Method for separation, purification and immunogenicity detection of cysticercosis pisiformis cyst fluid protein

A technology for the separation and purification of cysticerci lentiformis, applied in the field of cystic fluid protein separation and immunization, can solve the problems of complex structure, many antigenic components, poor specificity, etc., and achieve the effect of high concentration, strong antigenicity and short time course

Inactive Publication Date: 2015-11-18
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This may be related to the large number of antigenic components, complex structu

Method used

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  • Method for separation, purification and immunogenicity detection of cysticercosis pisiformis cyst fluid protein
  • Method for separation, purification and immunogenicity detection of cysticercosis pisiformis cyst fluid protein
  • Method for separation, purification and immunogenicity detection of cysticercosis pisiformis cyst fluid protein

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Embodiment 1

[0024] A Method for Separating Cysticercus lentiform Cystic Fluid Proteins Using Ultrafiltration Tubes

[0025] S1. Take two 100kDa ultrafiltration tubes and two 50kDa ultrafiltration centrifuge tubes, wash them with ultrapure water, fill them with ultrapure water, put them in a refrigerator at 4°C, and pre-cool for 5-10 minutes;

[0026] S2. Take 1200 μL of whole cyst fluid protein and mix it with an equal volume of ultrapure water to obtain a 2400 μL sample;

[0027] S3. Turn on the centrifuge in advance to cool down to 4°C, take out the two pretreated 100kDa ultrafiltration centrifuge tubes, discard the ultrapure water in the tubes, add 1200 μL samples to each, and centrifuge for 30-35 minutes;

[0028] S4. After centrifugation, take out the ultrafiltration centrifuge tube, draw 500 μL of ultrapure water and pipette 20-30 times, collect the protein greater than 100 kDa in the inner tube of the ultrafiltration tube into a clean small centrifuge tube, store it at -80°C for la...

Embodiment 2

[0036] Detection of Antigenicity of Purified Cystic Fluid Protein by PVC-Dot-ELISA

[0037] The antigenicity of the purified protein in this specific implementation was screened by PVC-Dot-ELISA detection method. Purified proteins of different molecular weights separated by ultrafiltration centrifuge tubes were used as coating sources for coating. The primary antibody was antiserum prepared from mice immunized with whole cyst fluid proteins, and the secondary antibody was labeled with horseradish peroxidase (HRP) goat anti-mouse IgG.

[0038] The basic operation steps are:

[0039] S1. Coat a 96-well PVC plate at a concentration of 10 μg / mL (diluted with PBS), 50 μL per well, coat one row (12 wells) of each purified protein, overnight at 4°C, wash 3 times with PBST, each time 5 minutes apart ;

[0040] S2. Block with PBST blocking solution containing 5% skimmed milk powder, 200 μL per well, 37°C, block for 2 hours, wash with PBST 3 times, each interval is 5 minutes;

[004...

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Abstract

The invention discloses a method for separation, purification and immunogenicity detection of cysticercosis pisiformis cyst fluid protein. The method comprises steps of pretreatment of ultrafiltration centrifugation tubes, treatment of cyst fluid protein, centrifugal treatment, collection of high molecular weight target protein and small molecular weight target protein, antigenic detection of purified cyst fluid protein with different molecular weights and the like. The method is simple to operate, the time course is short, the efficiency is high, action of ultrafiltration membranes is more sensitive and accurate, the concentration of the obtained purified protein is higher and purer, the protein recovery amount is large, and the purified protein with high antigenicity can be obtained. The method is a more ideal new method for separating and purifying the cyst fluid protein and detecting the immunogenicity of the purified protein.

Description

technical field [0001] The invention relates to the field of cyst fluid protein separation and immunity, in particular to a method for separation, purification and immunogenicity detection of cysticercus lentiform cyst fluid protein. Background technique [0002] Cysticercosis pisiformis in rabbit is a common and frequently-occurring disease caused by cysticercosis pisiformis parasitizing in the liver, omentum, mesentery and abdominal cavity of rodents such as rabbits. The disease is distributed worldwide, and it is more serious in my country. According to reports, from Heilongjiang and Liaoning in the northeast, to Qinghai and Xinjiang in the northwest, from Sichuan, Chongqing and Guizhou in the southwest, to Fujian in the southeast and Guangxi in south China, to Henan in central China, Zhejiang and Shandong in east China, etc. Province, all have the generation of this disease, and sickness rate varies from 5% to 100%, and average infection rate reaches about 40%, and mort...

Claims

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Application Information

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IPC IPC(8): C07K1/34G01N33/68G01N33/543
Inventor 潘耀谦潘博刘兴友李瑞珍孙玉倩银梅唐海蓉张慧辉
Owner HENAN INST OF SCI & TECH
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