CN100398150C - Toxoplasma metabolic secretion antigen vaccine and its prepn process - Google Patents
Toxoplasma metabolic secretion antigen vaccine and its prepn process Download PDFInfo
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- CN100398150C CN100398150C CNB2005100960469A CN200510096046A CN100398150C CN 100398150 C CN100398150 C CN 100398150C CN B2005100960469 A CNB2005100960469 A CN B2005100960469A CN 200510096046 A CN200510096046 A CN 200510096046A CN 100398150 C CN100398150 C CN 100398150C
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Abstract
The present invention is toxoplasma metabolic secretion antigen vaccine and its preparation process. The toxoplasma metabolic secretion antigen vaccine is prepared with toxoplasma metabolic secretion antigen. The preparation process includes the following steps: inoculating toxoplasma tachyzoite to abdominal cavity of mouse, killing the mouse and soaking in alcohol for surface sterilizing; collecting polypide; obtaining abdominal cavity liquid via centrifuging and collecting supernatant; inoculating toxoplasma tachyzoite to vero cell to culture and collecting the cultured liquid after cell falling off via centrifuging and collecting supernatant; thrice freezing and thawing abdominal cavity liquid and cultured liquid; mixing, measuring protein content of the mixed liquid; mixing the mixed liquid with M206 adjuvant, emulsifying and packing as the composite toxoplasma vaccine with antigen protein content of 10 mg/ml. The vaccine is for inducing humoral immunity and cellular immunity.
Description
Technical field
The present invention relates to toxoplasma vaccine field, is a kind of compound Toxoplasma metabolic secretion antigen vaccine specifically, the invention still further relates to the preparation method of this vaccine.
Background technology
Toxoplasma is the entozoic protozoon of a kind of special sexual cell, and this worm can infect all mammals that comprise human body, and human health and animal husbandry development are caused severe impairment.Up to the present, the development of toxoplasma vaccine is mainly contained (1) Scorpio vaccine, comprise killed vaccine, live vaccine, attenuated live vaccine.Killed vaccine does not have practical value for crowd and domestic animal, and there is diffusing malicious danger in live vaccine and can not applies, and the required strain of attenuated live vaccine needs tissue culture cells system to go down to posterity, and after long-term guarantor planted, its hereditary stability can not guarantee.(2) the special component vaccine of polypide, from polypide lysate or drainage and secretion antigen (ESA), the extraction specific components is a vaccine with immuno-chemical method, the special component vaccine of polypide has certain immunity, but has problems such as extracting difficulty.(3) molecular vaccine, monovalent molecule vaccine and compound polyvalent vaccine are arranged, the antigen of monovalent molecule vaccine such as P30 (SAG1) antigen vaccine, P22 (SAG2) antigen vaccine, P28 antigen vaccine, P23 antigen vaccine and other antigen such as bradyzoite 65kDa, hsp30/bag antigen, P18 antigen, PEF (invade and promote the factor), dense granule antigen, microneme antigen, 54kDa membrane antigen vaccine etc.Compound polyvalent vaccine is that the aminoacid sequence with the functional epi-position of different antigen molecules or different antigen molecules combines, the synthetic associating antigen of design or unite the epitope peptide section.Zoopery shows that unit price subunit vaccine effect is undesirable, is greatly developing compound polyvalent vaccine at present.(4) nucleic acid vaccine, aspect nucleic acid vaccine, the research of toxoplasma vaccine is not carried out as yet.Up to now, still there is not toxoplasma somatic antigen-Toxoplasma metabolic secretion antigen composite type vaccin.The document relevant with the present invention has:
Summary of the invention
The protectiveness that the objective of the invention is to avoid above-mentioned vaccine to have is weak, exist the poison that looses dangerous, make shortcomings such as difficulty; a kind of Toxoplasma metabolic secretion antigen vaccine and preparation method thereof is provided, and this vaccine can significantly improve the specific humoral immunity and the cellular immunization of body.
The objective of the invention is to realize by following technical solution.
A kind of Toxoplasma metabolic secretion antigen vaccine contains toxoplasma antigen, and described toxoplasma antigen adopts the following steps preparation:
(1) with 1 * 10^5 toxoplasma tachyzoite, abdominal cavity inoculation 20-25 gram body weight mice, through 72-96 hour, mice occur spirit depressed, during by the thick symptom such as disorderly of hair, take off the neck white mice that causes death, be soaked in body surface sterilization in the 70-75% ethanol; Every mice is washed abdomen with 2-3 milliliter PH7.2PBS buffer and collects polypide; Collected peritoneal fluid is changeed with per minute 3000-4000 the centrifugal 20-30 of speed minute, collect supernatant, centrifugal 3-5 time of the speed that supernatant changes with per minute 3000-4000 again, each centrifugal 20-30 minute, the collection supernatant ,-15--20 ℃ preservation is standby;
(2) with 2 * 10^7 toxoplasma tachyzoite, be inoculated in the vero African green monkey kidney cell, in incubator, continue to cultivate under 37 ℃ of conditions, after treating that cell all comes off, collect culture fluid, change with per minute 3000-4000 the centrifugal 20-30 of speed minute, centrifugal 3 times of the speed that supernatant changes with per minute 3000-4000 again, each centrifugal 20-30 minute, collect supernatant-15--20 ℃ preservation is standby;
(3) with peritoneal fluid and culture fluid multigelation 3 times;
(4) with peritoneal fluid and cell culture fluid with 1: the mixed of 8-10 obtains toxoplasma antigen.
