Purification method of Eimeria stigganii sporozoite

Abstract

The invention discloses a purification method of Eimeria stigganii sporozoites, which comprises the following steps: carrying out dissection detection to obtain lesion rabbit livers, and obtaining Eimeria stigganii oocysts; carrying out sporulation treatment to obtain a potassium dichromate sporulation oocyst suspension; adding sodium hypochlorite into the precipitate, and blowing and beating on ice; taking upper-layer floating foam and liquid after centrifugation, adding clear water, and centrifuging to obtain clean sporulated oocysts; adding glass beads for grinding and carrying out oscillation treatment; carrying out primary centrifugation, absorbing supernatant liquid, adding 50% percol liquid, and carrying out secondary centrifugation; adding a decapsulation buffer solution into the precipitate, and carrying out water bath digestion treatment for a certain time; and carrying out primary centrifugation on the digested suspension, taking the precipitate, adding a 55% percol solution, carrying out secondary centrifugation, taking the precipitate, diluting, fixing the volume, and naturally filtering to obtain the clean Eimeria stigmas sporozoites. The method has the remarkable effects of simplicity in operation, low cost, high sporozoite yield and time saving in the purification process.

Description

Technical Field

[0001] The invention relates to the technical field of biological extraction and purification, and in particular to a method for purifying sporozoites of Eimeria skrjabini. Background Art

[0002] Rabbit coccidiosis has brought great harm to the rabbit industry and seriously hindered the development of the rabbit industry. my country has classified it as a Class II animal epidemic disease, and together with rabbit plague, rabbit pasteurellosis, and rabbit scabies, it is called the four major rabbit diseases. According to reports, about 41.9% of rabbits in my country have been infected with coccidia, and the infection rate in northwest, northeast and southwest my country exceeds 60%. All breeds of rabbits are susceptible to the disease, among which the infection and morbidity are the most serious in young rabbits from weaning to 3 months old, with a mortality rate of up to 40% to 70%. It is a common disease in rabbit production. There are 11 generally recognized and detailedly identified pathogens of rabbit coccidiosis, among which Eimeria stehrenbergii, also known as rabbit liver coccidia, is the only coccidia parasitic on the bile duct epithelial cells of the rabbit liver and has the strongest pathogenicity. The mortality rate of young rabbits infected with Eimeria stehrenbergii from weaning to 2 months old can be as high as 100%, which seriously threatens the development of the rabbit industry. Therefore, many experts and scholars are engaged in research on molecular biology, immunology (vaccines), tissue culture and drug resistance of this type of coccidia.

[0003] Among them, sporozoites are the key stage for coccidia to invade host cells. Purification of sporozoites is the basis for subsequent research on coccidia virulence, screening of related antigens and drugs, and research on parasite-host interactions. At present, some scholars have established a method for purifying chicken Eimeria tenella sporozoites and applied for a patent. However, the inventor used this method to purify Eimeria skrjavinii sporozoites and found that it was difficult to obtain the required purified sporozoites. In addition, attempts to separate Eimeria skrjavinii sporozoites using other existing purification methods such as sucrose gradient centrifugation have also failed.

[0004] Therefore, a method for purifying Eimeria skrjabini sporozoites is needed. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for purifying sporozoites of Eimeria skrjabini, which can purify sporozoites of Eimeria skrjabini and has the advantages of simple operation, low cost and high sporozoite yield.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for purifying sporozoites of Eimeria skrjabini comprises the following steps:

[0008] Step 1: Obtain the diseased rabbit liver by autopsy to obtain the Eimeria skrjabini oocysts;

[0009] Step 2, dissolving the Eimeria skrjabini oocysts in a potassium dichromate solution for sporulation to obtain a potassium dichromate sporulated oocyst suspension;

[0010] Step 3, taking the potassium dichromate sporulated oocyst suspension and centrifuging it, adding sodium hypochlorite to the precipitate and blowing it on ice, adding clean water and centrifuging it;

[0011] Step 4, taking the upper foam and liquid after centrifugation, adding clean water, centrifuging, taking the precipitate, and obtaining clean sporulated oocysts;

