investigation of the dendrimer-DNA vaccine: the vaccine morphology was observed with a transmission electron microscope (TEM). Drop 10 μL of plasmid DNA and dendritic carrier-DNA vaccine solution on the copper grid respectively, and absorb the solvent for 2 minutes, and blot the solvent dry with filter paper; Blot dry with filter paper. After drying, place it under the electron microscope for observation. Results After the plasmid DNA was combined with the dendritic vector, the diameter of the pure plasmid became smaller, and the particle diameter of the dendritic vector-DNA vaccine tended ...

Embodiment 4

[0059] Example 4 Detection of cytokines in splenocyte culture supernatant

[0060] Three weeks after the last immunization, 2 mice in each group were killed by pulling the neck, and the spleen was aseptically taken out, and a single spleen cell suspension was prepared by conventional methods, and SjC23-large hydrophilic fragment protein (SjC23-HD) [Schistosome strain) 23kD molecule (SjC23) large hydrophilic peptide (HD) was expressed in PGEX-5X-1. Yu Chuanxin. Zhu Yinchang. Yin Xuren. He Wei. Hua Wanquan. Journal of Practical Parasitic Diseases 2000 (4)] as a stimulus to induce cytokines. Specific steps:

[0061] 1) Kill the mice by pulling their necks, soak in 75% alcohol for 3-5 minutes, take them out and place them in a plate, and take the spleen aseptically.

[0062] 2) The spleen was ground with tweezers, filtered through a 200-mesh stainless steel mesh, and suspended to 5 mL with incomplete RPMI-1640 (GIBCO) culture medium.

[0063] 3) Centrifuge at 1000rpm for 5min; ...

Embodiment 5

[0070] Example 5 Immunoprotective Test

[0071] Fifty experimental mice were randomly divided into 4 groups, namely pJW4303 (control group 1), PAMAM-Lys (control group 2), pJW4303-SjC23 and PAMAM-Lys / pJW4303-SjC23, with 12-14 mice in each group. The mice in each group were injected with pJW4303, PAMAM-Lys, pJW4303-SjC23 and PAMAM-Lys / pJW4303-SjC23 into the quadriceps femoris, 100 μg per mouse, 50 μg on the left and right sides. Booster immunization once every 2 weeks, with the same dose and method, immunization 3 times in total. Four weeks after the third immunization, each mouse was infected with (40±1) cercariae through the abdominal skin. Mice were dissected 42 days after challenge infection, flushed with citrate saline, and adult worms were collected from the portal vein and counted. The liver was taken out, weighed, cut into pieces, placed in 5 mL of 5% KOH solution, digested overnight at 37°C, and eggs were counted. Calculate the insect reduction rate and egg reduction...

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Abstract

A schistosomiasis japonica arborization carrier-DNA vaccine and a preparation method thereof relate to a zoonosis nucleic acid vaccine, in particular to a nucleic acid vaccine for schistosomiasis, and belong to the biotechnology field. The invention is formed by combining schistosomiasis DNA vaccine pJW4303-SjC23 and modified arborization carrier PAMAM-Lys into a compound of schistosomiasis japonica arborization carrier-DNA vaccine PAMAM-Lys/pJW4303-SjC23. The preparation method comprises the following steps: (1) constructing and preparing DNA vaccine pJW4303-SjC23, (2) preparing polyamide-amine arborization macromolecule, wherein quadrol performs modification on the terminal group of the core PAMAM, and (3) preparing the arborization carrier-DNA vaccine. The arborization carrier-DNA vaccine constructed by combining schistosomiasis japonica DNA vaccine pJW4303-SjC23 and PAMAM-Lys has the functions of obviously improving host immunoreactivity and immunoprotection, and has excellent biocompatibility with the host histiocyte. The vaccine has a very wide application prospect.

Description

technical field [0001] The invention discloses a schistosomiasis dendritic carrier-DNA vaccine and a preparation method thereof, which relate to a nucleic acid vaccine for zoonoses, in particular to a nucleic acid vaccine for schistosomiasis, and belong to the field of biotechnology. Background technique [0002] Schistosomiasis is a zoonotic parasitic disease that seriously endangers human health and affects social and economic development. Currently, schistosomiasis is prevalent in 76 countries and regions around the world, threatening the health of more than 700 million people, and it is estimated that about 200 million people are infected. my country is the most prevalent country of Schistosomiasis japonicum. After years of active control, the prevention and control of schistosomiasis in my country has achieved world-renowned achievements. However, because Schistosoma japonicum has more than 40 kinds of parasitic hosts, the intermediate host Oncomelania is widely distrib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K48/00A61K47/34A61P33/12C12N15/11
CPCY02A50/30
Inventor 王晓婷李新松朱荫昌郭玲香赵松高秋端
Owner JIANGSU INST OF PARASITIC DISEASES
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