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Preparation method and application of anti-helicobacter pylori egg yolk antibody

A technology against Helicobacter pylori and Helicobacter pylori, applied in the biological field, can solve the problems of lack of gastric cancer and complex prevention and treatment of Helicobacter pylori, and achieve the effect of low cost and simple operation

Inactive Publication Date: 2018-11-13
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main cause of gastric cancer is considered to be Helicobacter pylori (Helicobacter pylori) infection, but there is a lack of effective methods or drugs for the treatment of gastric cancer; on the other hand, due to the continuous spread and enhancement of antibiotic resistance, the prevention and treatment of Helicobacter pylori has changed more complicated

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Preparation of Helicobacter pylori 5'-methylthioadenosine nuclease antigen

[0031] (1) The genetically engineered bacterium HpMTAN was inoculated in LB liquid medium containing Amp and chloramphenicol (final concentration: 100 µg / mL), cultured with shaking at 37°C overnight, and transferred at an inoculum volume of 1% by volume the next day, Cultivate at 37°C until OD600 is 0.6-0.8 (the temperature of fermentation culture is preferably 35-37°C for fermentation culture), then add IPTG with a final concentration of 0.1mmol / L at 16°C (the condition for IPTG-induced protein expression is preferably 25°C- 0.1~1.0mM IPTG induces protein expression under the condition of 30℃) Induce expression for 32 hours, ice-bath for 15min, and collect the bacteria by centrifugation; use buffer A (20 mM HEPES pH 7.5, 0.5M NaCl, 5 mM mM imidazole) to suspend and centrifuge the collected bacteria, and ultrasonically disrupt them under the following conditions: 200W, ultrasonic 3S, intermi...

Embodiment 2

[0036] Example 2 Bacterial enzyme-labeled staining of anti-Helicobacter pylori yolk antibody

[0037] Take Helicobacter pylori J99 (ATCC700824) for enzyme-labeled staining, smear and heat fix. The yolk antibody against Helicobacter pylori prepared in Example 1 was distributed into 3 concentrations (1:10, 1:100, 1:1000) (mg:mL) with pH 7.4, 0.01mol / L PBS and added dropwise After acting on the glass slide at 37°C for 30 minutes, it was fully washed. The secondary antibody (HRP enzyme-labeled rabbit anti-chicken IgY) (Promega Corporation) was added dropwise with DAB chromogenic solution and observed under a microscope, and the IgY of unimmunized chicken (purified from egg yolk before immunization) was used as a negative control. The results showed that the negative control was not stained, and the staining intensities of the three concentrations (1:10, 1:100, 1:1000) were +++, +++, + respectively, indicating that the yolk antibody against Helicobacter pylori can bind to pylori ...

Embodiment 3

[0038] Example 3 Titer determination of anti-Helicobacter pylori Helicobacter pylori egg yolk antibody

[0039] The egg yolk antibody against Helicobacter pylori prepared in Example 1 was diluted from 1:10 times to 1:51200 to detect its titer.

[0040] ELISA indirect enzyme-linked immunoassay: Dilute the recombinant HpMTAN protein prepared in Example 1 to 10 µg / mL with coating solution, coat each well of the ELISA plate with 100 µL, cover the 96-well plate with sealing film, and incubate at 37°C for 2 hours , then dry the liquid in the well, wash once with PBST, and pat dry with absorbent paper; add 100 µL 30 mg / mL skimmed milk powder to each well to seal, incubate at 37°C for 1 hour, then dry the liquid in the well, and wash three times with PBST (Leave for 4 minutes after each wash), pat dry with absorbent paper; add egg yolk antibody dilution (1:160-1:10240) after washing the plate with PBST, add 100 µL / well to the enzyme-labeled reaction plate, and add PBS was used as bla...

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PUM

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Abstract

The invention discloses application of helicobacter pylori 5'-methylthioadenosine nucleosidase to the preparation of an anti-helicobacter pylori egg yolk antibody. The specific anti-helicobacter pylori 5'-methylthioadenosine nucleosidase is prepared by using the egg yolk antibody helicobacter pylori 5'-methylthioadenosine nucleosidase as an antigen, a conversion passage for inhibiting the helicobacter pylori 5'-methylthioadenosine nucleosidase can be resisted specifically, and the growth of helicobacter pylori is blocked, so that the infection of helicobacter pylori and related diseases causedby the infection of helicobacter pylori are prevented and / or cured effectively. Moreover, the preparation of the anti-helicobacter pylori egg yolk antibody uses the technologies of genetic engineering and protein recombinant, the operation is simple, the cost is low, and industrial large-scale production is utilized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the prevention and treatment of Helicobacter pylori infection, more specifically to a preparation method and application of an anti-Helicobacter pylori yolk antibody. Background technique [0002] Helicobacter pylori (Helicobacter pylori) was discovered by scientists Barry Marshall and Robin Warren, which can infect stomach tissue and cause ulcers, and the two researchers also won the 2005 Nobel Prize in Physiology and medical awards. [0003] Helicobacter pylori, the causative agent of gastric ulcer and gastric cancer. It is estimated that half of the world's population is infected with Helicobacter pylori. Many infected people develop gastritis symptoms, and 1 in 5 infected people is very likely to develop gastric ulcers. In many cases, it is a rapidly progressive and fatal disease. Gastritis and stomach ulcers can be treated with antibiotics, but usually two or thre...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N9/24A23L33/10C07K16/02A61K39/40A61P31/04
CPCA61P31/04A23L33/10C12N9/2497C12Y302/02016C07K16/121A23V2002/00C07K2317/11
Inventor 赵肃清陈莉莉肖益热崔锡平贺攀王甜甜
Owner GUANGDONG UNIV OF TECH
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