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Haemonchus contortus recombinant ARF1 protein nanometer subunit vaccine and application thereof

A technology of Haemonchus contortus and subunit vaccine, which is applied in recombinant DNA technology, vaccines, applications, etc., can solve the problems of difficulty in vaccine research and development, achieve strong immune protection ability, reduce the reduction rate of adult worms, and induce apoptosis. Effect

Active Publication Date: 2020-08-14
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the complex reaction mechanism between the parasite and the host also makes the development of vaccines difficult.
However, there is no relevant report on the preparation of vaccines using ARF1 as the antigen

Method used

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  • Haemonchus contortus recombinant ARF1 protein nanometer subunit vaccine and application thereof
  • Haemonchus contortus recombinant ARF1 protein nanometer subunit vaccine and application thereof
  • Haemonchus contortus recombinant ARF1 protein nanometer subunit vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Preparation of recombinant ARF1 protein nanomaterial subunit vaccine of Haemonchus contortus

[0047] 1. Extraction of total RNA from Haemonchus contortus

[0048] According to the method of Inviotrgen TRIzol instructions, the extraction of total RNA of Haemonchus contortus is carried out, and the specific steps are as follows: (1) add 1 mL TRIzol reagent to the centrifuge tube containing adult worms of Haemonchus contortus, transfer to 0.1% DEPC soaked In the homogenizer, grind thoroughly on ice, transfer to a new centrifuge tube and let stand at room temperature for 5 minutes;

[0049] (2) Add 200 μL of chloroform to the centrifuge tube, shake the tube vigorously by hand for 15 seconds, and let it stand at room temperature for 3 minutes;

[0050] (3) Centrifuge at 12,000 rpm for 15 minutes at 4°C, and carefully transfer the upper aqueous phase to a new centrifuge tube;

[0051] (4) Add 500 μL of isopropanol to the centrifuge tube, mix it upside down, and ...

Embodiment 2

[0128] Example 2. Detection of the immune effect of a recombinant protein ARF1 from Haemonchus contortus

[0129] 1. Experimental Design

[0130] ICR mice were randomly assigned to 3 groups of 8 and vaccinated only once on day 0. Mice were sacrificed on day 14. The three groups were PBS group, rARF1 group and rARF1-PLGA group. Multiple subcutaneous (SC) injections were performed using 1 mL of vaccine (20 μg rARF1 protein content). .

[0131] 2. Observation of immune protection effect

[0132] 2.1 Detection of antibodies in serum

[0133] (1) The mouse antiserum was collected on the 14th day and sacrificed. Antibody testing by ELISA. The levels of IgG1, IgM and IgG2a in serum samples were determined with a mouse ELISA kit according to the manufacturer's instructions (HengYuan, Shanghai, China).

[0134] (2) Briefly, the wells of a 96-well microtiter plate were coated with purified antibodies (anti-mouse IgG1, IgG2a or IgM), and mouse serum samples diluted in PBS were ad...

Embodiment 3

[0146] Example 3. Detection of immune protection of a recombinant protein ARF1 nanomaterial subunit vaccine of Haemonchus contortus

[0147] 1. Experimental Design

[0148] Divide 15 goats (3-6 months old) into 3 groups, 5 goats in each group, respectively the rARF1 group of Haemonchus contortus nanomaterial subunit vaccine (both immunization and challenge), positive control group (only challenge not immunized), negative control group (not challenged nor immunized).

[0149] The experimental group was first immunized and boosted with 500 μg of Haemonchus contortus nanomaterial subunit vaccine dissolved in 1 mL of PBS on the 0th day and the 14th day, respectively. On day 28, goats in the experimental group and the positive control group were infected with 8000 third-stage larvae of Haemonchus contortus. Goats were slaughtered on day 63. After attacking the worms, closely observe the sheep's feeding situation, coat and mental state.

[0150] 2. Observation of immune protecti...

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Abstract

The invention discloses a haemonchus contortus recombinant ARF1 (ADP-ribosylation factor) protein nanometer subunit vaccine and application thereof. The haemonchus contortus nanometer material subunitvaccine is formed in a way that the recombinant ARF1 protein is coated with a PLGA (poly (lactic-co-glycolic acid)) nanometer material. The experiment utilizes the rARF1-PLGA nanometer subunit vaccine constructed by the ARF1 protein to act in the body of a mouse to induce the immune response effect of the mouse, and an experiment in the body of a goat proves that the rARF1-PLGA nanometer subunitvaccine has an immunoprotection function for an organism. The recombinant ARF1 protein is coated by the PLGA nanometer material to form the nanometer subunit vaccine so as to provide a reference for the preventive utilization of the haemonchus contortus infection of the goat.

Description

technical field [0001] The invention relates to the technical field of biological veterinary medicine, and relates to a recombinant ARF1 protein nano-subunit vaccine of Haemonchus contortus and its application. Background technique [0002] Haemonchus contortus mainly parasitizes the abomasum of ruminants such as camels, cattle, sheep, and deer, and feeds on the blood of the host. The survey found that the annual cost of controlling Haemonchus contortus has reached several million dollars in some European countries. In the current market, chemical drugs are mainly used to prevent and control the infection of Haemonchus contortus, but due to people's over-reliance on chemical drugs, drug-resistant strains of Haemonchus contortus appear, making the effect of drugs not ideal. In addition, due to widespread concerns about drug residues and some biosafety factors, it is difficult for some anti-nematode drugs to be widely used in the prevention and control of Haemonchus contortus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K9/51A61K47/34A61P33/10C12N15/70C12N15/55C12R1/19
CPCA61K39/00A61K9/5146A61P33/10C12N15/70C12N9/14C12Y306/05A61K2039/552
Inventor 李祥瑞徐立新宋小凯严若峰穆罕默德·瓦卡斯·哈桑
Owner NANJING AGRICULTURAL UNIVERSITY
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