CN108715610B - Preparation and purification method of schistosome egg antigen - Google Patents
Preparation and purification method of schistosome egg antigen Download PDFInfo
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Abstract
The invention relates to a preparation and purification method of schistosome ovum antigen, adopting an abdominal slide method to infect New Zealand white rabbits with schistosoma japonicum cercaria, feeding and dissecting, taking out liver tissues, shearing into fine blocks by scissors, adding normal saline to break and homogenate, sieving through a 60-120 mesh copper sieve with phi 20cm, sieving through a 160-300 mesh nylon silk with phi 20cm, and collecting schistosome ovum on 300 meshes; adding physiological saline, stirring, standing, precipitating, removing impurities with a suction tube, washing with 200 mesh and 300 mesh nylon silk of phi 20cm, washing with distilled water, collecting eggs of 300 mesh, grinding, lyophilizing at low temperature, grinding with quartz grinding bowl, sieving with 300-400 mesh copper sieve of phi 20cm, crushing, homogenizing, centrifuging at low temperature with high-speed centrifuge, and collecting supernatant to obtain milky white schistosome egg antigen. The pure schistosome ovum has high yield, and the obtained schistosome ovum does not contain broken liver tissue and broken ovum with similar size, and the content of foreign protein in the ovum antigen is low.
Description
Technical Field
The invention relates to the technical field of antigen preparation, in particular to a preparation and purification method of schistosome egg antigen.
Background
Schistosomiasis is a parasitic disease which can be infected by both human beings and animals, and has larger harm degree. At present, the diagnostic standard (WS261-2006) for schistosomiasis in China mainly comprises an enzyme-linked immunosorbent assay, a colloidal gold immunochromatography, an indirect hemagglutination method and a percolation method. In SEA prepared from pure eggs, specific protein components are mainly concentrated at 10-20kDa, and the liver tissue cell protein, broken egg denatured protein and pure egg protein can be separated by adopting treatment modes such as macroporous resin, dialysis bag and the like, so that the purification of protein with specific molecular weight is realized, but the problems of long time consumption and low SEA yield exist.
The general steps for preparing the schistosome egg antigen are as follows: infecting New Zealand white rabbit with Schistosoma japonicum cercaria by abdominal slide method, feeding for 45 days, dissecting, and taking out liver tissue; cutting liver tissue into fine blocks with scissors, adding normal saline, crushing and homogenizing, passing through nylon silk of 160, 200 and 300 meshes with diameter of 20cm, and collecting schistosome eggs retained on the nylon silk of 300 meshes; adding physiological saline, stirring, standing, precipitating, removing suspended impurities with a suction tube, washing with 20 cm-diameter nylon silk of 200 and 300 meshes, washing with distilled water, collecting ovum retained on the nylon silk of 300 meshes, pouring into a cell homogenizer, crushing, homogenizing, centrifuging with a high-speed centrifuge, and collecting supernatant to obtain milky schistosome ovum antigen.
When pure schistosome eggs are directly filtered by using 160-300-mesh nylon silk cloth step by step, large pieces of broken liver tissues contained in the solution cannot smoothly pass through the nylon silk cloth, and meanwhile, the eggs are blocked from passing through, so that the filtering time is long, and the yield of the pure schistosome eggs is low, generally 1.78g +/-0.16 g (per 1kg of raw materials). Pure eggs in eggs obtained by filtering 300-mesh nylon silk cloth are low in yield, and broken liver tissues and broken eggs are not completely separated, so that the prepared SEA component contains hybrid proteins such as liver tissue cell protein and broken egg protein. Because the protein in the broken eggs can be oxidized and denatured or naturally degraded in the long-time preparation process, and the broken eggs have certain similarity with the protein prepared from pure eggs. The SEA containing the hybrid protein causes low specificity and sensitivity of a subsequent detection method, and false positive may occur, such as cross reaction with other parasitoses such as paragonimiasis, clonorchiasis sinensis and the like to a certain extent; there may also be missed tests, if the patient is at a lower level of infection, and if impure antigen is used, the result is negative.
