CN100569937C - The separation and purification of coccidian oocyst and preparation method of sporozoite - Google Patents
The separation and purification of coccidian oocyst and preparation method of sporozoite Download PDFInfo
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- CN100569937C CN100569937C CNB2006100501937A CN200610050193A CN100569937C CN 100569937 C CN100569937 C CN 100569937C CN B2006100501937 A CNB2006100501937 A CN B2006100501937A CN 200610050193 A CN200610050193 A CN 200610050193A CN 100569937 C CN100569937 C CN 100569937C
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Abstract
A kind of coccidian oocyst separates, purifying and preparation method of sporozoite, handle the chicken caecum through routine, content or ight soil, obtain the roughing out thing of egg capsule, add 2.5% potassium bichromate, put into incubator spore cultivation 2～4 days, get spore egg capsule deposit suspension, centrifuge washing is removed potassium bichromate, adds 30%NaClO solution, centrifugal, get white floating matter in the supernatant, add the aquae destillata mixing, the centrifugal supernatant of abandoning, again with distilled water centrifuge washing precipitation, remove NaClO and other organic substance, keep throw out, move in the tissue homogenizer after the spore egg capsule throw out after the purification process is suspended with physiological saline, ground broken the getting final product of sediments microscope inspection 90% above sporozoite capsule 15-20 minute.The inventive method is reasonable in design, compares with traditional method for separating and preparing, have (1) easy and simple to handle, quick, egg capsule purity height, the impurity that extracts (2) are few; (3) the active advantages of higher of sporozoite.
Description
Technical field:
The invention belongs to biotechnology, relate generally to coccidian oocyst separation and purification and preparation method of sporozoite.
Background technology:
The chicken coccidia distributes extensively, harm is big, the development of serious threat aviculture, and is especially for chick, destructive often.The intensification poulty house is the optimum place of coccidiosis outburst, and its sickness rate is 50%-70%, mortality ratio 20-30%, when serious up to 80%.In various chicken diseases, the sickness rate of coccidiosis is the highest, accounts for 1/6-1/5.According to Bhogal etc., whole world every year because of coccidiosis of chicken cause with a toll of 2,000,000,000 dollars, the year consumption of anticoccidial drug is 3.2 hundred million dollars.Along with the continuous development of poultry husbandry, these data also are and double ascendant trend.Hundred million yuan of China's year expenditure anticoccidial drug expense 6-18, up to now, the main means of control coccidiosis remain the chemicals control.Because being on the rise of resistance and drug residue two large problems, immunoprophylaxis research has caused extensive attention.Along with going deep into of coccidiosis research, vitro culture technology and Protocols in Molecular Biology are used widely in this field.The preparation of the separation of egg capsule, purifying and sporozoite is the basis and the prerequisite of vitro culture and molecular biology research.More time-consuming, the effort of traditional method.
Summary of the invention:
The purpose of this invention is to provide a kind of coccidian oocyst separation, purifying and preparation method of sporozoite, realize by following steps:
1. the separation of egg capsule
(1) from chicken caecum and content, separates
Cut open extremely chicken, get caecum and content and put into that to organize stirrer to add water an amount of, smash some minutes (100 grams were organized 3-5 minute) to pieces.In suspension, add 39 ℃ of digestion of 2mg/ml trypsinase 1 hour, remove enzyme 3 times with distilled water wash then.In 40 orders, 200 order copper sieve, filter successively, centrifugal 5 minutes of filtered liquid 5000rpm, precipitation is the roughing out thing of egg capsule.
(2) from chicken ight soil, separate
Gather an amount of ight soil and suspend, left standstill 15 minutes, in 40 orders, 200 order copper sieve, filter successively with distilled water, centrifugal 5 minutes of filtered liquid 5000rpm, precipitation is the roughing out thing of egg capsule.
