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Silkworm hemolymph with anti-helicobacter-pylori activity, preparation method of silkworm hemolymph and application of silkworm hemolymph

A technology of Helicobacter pylori and silkworm blood, applied in the field of silkworm hemolymph and its preparation, can solve the problems of bacterial drug resistance, limited radical cure, and poor treatment compliance

Inactive Publication Date: 2018-03-09
ANHUI SCI & TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the increase of antibiotic-resistant strains, re-infection, complications, high treatment costs and poor treatment compliance, the eradication of antibiotics to Helicobacter pylori infection is limited, and the main reason is bacterial resistance

Method used

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  • Silkworm hemolymph with anti-helicobacter-pylori activity, preparation method of silkworm hemolymph and application of silkworm hemolymph
  • Silkworm hemolymph with anti-helicobacter-pylori activity, preparation method of silkworm hemolymph and application of silkworm hemolymph
  • Silkworm hemolymph with anti-helicobacter-pylori activity, preparation method of silkworm hemolymph and application of silkworm hemolymph

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] This embodiment provides an antibacterial peptide with anti-H. pylori activity, the amino acid sequence of which is shown in SEQ ID NO:1.

[0124] The screening and experimental process of this antimicrobial peptide are as follows:

[0125] a. Screening of antimicrobial peptides: Antimicrobial peptides have their own unique antibacterial spectrum just like antibiotics. Not all antimicrobial peptides have antibacterial activity against Helicobacter pylori. There are 25 kinds of antibacterial peptides against Gram-negative bacteria, which are synthesized by chemical methods.

[0126] b. Antimicrobial peptide preparation: According to the minimum inhibitory concentration test method, the chemically synthesized antimicrobial peptides were diluted to 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL.

[0127] c. Detect the minimum inhibitory concentration: add the logarithmic Helicobacter pylori ATCC34504 standard bacterial strain in each test tube to re...

Embodiment 2

[0130] The present embodiment simulates the gastric environment to carry out antibacterial effect test to the antimicrobial peptide PGQ that embodiment 1 provides:

[0131] a. Preparation of artificial gastric juice (containing pepsin and gastric acidic substances, pH 1.3-1.8);

[0132] b. Culture different strains of Helicobacter pylori, including: ATCC34504 standard strain and clinical strains isolated from gastric ulcer and gastric cancer patients.

[0133] c. Add log phase Helicobacter pylori 10 to artificial gastric juice 6 CFU / mL, then add antimicrobial peptide PGQ to achieve a concentration of 4 μg / mL (4×MIC) and no antimicrobial peptide as the control group, take 10 μL of the mixed solution at 15 min, 30 min, 60 min and 120 min to dilute to 50 μL, and apply In solid medium, count the colonies on each plate after culturing for 36 h. Calculate the sterilization rate according to the formula:

[0134] Bactericidal rate=(1-the number of colonies on the bacteria liquid p...

Embodiment 3

[0137] The antibacterial peptide PGQ provided by Example 1 with anti-H. If it is directly expressed in a large amount in the host cell, it will have a great impact on the physiological pH of the host cell, and then affect the survival of the host cell, which is not conducive to the high-level expression of the antimicrobial peptide PGQ. In fact, the inventors found in experiments that if the antimicrobial peptide is directly expressed without fusion, the antimicrobial peptide cannot be expressed in the host cell at all.

[0138] In view of this, this embodiment provides a fusion protein for expressing the antimicrobial peptide PGQ, the fusion protein has the following primary amino acid sequence structure:

[0139] Carrier protein-connecting peptide-protease action site peptide-antibacterial peptide;

[0140] Wherein, the carrier protein includes Bombyx mori baculovirus nuclear polyhedrin, glutathione sulfhydryl transferase, green fluorescent protein, or ubiquitin-like small ...

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Abstract

The invention provides silkworm hemolymph with anti-helicobacter-pylori activity, a preparation method of the silkworm hemolymph and an application of the silkworm hemolymph, and belongs to the fieldof pharmacy. The method includes the step: co-expressing fusion protein and enterokinase by a bombyx mori baculovirus double-promoter expression system, wherein the fusion protein comprises antibacterial peptides as shown in SEQ ID NO:1 and specific protein acting sites of the enterokinase. The silkworm hemolymph simultaneously contains the fusion protein and the enterokinase, the antibacterial peptides with natural structures are acquired after the fusion protein is subjected to specific enzyme digestion by the enterokinase, so that the silkworm hemolymph has anti-helicobacter-pylori activity, can be directly taken orally or freeze-dried and accordingly can be used for treating helicobacter pylori infection, and the production cost of the antibacterial peptides is greatly reduced.

Description

technical field [0001] The invention relates to the field of pharmacy, in particular to silkworm hemolymph with anti-helicobacter pylori activity and its preparation method and application. Background technique [0002] Helicobacter pylori was discovered by two Australian scientists in the 1980s and won the Nobel Prize in Physiology in 2005. At present, more than 50% of the world's population is infected with this pathogen, and 20% of those infected will cause clinical symptoms of chronic gastritis, peptic and duodenal ulcers, gastric cancer, and mucosa-associated lymphoid tissue tumors. The World Health Organization has listed it as a class I carcinogen. It is more popular in developing countries, and the infection rate of the population in some areas of our country is as high as 60%. [0003] Antibiotics are an effective way to treat Helicobacter pylori infection, but Helicobacter pylori is resistant to multiple antibiotics, and even the same infected patient is resistan...

Claims

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Application Information

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IPC IPC(8): C12N15/866C07K19/00C07K14/00C12N5/10A61K35/64A61P1/04A61P31/04A61P35/00
CPCA61K35/63C07K14/00C07K2319/00C07K2319/50C12N5/0601C12N9/6424C12N15/86C12N2510/00C12Y304/21009
Inventor 张孝林汪建飞蒋安民贡成良范涛张钦元
Owner ANHUI SCI & TECH UNIV
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