CN103076447B A method for purifying crude antigen of schistosome ovum, which adopts Sephacryl S-300HR column chromatography to separate crude antigen of schistosome ovum and removes component with molecular weight less than 10 kDa. Preferably, the final product is further separated and purified by using an ultrafiltration tube with the molecular weight cut-off of 10 kDa. Collecting schistosome ovum, adding normal saline to make homogenate, then ultrasonic pulverizing and centrifuging to obtain supernatant as crude antigen of schistosome ovum. Also provides a schistosome ovum purified antigen, the application thereof and a schistosome antibody detection colloidal gold immunoassay kit
CN102079781A discloses an immunogenic protein in a group of schistosoma japonicum soluble egg antigens, which is selected from amino acid sequences shown in SEQ ID NO. 2-SEQ ID NO. 14. Also discloses a screening method and application of the immunogenic protein in the schistosoma japonicum soluble egg antigen, mainly screening the immunogenic protein in the schistosoma japonicum soluble egg antigen by combining a proteomics technology and a serology method, in particular to a method for analyzing the immunogenic protein in the schistosoma japonicum soluble egg antigen by utilizing two-dimensional electrophoresis, Westernblot (2D-WB) and mass spectrum, and taking the immunogenic protein as a potential target spot for diagnosing schistosomiasis japonica
Disclosure of Invention
The invention aims to solve the problems and aims to provide a method for preparing and purifying schistosome egg antigen, which can improve the yield of pure schistosome eggs, and the obtained schistosome eggs do not contain broken liver tissues and broken eggs with similar sizes, and the prepared crude schistosome egg antigen has low content of foreign protein and the subsequent product is stable in storage period.
The invention aims to realize a preparation and purification method of schistosome egg antigen, which comprises the following steps:
1) placing Schistosoma japonicum cercaria by using field collected positive oncomelania at 30 ℃ for 6h, artificially infecting New Zealand white rabbits by 1800 plus 2000 cercarias/rabbit by adopting an abdomen slide method, feeding for 45d, dissecting, and taking out liver tissues; the weight of each new Zealand white rabbit is 10 kg;
2) cutting 1/4 of the liver tissue obtained in the step 1) into a fine block by using scissors, then adding the fine block into a 500mL tissue triturator cup, adding 400mL of normal saline, crushing homogenate at 5000r/min, stopping the homogenate for 1min every 1min, and repeating for 3 times to obtain homogenate;
3) filtering the homogenate prepared in the step 2) by sequentially passing through 60, 80, 100 and 120-mesh copper sieves with the diameter of 20cm, and collecting liver tissue filtrate passing through the 120-mesh copper sieve;
4) sequentially passing the liver tissue filtrate collected in the step 3) through nylon silk of 160 meshes, 200 meshes and 300 meshes with the diameter of 20cm, and collecting schistosome eggs reserved on the nylon silk of 300 meshes;
5) putting 5g of the schistosome eggs collected in the step 4) into a 500mL measuring cup, adding 500mL of normal saline, stirring for 1-2min, standing for 15min, taking out the normal saline by using a suction pipe, removing suspended impurities, repeating for 3 times, and repeating for 3 times;
6) enabling the blood fluke eggs processed in the step 5) to sequentially pass through 200-mesh nylon silk and 300-mesh nylon silk with the diameter of 20cm, washing with distilled water with the volume 10-20 times that of the nylon silk, and collecting the insect eggs reserved on the 300-mesh nylon silk;
7) freezing the schistosome ovum treated in the step 6) in a low-temperature refrigerator at-80 ℃ for 24h, and then freeze-drying for 24h to obtain schistosome ovum freeze-dried powder;
the freeze-drying conditions are as follows: the temperature of the cold well is lower than-55 ℃, and the vacuum degree is lower than 0.001 MPa;
8) 1g of the schistosome egg freeze-dried powder prepared in the step 7) is put into a quartz grinding bowl; grinding into schistosome ovum powder under air purification level of one hundred thousand or above;
the indoor temperature of the quartz grinding bowl is 25 +/-1 ℃, and the indoor humidity is less than 20%;
9) sequentially passing the schistosome ovum powder prepared in the step 8) through 300-mesh and 400-mesh copper sieves, and collecting the ovum remained on the 400-mesh copper sieve to obtain pure schistosome ovum powder;
10) preparing the pure schistosome egg powder prepared in the step 9) into a suspension with the volume weight ratio of 1% by using physiological saline, pouring the suspension into a cell homogenizer to break and homogenate for 1min until the golden yellow egg powder becomes milk white liquid, then centrifuging the milk white egg powder for 30min at the temperature of 4 ℃ by using a high-speed centrifuge, and sucking supernatant fluid to obtain the milk white schistosome egg antigen.