[0012] Step 5, add 1×PBS buffer to the clean sporulated oocysts, transfer to a centrifuge container, add grinding glass beads, and shake for a certain period of time;

[0013] Step 6: Use a pipette to suck out the suspension after shaking into a new centrifuge container, centrifuge once, suck off the upper liquid, add 50% percol solution, and centrifuge again;

[0014] Step 7, taking the precipitate obtained by the secondary centrifugation in step 6, adding decysting buffer to the precipitate, and digesting it in a water bath for a certain period of time;

[0015] Step 8, centrifuge the digested suspension once, take the precipitate, add a certain amount of 55% percol solution, centrifuge twice, take the precipitate, add 1×PBS buffer to dilute, and transfer to a new centrifuge tube, use 1×PBS buffer to make up the volume, and after making up the volume, use a sand core funnel to naturally filter the PBS suspension containing sporozoites, and filter repeatedly to obtain clean sporozoites of Eimeria skrjejuni.

[0016] Furthermore, in step 1, the diseased rabbit liver is obtained by autopsy and then crushed, filtered and purified to obtain the Eimeria skrjabini oocysts.

[0017] Furthermore, the mass concentration of the potassium dichromate solution in step 2 is 2.5%.

[0018] Furthermore, in step 3, the sporulation time is 5 days; the added volumes of clean water and potassium dichromate sporulated oocyst suspension are registered equal; the speed during centrifugation is 2500-2700 rpm, and the duration is not less than 6 minutes.

[0019] Furthermore, in step 4, the volume of clean water added is 5-6 times the volume of the upper foam and liquid, the speed during centrifugation is 3800 rpm, and the duration is not less than 6 minutes.

[0020] Furthermore, in step 5, the diameter of the grinding glass beads is 1.5-2.0 mm, and after the glass beads are added, the liquid level is above the glass beads, and the shaking treatment time is not less than 40 seconds.

[0021] Furthermore, in step 6, the rotation speed during one centrifugation is 2500-2700 rpm, and the duration is not less than 6 minutes; the centrifugal force during one centrifugation is 10000g, and the duration is not less than 1 minute.

[0022] Furthermore, in step 7, the decystation buffer is a mixture of 0.75% pancreatic enzyme and 10% rabbit bile; the temperature during the water bath digestion treatment is 41° C., and the duration is 10-60 min.

[0023] Furthermore, in step 8, the rotation speed during the first centrifugation is 3800 rpm, and the duration is 6 minutes; the centrifugal force during the second centrifugation is 10000 g, and the duration is 1 minute.

[0024] Furthermore, in step 8, the pore size of the sand core funnel is 16-30 μm, and the number of repeated filtrations is no less than 2 times.

[0025] The remarkable effects of the present invention are:

[0026] 1. Compared with the existing method for purifying sporozoites of Eimeria tenella, the method of the present invention is simple to operate, low in cost, and high in sporozoite yield (the existing method requires the preparation of a cellulose column, which has high cost and requires acid-base treatment, which is cumbersome and time-consuming to operate, and the suspension must be heated to allow the sporozoites to remain active and move through the cellulose column, resulting in a small number of sporozoites after filtration).

[0027] 2. The method of the present invention uses a glass bead oscillator for oscillation and does not require the processing time of a cellulose column, which greatly reduces the time required to break the oocyst wall and saves time during the purification process.

[0028] 3. Since Eimeria skrjabini is obtained from the liver, it contains a lot of immature oocysts, while chicken coccidia oocysts are obtained from feces, which are basically mature oocysts. This method uses sodium hypochlorite to treat Eimeria skrjabini oocysts, removes a large amount of impurities and immature oocysts, and is convenient for subsequent experimental operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a flow chart of the method of the present invention;

[0030] Figure 2 is a photomicrograph of a suspension of potassium dichromate-sporulated oocysts before sodium hypochlorite treatment;

[0031] Figure 3 is a micrograph of sporulated oocysts after sodium hypochlorite treatment;

[0032] Figure 4 is a micrograph of the sporulated oocyst suspension after cell wall rupture;

[0033] Figure 5It is a microscopic photograph of purified sporozoites obtained after filtration through the sand core filter bucket of the present invention. DETAILED DESCRIPTION

[0034] The specific implementation manner and working principle of the present invention are further described in detail below with reference to the accompanying drawings.

