CN103881941A - Separation and purification method of eperythrozoon suis - Google Patents

Abstract

The invention discloses a separation and purification method of eperythrozoon suis. The method includes: separating and purifying a pathogen, dissolving blood at 37 DEG C, performing chromatography by utilization of cane sugar, adding a PBS buffer solution into the collected purified substance, centrifuging at 4 DEG C at a rate of 10000 r/min for 2 h to remove the cane sugar, removing the liquid supernatant, suspending the precipitate with 2 mL of the PBS buffer solution, subpackaging, storing at -20 DEG C and reserving for further use. According to the method, the blood sample collected under a sterilized state is firstly washed by utilization of the PBS buffer solution so as to primarily separate and purify the blood sample and to eliminate interfering substances, thus laying a base for subsequent separation and purification. A physical method of heating with a water bath having a temperature of 56 DEG C to separate eperythrozoon from erythrocytes is adopted, the method is free of influences on activity of the eperythrozoon, and separation can be performed for a plurality of times, thus obtaining a larger amount of an antigen. After the eperythrozoon is separated from the erythrocytes, a cane sugar chromatography method is adopted to further purify the eperythrozoon, thus increasing the purity of the obtained antigen.

Description

The method of eperythrozoon suis separation and purification

 

Technical field

The invention belongs to biological technical field, be specifically related to a kind ofly make Eperythrozoon separation and purification method out from red corpuscle by physical method.

 

Background technology

Eperythrozoonosis (Eperythrozoonsis) is by Eperythrozoon (Eperythmzoon, EH) parasitize in people, animal erythrocyte surface, blood plasma and marrow a kind of zoonosis taking anaemia, jaundice, heating etc. as main clinic symptoms causing.

At present to the separation and purification of Eperythrozoon being adopted to chemical reagent or the medicine method of dissociating mostly in this sick biological study, but chemical reagent dissociates, after method, the reactivity of Eperythrozoon weakens greatly, and an Eperythrozoon scale of construction of purifying is also less, medicine dissociates rule because the difference of drug dose produces different impacts to red corpuscle, even cause erythrocyte hemolysis, cause antigen purity to reduce, thereby affect test-results.

 

Summary of the invention

The above-mentioned defect existing in order to overcome prior art field, the object of the invention is to, a kind of method of eperythrozoon suis separation and purification is provided, solving prior art adopts the reactivity of chemical-agent technique Eperythrozoon greatly to weaken, and an Eperythrozoon scale of construction of purifying is also less, medicine dissociates rule because the difference of drug dose produces different impacts to red corpuscle, even causes erythrocyte hemolysis, cause antigen purity to reduce, thereby affect the problem of test-results.

The method of eperythrozoon suis separation and purification provided by the invention, comprises the following steps:

One, the separation and purification of cause of disease, 10 parts of strict aseptic collection can hypochondriasis pig blood sample 1ml, 4 DEG C or freezing preservation, in the clinical blood sample gathering, add the PBS that 3 times of 0.01mol/L, pH are 7.4 to clean, the centrifugal 10min of 2000r/min, remove supernatant liquor, in precipitation, add the PBS dilution of 1 times, 56 DEG C of water-bath 3min, the immediately centrifugal 10min of 2000r/min, abandon precipitation, get supernatant in another centrifuge tube, the centrifugal 2h of 15000r/min, removes supernatant, in precipitation, add 1ml normal saline dilution, 4 DEG C or-20 DEG C save backup;

Two, to adding sterile purified water in 1:2 ratio in the precipitation that is rich in Eperythrozoon separating through 56 DEG C of water-baths, be placed in 37 DEG C of haemolysis 10min;

Three, the sucrose of sterilizing is mixed with to 2 gradients with PBS damping fluid, in centrifuge tube, add successively from top to bottom 55% and 20% sucrose solution, by through surpassing from crude extract 3ml be carefully added in above 20% sucrose liquid, 4 DEG C of centrifugal 2h of 12000r/min, finally with the Eperythrozoon band between the sucrose section of the careful absorption 55% and 20% of sterilizing syringe;

Four, in the purification thing of collecting, add PBS damping fluid, 4 DEG C of centrifugal 2h of 10000r/min remove sucrose, abandon supernatant, and precipitation suspends with the PBS damping fluid of 2ml, and after packing ,-20 DEG C save backup.