The invention has the beneficial effects that:
1. when a two-step filtering method of firstly passing through a 60-120-mesh copper sieve and then passing through 160-plus-300 nylon silk cloth is adopted, the copper sieve can quickly remove large broken liver tissues by primary filtration, the subsequent filtering efficiency of the nylon silk cloth is improved, the yield of pure schistosome eggs is high and is generally 2.06 +/-0.08 g (per 1kg of raw materials);
2. the method of freezing and freeze-drying at-80 ℃ is adopted, the liver tissue protein can be freeze-dried into solid, the further crushing of the crushed egg is promoted (because the crushed egg is exposed outside, the water content is continuously lost in the freeze-drying process, and the egg wall is dehydrated, becomes brittle and fragile), and in the subsequent grinding process, the liver tissue protein, the crushed egg and the crushed egg protein are crushed to the particle size of less than 400 meshes by controlling certain grinding strength and grinding time; the egg wall of the complete egg has a certain protection function, so that the egg is kept complete during grinding, and the particle size of the egg is between 300 meshes and 400 meshes, so that the separation of pure egg from the crushed liver tissue and the crushed egg is realized; meanwhile, the prepared pure worm egg freeze-dried powder is easy to store, and the protein structure in the worm eggs is stable, so that the specificity and sensitivity of the worm eggs can be ensured;
3. the preparation method of the SEA uses normal saline as a solvent, does not use other chemical reagents, only uses physical processing methods such as crushing, filtering, freeze-drying and centrifuging to realize the preparation of the SEA, can avoid the influence of residual chemical reagents on the SEA, ensures the specificity and sensitivity of the SEA, and improves the stability of subsequent products in a storage period.
Drawings
FIG. 1 is a diagram showing the results of SDS-PAGE gel electrophoresis experiments of a Marker (labeled M) as a standard protein sample and SEA prepared according to the present invention (labeled as sample).
Detailed Description
The method comprises the following specific steps:
1) placing Schistosoma japonicum cercaria by using field collected positive oncomelania at 30 ℃ for 6h, artificially infecting New Zealand white rabbits by 1800 plus 2000 cercarias/rabbit by adopting an abdomen slide method, feeding for 45d, dissecting, and taking out liver tissues; a single New Zealand white rabbit weighs 10 kg.
2) Cutting 1/4 of the liver tissue obtained in the step 1) into a fine block by using scissors, then adding the fine block into a 500mL tissue triturator cup, adding 400mL of normal saline, crushing homogenate at 5000r/min, stopping the homogenate for 1min every 1min to avoid the blade temperature from being overhigh, and repeating for 3 times to obtain homogenate.
3) Filtering the homogenate prepared in the step 2) by sequentially passing through 60, 80, 100 and 120-mesh copper sieves with the diameter of 20cm, and collecting the liver tissue filtrate passing through the 120-mesh copper sieve.
4) Sequentially passing the liver tissue filtrate collected in the step 3) through nylon silk of 160 meshes, 200 meshes and 300 meshes with the diameter of 20cm, and collecting schistosome eggs reserved on the nylon silk of 300 meshes.
When the invention adopts a two-step filtering method of firstly passing through a 60-120-mesh copper sieve and then passing through 160-plus-300 nylon silk cloth, the copper sieve can quickly remove large broken liver tissues by primary filtration, the subsequent filtering efficiency of the nylon silk cloth is improved, the yield of pure schistosome eggs is high, and the yield is generally 2.06 +/-0.08 g (per 1kg of raw materials).
5) Putting 5g of the schistosome eggs collected in the step 4) into a 500mL measuring cup, adding 500mL of normal saline, stirring for 1-2min, standing for 15min, taking out the normal saline by using a suction tube, removing suspended impurities, repeating for 3 times, and repeating for 3 times.
6) Enabling the blood fluke eggs treated in the step 5) to pass through 200-mesh and 300-mesh nylon silk with the diameter of 20cm in sequence, washing with distilled water with the volume 10-20 times that of the silk, and collecting the eggs remained on the 300-mesh nylon silk.
7) Freezing the schistosome ovum treated in the step 6) in a low-temperature refrigerator at-80 ℃ for 24h, and then freeze-drying for 24h to obtain the schistosome ovum freeze-dried powder.
The freeze-drying conditions are as follows: the temperature of the cold well is less than-55 ℃, and the vacuum degree is lower than 0.001 MPa.
8) 1g of the schistosome egg freeze-dried powder prepared in the step 7) is put into a quartz grinding bowl; grinding into schistosome ovum powder under air purification level of one hundred thousand or above.
The indoor temperature of the quartz grinding bowl is 25 +/-1 ℃, and the indoor humidity is less than 20%.
Note that: the complete single eggs of the schistosome can not be broken.
The milling intensity is controlled in such a way that the milling rod mills in the grinding bowl for one circle for 1-2min, and the total milling time is 1 h. By controlling the grinding intensity and the grinding time, the liver tissue protein, the broken egg and the protein are crushed to the particle size of less than 400 meshes, and the particle size of the schistosoma egg powder is between 300 meshes and 400 meshes.