[0035] Embodiment 1:

[0036] like Figure 1 As shown, this embodiment provides a method for purifying Eimeria skrjabini sporozoites, and the specific steps are as follows:

[0037] Step 1, dissecting and obtaining the diseased rabbit liver, crushing it, and obtaining the Eimeria skrjabini oocysts by filtering and purification;

[0038] Step 2, dissolving the Eimeria skrjabini oocysts in a 2.5% potassium dichromate solution for sporulation to obtain a potassium dichromate sporulated oocyst suspension, and stopping when no more sporulation occurs after about 5 days;

[0039] Step 3, take 10mL of potassium dichromate sporulated oocyst suspension and centrifuge at 2500rpm for 6min, add sodium hypochlorite to the precipitate and blow on ice for 15min, then add an equal amount of clean water and centrifuge at 2500rpm for 6min; the micrographs before and after sodium hypochlorite treatment are as follows Figure 2 and Figure 3 As shown;

[0040] Step 4, aspirate the upper foam and liquid after centrifugation (spore-forming oocysts are mostly concentrated in the foam, and the precipitate is mostly other impurities or immature oocysts), add 5 times of clean water, centrifuge at 3800rpm for 6min, take the precipitate, and obtain clean sporulated oocysts treated with sodium hypochlorite;

[0041] Step 5: Add 1 mL of 1×PBS buffer to the clean sporulated oocysts, pipette 0.5 mL into a centrifuge tube, add 1.5 mm diameter grinding glass beads, and require that the sporulated oocyst suspension is submerged after the glass beads are added. Place in an oscillator for 40 seconds of shaking. The micrograph of the sporulated oocyst suspension after shaking is as follows: Figure 4 As shown;

[0042] Step 6, use a pipette to suck out the suspension after the shock treatment, the suspension contains broken oocyst walls, sporangia, unbroken oocysts and a very small amount of sporozoites that are over-shocked, the sucked suspension is transferred to a new 1.5 mL centrifuge tube, centrifuged at 3800 rpm for 6 min, the upper liquid is sucked off, 50% percol solution is added, and centrifuged at 10000 g for 1 min, the upper foam is the unbroken oocysts and oocyst walls, and the lower sediment is sporangia, a small amount of sporozoites and a small amount of egg shells;

[0043] In this case, 50% percol solution was prepared by mixing 100% percol solution and 1× PBS buffer at a ratio of 1:1, while 100% percol solution was prepared by mixing percol stock solution and 10× PBS buffer at a ratio of 9:1;

[0044] Step 7, take the precipitate obtained by the second centrifugation in step 6, add 1 mL of excystation buffer to the precipitate, and digest it in a water bath at 41°C for 20 minutes;

[0045] In this example, the decystation buffer was a mixture of 0.75% pancreatin and 10% rabbit bile;

[0046] Step 8, centrifuge the digested suspension at 3800rpm for 6min, take the precipitate, add 1mL55% percol solution, the 55% percol solution is mixed with 100% percol solution and 1×PBS buffer in a ratio of 11:9; centrifuge at 10000g for 1min, remove the upper foam and liquid, retain the lower precipitate (mostly sporozoites, a few have egg shell impurities), take a small amount of 1×PBS buffer to dilute, and transfer to a new 5mL centrifuge tube, use 1×PBS buffer to make up to 3mL, and after constant volume, naturally filter the PBS suspension containing sporozoites with a sand core funnel with a pore size of 16μm, and filter twice repeatedly to obtain clean sporozoites of Eimeria skrjejuni. Figure 5 shown.

