Method for extracting and purifying eimeria tenella merozoite

A technology for Eimeria merozoites and merozoites, applied in the field of extraction and purification of Eimeria tenella merozoites, can solve the problems of numerous test steps, high condition requirements, low recovery rate, etc., and achieve good purification effect , simple operation and high survival rate

Active Publication Date: 2012-11-28
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have disadvantages such as inability to remove impurities such as red blood cells, low recovery rate, difficult control of enzyme digestion time or complicated operation, poor purification effect or expensive instruments and reagents.
[0003] At present, some researchers have improved the chromatography method. For example, Anjian improved the DEAE-cellulose 52 chromatography column method in the literature of "Isolation and Purification of Eimeria tenella merozoites" to improve the The eluent is purified to obtain second-generation merozoites with higher purity, but it has higher requirements on the conditions of the eluent, such as the pH value and ion concentration of the eluent, and is also affected by external conditions such as elution. Influenced by hydrostatic pressure, temperature, and tightness of the chromatography column, there are many test steps at the same time
The average number of merozoites obtained per chicken in the experiment was 6.17×10 5 One, can not meet the test requirements of drug susceptibility determination, and DEAE-cellulose 52 chromatography column is more expensive, the cost is higher
Liu Liheng and others combined enzyme digestion, centrifugal washing, erythrocyte lysis and Percoll density gradient centrifugation in the literature "Isolation and Purification of the Second Generation Merozoite of Eimeria tenella" to obtain higher purity and recovery merozoites (7.25 x 10 6 Individuals / only), the number is still not enough for drug susceptibility testing

Method used

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  • Method for extracting and purifying eimeria tenella merozoite

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Experimental program
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Effect test

Embodiment 1

[0024] The method for extracting and purifying Eimeria tenella merozoites, the steps are as follows

[0025] (1) Release of merozoites

[0026] a) Mechanical grinding release: mechanically scrape the isolated chicken cecum mucosa infected with sporulated oocysts and develop to the merozoite stage, and then fully grind the mucosal tissue to free the merozoites; take some tissue fluid microscopic examination of the figure 1 and figure 2 shown, ( figure 1 and figure 2 The tissue liquid was dropped on a glass slide for microscope observation and photographing), and it can be observed from the picture that merozoites are mixed with a large number of impurities such as intestinal epithelial cells, fecal residues, and red blood cells.

[0027] b) Enzyme digestion and release: add the mucosal tissue released by mechanical grinding in step a) into PBS solution with a pH value of 7.2 at a volume ratio of 1:1, grind and mix well, and then coarsely filter through a 80-100 mesh sieve...

Embodiment 2

[0034] The method for extracting and purifying Eimeria tenella merozoites, the steps are as follows

[0035] (1) Release of merozoites

[0036] c) Mechanical release by grinding: mechanically scrape the isolated chicken cecum mucosa infected with sporulated eggs and develop to the merozoite stage, and then fully grind the mucosal tissue to free the schizonts.

[0037] d) Enzyme digestion and release: add the mucosal tissue released by mechanical grinding in step a) into PBS solution with a pH value of 7.2 at a volume ratio of 1:1, grind and mix well, and then coarsely filter through a 80-100 mesh sieve. Take out 10ml of each filtrate and add to the digestive solution in Table 1, shake well, place on a shaker at 37°C for digestion, and perform a microscopic examination every 10 minutes. The microscopic examination conditions are shown in Table 1.

[0038] Table 1

[0039]

[0040]

[0041] It can be seen that the digestion treatment with 0.625‰ trypsin and 1.75‰ sodium ta...

Embodiment 3

[0043] The method for extracting and purifying Eimeria tenella merozoites, the steps are as follows

[0044] (1) Release of merozoites

[0045] a) Mechanical grinding release: mechanically scrape the isolated chicken cecum mucosa infected with sporulated oocysts and develop to the merozoite stage, then fully grind the mucosal tissue to free the schizonts, and take some tissue fluid Microscopic examination revealed that the merozoites were mixed with a large number of impurities such as intestinal epithelial cells, fecal debris, and red blood cells.

[0046] b) Enzyme digestion and release: the mucosal tissue released by mechanical grinding in step a) is added to the PBS solution with a pH value of 7.4 at a volume ratio of 1:2, ground and mixed, and then coarsely filtered with an 80-100 mesh sieve. Take out 10ml of filtrate and add 15ml of 0.625‰pancreatin and 1.75‰sodium taurodeoxycholate mixture, shake well, put it on a shaker at 37~38℃ for digestion, and check under microsc...

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Abstract

The invention discloses a method for extracting and purifying eimeria tenella merozoite. The method comprises the steps of: releasing the merozoite and purifying the merozoite, wherein the merozoite release comprises mechanical grinding release and enzymic digestion release, 0.0625% pancreatin and 0.175% sodium taurodeoxylate are adopted as digestive juice in the enzymic digestion release process; digestion is moderate, which is beneficial to the activation of the merozoite, can eliminate erythrocytes to obtain pure merozoite. The method is convenient and simple to operate, is low in cost, and is ideal in separation and purification effects; the separated merozoites are large in quantity, are pure with few impurities and keep vigorous energy; and the method is suitable for research on drug susceptibility detection, vaccine research and other biological researches.

Description

technical field [0001] The invention belongs to biological extraction and purification technology, in particular to a method for extraction and purification of Eimeria tenella merozoites. Background technique [0002] Chicken coccidiosis is mainly a kind of soil-borne intestinal parasitic protozoan disease caused by a variety of coccidia of the genus Eimeria. The disease is transmitted through coccidiosis oocysts, and chickens are infected by swallowing a large number of sporulated oocysts. The disease is widely distributed and occurs widely. Among various poultry diseases of chickens, the incidence of coccidiosis is the highest, accounting for 1 / 6~ 1 / 5, often showing a fulminant epidemic, with the highest incidence rate in 15-50-day-old chicks, and the mortality rate can be as high as 80%. Because chicken coccidiosis is widely distributed and harmful, which seriously threatens the development of chicken industry, many experts and scholars are engaged in research on molecul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/10C12R1/90
Inventor 谢永平彭昊马春霞李军杨威陈泽祥潘艳许力干禤雄标胡帅谢宇舟
Owner GUANGXI VETERINARY RES INST
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