2. the sporeization of egg capsule
Get egg capsule roughing out thing, add the 2.5% potassium bichromate solution back that stirs in 1: 10 ratio and divide the plate (solution deep does not surpass 1 centimetre) of packing into, putting 28 ℃ of incubators cultivates, every ventilation in 1 hour once, sporeization 2～4 days, get a little nutrient solution microscopy, can gather in the crops if any the egg capsule sporeization more than 85%, it is standby to put 4 ℃ of preservations.
3. the purifying of spore egg capsule
(1) centrifuge washing
Get an amount of spore egg capsule deposit suspension and put into centrifuge tube, centrifugal 2 minutes of 5000rpm removes supernatant, adds an amount of distilled water wash, and centrifuge washing 3-5 time is to remove potassium bichromate repeatedly.
(2) clorox rinsing purifying
After removing potassium bichromate, in centrifugal sediment, add 30%NaClO solution, the piping and druming mixing is placed after 10 minutes with 5000rpm, is drawn white floating matter in the supernatant for 4 ℃, change another new centrifuge tube over to, the distilled water mixing that adds 5 times of volumes, the centrifugal supernatant of abandoning of 12000rpm precipitates 2 times with the distilled water centrifuge washing again, remove NaClO and other organic substance, standby.
4. the preparation of sporozoite suspension
Spore egg capsule throw out after the purification process is moved in the tissue homogenizer with physiological saline suspension back, grind 15-20min, broken the getting final product of sediments microscope inspection 90% above sporocyst.
The inventive method is compared with traditional method for separating and preparing, has the following advantages: (1) is easy and simple to handle, quick; (2) egg capsule purity height, the impurity of Ti Quing is few; (3) sporozoite is active high.Using the inventive method can prepare high purity egg capsule or sporozoite easy, apace, is used for molecular biology research and vitro culture etc.
Description of drawings
Fig. 1 is the spore egg capsule behind the purifying.
Fig. 2 is the sporozoite of preparation.
The egg capsule that Fig. 3 goes out for vitro culture in the chicken embryo.
Fig. 4 is tender eimeria tenella TA4 expression of gene and detection.
Embodiment:
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Get an amount of spore egg capsule deposit suspension and put into centrifuge tube, centrifugal 2 minutes of 5000rpm removes supernatant, adds an amount of distilled water wash, and centrifuge washing 3-5 time is to remove potassium bichromate repeatedly.In centrifugal sediment, add 30%NaClO solution then, the piping and druming mixing, place after 10 minutes with 5000rpm for 4 ℃, draw white floating matter in the supernatant, change another new centrifuge tube over to, add the distilled water mixing of 5 times of volumes, the centrifugal supernatant of abandoning of 12000rpm, precipitate 2 times with the distilled water centrifuge washing again, remove NaClO and other organic substance, standby.
With the spore egg capsule of this law purifying referring to Fig. 1.
The spore egg capsule throw out of example 1 method preparation is moved in the tissue homogenizer with physiological saline suspension back, grind 15-20min, broken the getting final product of sediments microscope inspection 90% above sporocyst.
With the sporozoite of this law preparation referring to Fig. 2.
Get the sporozoite of embodiment 2 methods preparation, with 3 * 10
4Individual amount is inoculated the non-immunity to coccidiosis AA of 10 ages in days meat embryonated chicken, infects the back the 7th day, gets chorioallantoic membrane and finds egg capsule through 39 ℃ of digestion of 2mg/ml trypsinase after 1 hour in suspension, and the result is referring to Fig. 3.
Get the sporozoite of embodiment 2 preparations, with Trizol reagent (20 ℃ of following precoolings), operate with reference to TrizolReagent Total RNA Isolation Kit (total RNA extraction reagent box) specification sheets step, the total RNA of tender eimeria tenella Zhejiang strain is extracted in success, successfully clone its TA4 sporozoite surface antigen gene by RT-PCR, and in prokaryotic expression carrier successful expression, the result is referring to Fig. 4.
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the preferred specific embodiments of front is interpreted as only illustrating, but not limits the scope of the invention by any way.