[0047] Embodiment 2:

[0048] The difference from Example 1 is:

[0049] The speed during centrifugation in step 3 is 2600 rpm;

[0050] Add 6 times the amount of clean water to the upper foam and liquid in step 4;

[0051] In step 5, 1.0 mL was pipetted into a centrifuge tube and ground with 2.0 mm diameter glass beads;

[0052] In step 7, the digestion was carried out in a water bath for 10 min;

[0053] In step 8, 1×PBS buffer was used to make up to 4 mL, and after the volume was fixed, the PBS suspension containing sporozoites was naturally filtered using a sand core funnel with a pore size of 20 μm, and the filtration was repeated 3 times to obtain Figure 5 Clean Eimeria skrjabini sporozoites are shown.

[0054] Embodiment 3:

[0055] The difference from Example 1 is:

[0056] The centrifugal speed in step 3 is 2700 rpm;

[0057] In step 4, add 5.5 times the amount of clean water to the upper layer of foam and liquid;

[0058] In step 5, 1.5 mL was pipetted into a centrifuge tube and ground using 1.6 mm diameter glass beads;

[0059] In step 7, the digestion was carried out in a water bath for 30 min;

[0060] In step 8, 1×PBS buffer was used to make up to 3.5 mL. After the volume was fixed, the PBS suspension containing sporozoites was naturally filtered using a sand core funnel with a pore size of 25 μm. The filtration was repeated 3 times to obtain Figure 5 Clean Eimeria skrjabini sporozoites are shown.

[0061] Embodiment 4:

[0062] The difference from Example 1 is:

[0063] The speed during centrifugation in step 3 is 2550 rpm;

[0064] Add 5 times the amount of clean water to the upper foam and liquid in step 4;

[0065] In step 7, the digestion was carried out in a water bath for 45 min;

[0066] In step 5, 1.0 mL was pipetted into a centrifuge tube and ground with 2.0 mm diameter glass beads;

[0067] In step 8, 1×PBS buffer was used to make up to 4 mL, and after the volume was fixed, the PBS suspension containing sporozoites was naturally filtered using a sand core funnel with a pore size of 28 μm, and the filtration was repeated twice to obtain Figure 5 Clean Eimeria skrjabini sporozoites are shown.

[0068] Embodiment 5:

[0069] The difference from Example 1 is:

[0070] The centrifugal speed in step 3 is 2650 rpm;

[0071] Add 6 times the amount of clean water to the upper foam and liquid in step 4;

[0072] In step 5, 1.5 mL was pipetted into a centrifuge tube and ground using 1.8 mm diameter glass beads;

[0073] In step 7, the digestion was carried out in a water bath for 60 min;

[0074] In step 8, 1×PBS buffer was used to make up to 3 mL, and after the volume was fixed, the PBS suspension containing sporozoites was naturally filtered using a sand core funnel with a pore size of 30 μm, and the filtration was repeated 3 times to obtain Figure 5 Clean Eimeria skrjabini sporozoites are shown.

[0075] from Figure 5 It can be seen from the obtained microphotographs that the method of the present invention achieves the purification of Eimeria skrjabini sporozoites. In addition, in the specific implementation process, by counting the clean Eimeria skrjabini sporozoites obtained in Examples 1 to 5, it was found that after the duration of the water bath digestion treatment in step 7 exceeded 30 minutes, excessive digestion began, which greatly reduced the number of sporozoites, while if it was less than 15 minutes, the digestion was not completed and the number of sporozoites did not reach the maximum value. It can be seen that the optimal duration of the water bath digestion treatment is 15 minutes.

[0076] In summary, the purification method of the present invention is simple to operate, low in cost and high in sporozoite yield compared with the existing Eimeria tenella sporozoite purification method; at the same time, the method of the present invention uses a glass bead oscillator for oscillation, and there is no processing time for a cellulose column, which greatly reduces the time for breaking the oocyst wall, and the purification process is more time-saving; in addition, since Eimeria swirlenni is obtained from the liver, it contains a lot of immature oocysts, while the chicken coccidia oocysts are obtained from feces and are basically mature oocysts, so the method uses sodium hypochlorite to process the Eimeria swirlenni oocysts, removes a large amount of impurities, and is convenient for subsequent experimental operations.