9) And (3) sequentially passing the schistosome ovum powder prepared in the step (8) through 300-mesh and 400-mesh copper sieves, and collecting the ovum remained on the 400-mesh copper sieve to obtain pure schistosome ovum powder.
10) Preparing the pure schistosome ovum powder prepared in the step 9) into a suspension with the volume weight ratio of 1% by using physiological saline, pouring the suspension into a cell homogenizer for crushing and homogenizing for 1min at 5000r/min until the golden yellow ovum powder becomes milk white liquid, then centrifuging for 30min at 4 ℃ by using a high-speed centrifuge, and absorbing supernatant fluid to obtain the milk white schistosome ovum antigen.
As can be seen from FIG. 1, the schistosoma ovum antigen prepared by the invention has a stronger band only at the 15kD position, which indicates that the schistosoma ovum antigen prepared by the invention has low content of hetero-protein, so that the subsequent product is stable in storage period.
Claims (3)
1. A method for preparing and purifying schistosome ovum antigen comprises the following steps:
1) placing Schistosoma japonicum cercaria by using field collected positive oncomelania at 30 ℃ for 6h, artificially infecting New Zealand white rabbits by 1800 plus 2000 cercarias/rabbit by adopting an abdomen slide method, feeding for 45d, dissecting, and taking out liver tissues; the weight of each new Zealand white rabbit is 10 kg;
2) cutting 1/4 of the liver tissue obtained in the step 1) into a fine block by using scissors, then adding the fine block into a 500mL tissue triturator cup, adding 400mL of normal saline, crushing homogenate at 5000r/min, stopping the homogenate for 1min every 1min, and repeating for 3 times to obtain homogenate;
3) filtering the homogenate prepared in the step 2) by sequentially passing through 60, 80, 100 and 120-mesh copper sieves with the diameter of 20cm, and collecting liver tissue filtrate passing through the 120-mesh copper sieve;
4) sequentially passing the liver tissue filtrate collected in the step 3) through nylon silk of 160 meshes, 200 meshes and 300 meshes with the diameter of 20cm, and collecting schistosome eggs reserved on the nylon silk of 300 meshes;
5) putting 5g of the schistosome eggs collected in the step 4) into a 500mL measuring cup, adding 500mL of normal saline, stirring for 1-2min, standing for 15min, taking out the normal saline by using a suction pipe, removing suspended impurities, and repeating for 3 times;
6) enabling the blood fluke eggs processed in the step 5) to sequentially pass through 200-mesh nylon silk and 300-mesh nylon silk with the diameter of 20cm, washing with distilled water with the volume 10-20 times that of the nylon silk, and collecting the insect eggs reserved on the 300-mesh nylon silk;
7) freezing the schistosome ovum treated in the step 6) in a low-temperature refrigerator at-80 ℃ for 24h, and then freeze-drying for 24h to obtain schistosome ovum freeze-dried powder;
the freeze-drying conditions are as follows: the temperature of the cold well is lower than-55 ℃, and the vacuum degree is lower than 0.001 MPa;
8) 1g of the schistosome egg freeze-dried powder prepared in the step 7) is put into a quartz grinding bowl; grinding into powder with particle size of 300 mesh and 400 mesh under the condition of air purification grade of one hundred thousand or above;
the indoor temperature of the quartz grinding bowl is 25 +/-1 ℃, and the indoor humidity is less than 20%;
the grinding intensity is controlled in the way that the grinding rod grinds in the grinding bowl for one circle for 1-2min, and the total grinding time is 1 h;
9) sequentially passing the schistosome ovum powder prepared in the step 8) through 300-mesh and 400-mesh copper sieves, and collecting the ovum remained on the 400-mesh copper sieve to obtain pure schistosome ovum powder;
10) preparing the pure schistosome egg powder prepared in the step 9) into a suspension with the volume weight ratio of 1% by using physiological saline, pouring the suspension into a cell homogenizer to break and homogenate for 1min until the golden yellow egg powder becomes milk white liquid, then centrifuging the milk white egg powder for 30min at the temperature of 4 ℃ by using a high-speed centrifuge, and sucking supernatant fluid to obtain the milk white schistosome egg antigen.
2. The method for preparing and purifying schistosome egg antigen according to claim 1, wherein the method comprises the following steps: step 8) requires: the complete single eggs of the schistosome can not be broken.
3. The method for preparing and purifying schistosome egg antigen according to claim 1, wherein the method comprises the following steps: step 10) pouring the mixture into a cell homogenizer for crushing and homogenizing at 5000 r/min.
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