Claims (2)
1. a coccidian oocyst separation, purifying and preparation method of sporozoite, realize by following steps: the separation of (1) egg capsule: get chicken caecum, content or ight soil, handle through routine, obtain the roughing out thing of egg capsule, (2) sporeization of egg capsule, (3) purifying of spore egg capsule, the preparation of (4) sporozoite suspension;
It is characterized in that:
Step (2) is to get egg capsule roughing out thing, adds 2.5% potassium bichromate solution in 1: 10 ratio and stirs, put into 28 ℃ of incubators and cultivate, and sporeization 2～4 days, 4 ℃ of preservations are standby;
Step (3) is to get spore egg capsule deposit suspension, centrifuge washing, remove potassium bichromate, add 30%NaClO solution, centrifugal, get white floating matter in the supernatant, add the distilled water mixing, the centrifugal supernatant of abandoning is again with distilled water centrifuge washing precipitation, remove NaClO and other organic substance, keep throw out;
Step (4) is to move in the tissue homogenizer after the spore egg capsule throw out after the purification process is suspended with physiological saline, grinds broken the getting final product of sediments microscope inspection 90% above sporocyst 15-20 minute.
2. coccidian oocyst according to claim 1 separates, purifying and preparation method of sporozoite, it is characterized in that: the purifying of the described spore egg capsule of step (3), be to get spore egg capsule deposit suspension to put into centrifuge tube, centrifugal 2 minutes of 5000rpm, remove supernatant, add an amount of distilled water wash, centrifuge washing 3-5 time is to remove potassium bichromate repeatedly, add 30%NaClO solution at centrifugal sediment, the piping and druming mixing, 4 ℃ of placements are centrifugal with 5000rpm after 10 minutes, draw white floating matter in the supernatant, change another new centrifuge tube over to, add the aquae destillata mixing, the centrifugal supernatant of abandoning of 12000rpm precipitates 2 times with the distilled water centrifuge washing again, remove NaClO and other organic substance, it is standby to keep throw out.
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Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101659935B (en) * | 2009-09-01 | 2011-06-15 | 佛山市正典生物技术有限公司 | Application of adjustable hyperstatic oxygen-increasing machine in coccidian oocyst sporulation development |
| CN103374526A (en) * | 2012-04-18 | 2013-10-30 | 上海市农业科学院 | Coccidian oocyst culture preservative fluid and application method thereof |
| CN104862415A (en) * | 2015-06-19 | 2015-08-26 | 龙岩学院 | Isospora suis oocyst detection method based on LAMP real-time fluorescence and isospora suis oocyst detection primer |
| CN106244460B (en) * | 2015-09-30 | 2019-08-13 | 中国农业科学院兰州兽医研究所 | A kind of preparation method of the coccidian oocyst of no external source bacterium DNA pollution |
| CN108251436A (en) * | 2018-01-30 | 2018-07-06 | 广东省农业科学院动物卫生研究所 | A kind of Eimeria species autophagy GAP-associated protein GAP Etatg8 coding gene sequences and its acquisition methods |
| CN108085322A (en) * | 2018-01-30 | 2018-05-29 | 广东省农业科学院动物卫生研究所 | A kind of Eimeria species autophagy GAP-associated protein GAP Etatg6 coding gene sequences and its acquisition methods |
| CN110004104B (en) * | 2019-04-09 | 2022-06-07 | 华中农业大学 | Separation and purification method of Wuli iodiplodia shell petals |
| CN111154653B (en) * | 2020-01-16 | 2020-11-20 | 中国农业科学院兰州兽医研究所 | Method for purifying insect eggs through iodixanol density gradient centrifugation |
| CN111733079B (en) * | 2020-07-08 | 2021-09-07 | 佛山市正典生物技术有限公司 | Separation composition, preparation method thereof and chicken coccidian oocyst separation method using same |
| CN112400864B (en) * | 2020-11-27 | 2022-05-03 | 松山湖材料实验室 | Oocyst type biological wall breaking agent composition and preparation method and application thereof |
| CN112877215A (en) * | 2021-03-02 | 2021-06-01 | 华南农业大学 | Separation and purification method and inoculation method of Eimeria tenella sporozoites |
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2006
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