[0077] The technical solution provided by the present invention is described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for purifying sporozoites of Eimeria skrjabini, characterized in that: The steps include: Step 1: Obtain the diseased rabbit liver by autopsy to obtain the Eimeria skrjabini oocysts; Step 2, dissolving the Eimeria skrjabini oocysts in a potassium dichromate solution for sporulation to obtain a potassium dichromate sporulated oocyst suspension; Step 3, taking the potassium dichromate sporulated oocyst suspension and centrifuging it, adding sodium hypochlorite to the precipitate and blowing it on ice, adding clean water and centrifuging it; Step 4, taking the upper foam and liquid after centrifugation, adding clean water, centrifuging, taking the precipitate, and obtaining clean sporulated oocysts; Step 5, add 1×PBS buffer to the clean sporulated oocysts, transfer to a centrifuge container, add grinding glass beads, and shake for a certain period of time; Step 6: Use a pipette to suck out the suspension after shaking into a new centrifuge container, centrifuge once, suck off the upper liquid, add 50% percol solution, and centrifuge again; Step 7, taking the precipitate obtained by the secondary centrifugation in step 6, adding decysting buffer to the precipitate, and digesting it in a water bath for a certain period of time; Step 8, centrifuge the digested suspension once, take the precipitate, add a certain amount of 55% percol solution, centrifuge twice, take the precipitate, add 1×PBS buffer to dilute, and transfer to a new centrifuge tube, use 1×PBS buffer to make up the volume, and after making up the volume, use a sand core funnel to naturally filter the PBS suspension containing sporozoites, and filter repeatedly to obtain clean sporozoites of Eimeria skrjejuni.

2. The method for purifying Eimeria skrjabini sporozoites according to claim 1, characterized in that: In step 1, the diseased rabbit liver is obtained by autopsy and then crushed, filtered and purified to obtain the Eimeria skrjabini oocysts.

3. The method for purifying Eimeria skrjabini sporozoites according to claim 1, characterized in that: The mass concentration of the potassium dichromate solution in step 2 is 2.5%.

4. The method for purifying Eimeria skrjabini sporozoites according to claim 1, wherein: In step 3, the sporulation time is 5 days; the added volumes of clean water and potassium dichromate sporulated oocyst suspension are registered equal; the speed during centrifugation is 2500-2700 rpm, and the duration is not less than 6 minutes.

5. The method for purifying Eimeria skrjabini sporozoites according to claim 1, characterized in that: In step 4, the volume of clean water added is 5-6 times the volume of the upper foam and liquid, the speed during centrifugation is 3800 rpm, and the duration is not less than 6 minutes.

6. The method for purifying Eimeria skrjabini sporozoites according to claim 1, characterized in that: In step 5, the diameter of the grinding glass beads is 1.5-2.0 mm, and after the glass beads are added, the liquid level is above the glass beads, and the shaking treatment time is not less than 40 seconds.

7. The method for purifying Eimeria skrjabini sporozoites according to claim 1, characterized in that: In step 6, the rotation speed during one centrifugation is 2500-2700 rpm, and the duration is not less than 6 minutes; the centrifugal force during one centrifugation is 10000g, and the duration is not less than 1 minute.

8. The method for purifying Eimeria skrjabini sporozoites according to claim 1, characterized in that: In step 7, the decystation buffer is prepared by mixing 0.75% pancreatic enzyme and 10% rabbit bile; the temperature during the water bath digestion treatment is 41° C. and the duration is 10-60 min.

9. The method for purifying Eimeria skrjabini sporozoites according to claim 1, characterized in that: In step 8, the rotation speed during the first centrifugation is 3800 rpm, and the duration is 6 minutes; the centrifugal force during the second centrifugation is 10000 g, and the duration is 1 minute.

10. The method for purifying sporozoites of Eimeria skrjabini according to claim 1 or 9, characterized in that: In step 8, the pore size of the sand core funnel is 16-30 μm, and the number of repeated filtrations is no less than 2 times.

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