The method of eperythrozoon suis separation and purification provided by the invention, its beneficial effect is, the present invention in aseptic collection after blood sample, first rinse with PBS damping fluid, initial gross separation purifying blood sample, got rid of interfering substance, for follow-up separation and purification lays the foundation; Adopt 56 DEG C of heating in water bath that Eperythrozoon is departed from erythrocytic physics method, neither affected the activity of Eperythrozoon, simultaneously also can separating for several times, thus can obtain more antigen amount; Depart from after red corpuscle at Eperythrozoon, adopted again the method for sucrose chromatography to be further purified Eperythrozoon, the purity of the antigen obtaining is increased.

 

Embodiment

Below in conjunction with an embodiment, the method for eperythrozoon suis separation and purification provided by the invention is described in detail.

 

Embodiment

The method of the eperythrozoon suis separation and purification of the present embodiment, comprises the following steps:

One, the separation and purification of cause of disease, 10 parts of strict aseptic collection can hypochondriasis pig blood sample 1ml, 4 DEG C or freezing preservation, in the clinical blood sample gathering, add the PBS that 3 times of 0.01mol/L, pH are 7.4 to clean, the centrifugal 10min of 2000r/min, remove supernatant liquor, in precipitation, add the PBS dilution of 1 times, 56 DEG C of water-bath 3min, the immediately centrifugal 10min of 2000r/min, abandon precipitation, get supernatant in another centrifuge tube, the centrifugal 2h of 15000r/min, removes supernatant, in precipitation, add 1ml normal saline dilution, 4 DEG C or-20 DEG C save backup;

Two, to adding sterile purified water in 1:2 ratio in the precipitation that is rich in Eperythrozoon separating through 56 DEG C of water-baths, be placed in 37 DEG C of haemolysis 10min;

Three, the sucrose of sterilizing is mixed with to 2 gradients with PBS damping fluid, in centrifuge tube, add successively from top to bottom 55% and 20% sucrose solution, by through surpassing from crude extract 3ml be carefully added in above 20% sucrose liquid, 4 DEG C of centrifugal 2h of 12000r/min, finally with the Eperythrozoon band between the sucrose section of the careful absorption 55% and 20% of sterilizing syringe;

Four, in the purification thing of collecting, add PBS damping fluid, 4 DEG C of centrifugal 2h of 10000r/min remove sucrose, abandon supernatant, and precipitation suspends with the PBS damping fluid of 2ml, and after packing ,-20 DEG C save backup.

Claims (1)

1. a method for eperythrozoon suis separation and purification, is characterized in that: comprise the following steps:

One, the separation and purification of cause of disease, 10 parts of strict aseptic collection can hypochondriasis pig blood sample 1ml, 4 DEG C or freezing preservation, in the clinical blood sample gathering, add the PBS that 3 times of 0.01mol/L, pH are 7.4 to clean, the centrifugal 10min of 2000r/min, remove supernatant liquor, in precipitation, add the PBS dilution of 1 times, 56 DEG C of water-bath 3min, the immediately centrifugal 10min of 2000r/min, abandon precipitation, get supernatant in another centrifuge tube, the centrifugal 2h of 15000r/min, removes supernatant, in precipitation, add 1ml normal saline dilution, 4 DEG C or-20 DEG C save backup;

Two, to adding sterile purified water in 1:2 ratio in the precipitation that is rich in Eperythrozoon separating through 56 DEG C of water-baths, be placed in 37 DEG C of haemolysis 10min;

Three, the sucrose of sterilizing is mixed with to 2 gradients with PBS damping fluid, in centrifuge tube, add successively from top to bottom 55% and 20% sucrose solution, by through surpassing from crude extract 3ml be carefully added in above 20% sucrose liquid, 4 DEG C of centrifugal 2h of 12000r/min, finally with the Eperythrozoon band between the sucrose section of the careful absorption 55% and 20% of sterilizing syringe;

Four, in the purification thing of collecting, add PBS damping fluid, 4 DEG C of centrifugal 2h of 10000r/min remove sucrose, abandon supernatant, and precipitation suspends with the PBS damping fluid of 2ml, and after packing ,-20 DEG C save backup.