Preparation method of fusobacterium nucleatum neutrophile granulocyte activator protein

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method of fusobacterium nucleatum neutrophile granulocyte activator protein. The preparation method comprises the following steps of 1, culturing fusobacterium nucleatum, specifically, taking out a bacterium cryopreservation tube, placing the bacterium cryopreservation tube on ice, dissolving, inoculating an Fn strain on a blood plate, and culturing at 37 DEG C for 48-72 hours under extremely anaerobic conditions; step 2, extraction of bacterial DNA, specifically, scraping a proper amount ofFn bacterial colonies in a good growth state, placing the Fn bacterial colonies in a 1*PBS buffer solution, and extracting bacterial genome DNA; and step 3, NapA gene amplification, specifically, taking Fn bacteria DNA as a template, and carrying out the amplification according to the following condition that a reaction system is 50 [mu] L. The Fn NapA protein has a plurality of high-antigenicitypeptide fragments, has good antigenicity, has a small cross-immune reaction with human intestinal symbiotic bacteria, and has a good development prospect in Fn vaccine candidate antigens.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for preparing neutrophil activating protein from Fusobacterium nucleatum. Background technique [0002] Fusobacterium nucleatum (Fn), a common Gram-negative anaerobic bacterium, is one of the most abundant microorganisms in the oral cavity of patients with stomatitis diseases. Neutrophil-activating protein A (NapA) It was originally a soluble protein found in the water extract of H. pylori. Since this soluble protein can induce neutrophils to produce reactive oxygen species (ROI), it is named neutrophil activating protein. NapA exists in Hp and In most strains of Fn, it is a conserved and highly antigenic protein. In recent years, recombinant NapA is mainly used as a candidate vaccine against Hp infection. Most Hp infected patients contain high levels of NapA antibodies. Rossi et al. Using HP-NapA and the other two pathogenic factors of Hp, Cag and VacA, to immunize...

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Problems solved by technology

[0002] Fusobacterium nucleatum (Fn), a common Gram-negative anaerobic bacterium, is one of the most abundant microorganisms in the oral cavity of patients with stomatitis diseases. Neutrophil-activating protein A (NapA) It was originally a soluble protein found in the water extract of H. pylori. Since this soluble protein can induce neutrophils to produce reactive oxygen species (ROI), it is named neutrophil activating protein. NapA exists in Hp and In most strains of Fn, it is a conserved and highly antigenic protein. In recent years, recombinant NapA is mainly used as a candidate vaccine against Hp infection. Most Hp infected patients contain high levels of NapA antibodies. Rossi et al. Using HP-NapA and the other two pathogenic factors of Hp, Cag and VacA, to immunize Beagle dogs can reduce the colonization infection of Hp. Satin et al. found that immunizing mice with Hp-NapA can reduce the infection of Helicobacter pylori, and the protection rate is as high as 80%. Some studies have found that oral administration of Hp-NapA vaccine and immunomodulators can control Hp infection. Dong et al. found that combined oral administration of cholera toxin B (CTB) and NapA can inhibit allergic inflammation in mice. At present, there is still a lack of Fn-NapA gene as an antigen. application

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