Described antigen is Toxoplasma metabolic secretion antigen.
Described metabolic secretion antigen is from cell culture and the antigenic hybrid antigen of mouse peritoneal inoculation gained.
Be 8-10 from the antigenic ratio of cell culture and mouse peritoneal inoculation gained in the described antigen: 1.
The preparation method of described Toxoplasma metabolic secretion antigen vaccine is that toxoplasma antigen is finished according to the following steps:
(1) toxoplasma antigen is carried out determining the protein quantity, protein content is 10mg/ml;
(2) with mixed liquor and M206 adjuvant ratio mixing with 1: 1, emulsifying, packing is Toxoplasma metabolic secretion antigen vaccine.
Antigen protein content wherein is 10mg/ml.
The invention has the advantages that: in toxoplasma vaccine production process in the past, metabolic secretion antigens a large amount of in the mouse peritoneal liquid are passed into disuse after centrifugal (1), and the present invention is used for metabolic secretion antigen the preparation of resisting toxoplasmosis vaccine at home first.(2) more effectively induce humoral immunization and cellular immunization.Show as the higher specific IgG antibodies of generation, specificity lymphproliferation response positive rate raises, and the mononuclear cell that induces body under the stimulation of specific antigen produces more gamma interferon.(3) immune efficacy that is higher than weak malicious Seedling is arranged, but do not have the diffusing poison danger of weak malicious Seedling.
Behind the Toxoplasma metabolic secretion antigen vaccine immune mouse, the no abnormal reaction of mice.The strong malicious GJS strain counteracting toxic substances of toxoplasma after 42 days, morbidity in the 3rd day behind the contrast Mus counteracting toxic substances, is tiredly contracted, is increased by Mao Songluan, abdominal circumference down in spirits, and the 7th day is all dead, and abdomen liquid microscopy is found to contain the worm amount and is reached every visual field (40 * 10 times) 〉=50 tachyzoites.Behind the E/SA emulsifying Seedling immunized mice counteracting toxic substances, it is normal to survive before 6 days, and clinical symptoms appearred in indivedual mices in the 7th day, and beginning in the 8th day is dead, all death in the 12nd day.The results are shown in Table 1.
Table 1 Toxoplasma metabolic secretion antigen vaccine is to the protection test of mice
Immune antibody and lymphocyte responses reaction: it is all negative that each organizes sheep test front arcuate worm serum antibody, and the 1st immunity back 1-4 group goat-anti body titre raises in various degree, and (1: 4-1: 256), antibody titer rises to 1: 1024 behind the booster immunization.
Each is organized, and Sanguis caprae seu ovis smear ANAE detects and the statistical procedures result, and the T of immune sheep and immune sheep not, bone-marrow-derived lymphocyte immune response difference is remarkable (being respectively T lymphocyte P＜0.01, bone-marrow-derived lymphocyte P＜0.01) extremely.The results are shown in Table 2.
Table 2 toxoplasma E/SA vaccine is to the immune effect observation of sheep
Toxoplasma E/SA vaccine immunity flock of sheep abortion ratio be starkly lower than the abortion ratio of contrast flock of sheep of not carrying out immunity, its abortion ratio reduces about 50% (47.7-52.5%), 1323 of 6151 test sheep fecund lambs.The results are shown in Table 3.
Table 3 toxoplasma E/SA vaccine test group and the lambing of matched group sheep, miscarriage situation statistical table
Before the vaccinate, 9 pig toxoplasma IHA antibody are all negative; Annotated for the 1st time behind the Seedling 14 days, the immune swine serum antibody titer is 1: 16-1: 64, and behind the booster immunization 14 days, (back 28 days of immunity for the first time), serum antibody titer is 1: 64-1: 1024.Vaccinated 7 pigs, 6 occurred disposable body temperature reaction in 6 days behind counteracting toxic substances, and 1 the fervescence reaction do not occur, and significant change does not appear in spirit, appetite.Two contrast pig is the 3rd day occur generating heat (continuing 7-8 days) behind counteracting toxic substances, and body temperature is continued fever up to 42 ℃, loss of appetite even useless exhausted, down in spirits, 1 death.The results are shown in Table 4.
Table 4 toxoplasma E/SA vaccine is to the immune effect observation of pig
The specific embodiment
Embodiment 1,
On November 5th, 2003 is with 1 * 10^5 toxoplasma tachyzoite/only, 150 of abdominal cavity inoculation 20-25 gram body weight mices, November 8, mice occurred spiritual depressed, by the thick symptom such as disorderly of hair, white mice is taken off neck cause death, be soaked in and carry out the body surface sterilization in 70% ethanol.Every mice is washed abdomen with 2 milliliters of PH7.2PBS buffer and collects polypide, collect peritoneal fluid 400ml altogether, centrifugal 30 minutes of the speed that collected peritoneal fluid is changeed with per minute 3000, collect supernatant, centrifugal 5 times of the speed that supernatant changes with per minute 3000 again, each centrifugal 30 minutes, collect supernatant, standby-20 ℃ of preservations.
While is the amount with 2 * 10^7 toxoplasma tachyzoite/bottle on November 5th, 2003, be inoculated in well-grown and do not contain in the vero cell of serum culture fluid original fluid is toppled over and add new 100ml, inoculate 35 bottles altogether, in incubator, continue to cultivate under 37 ℃ of conditions, after November 10, cell all came off, collect culture fluid, centrifugal 30 minutes of the speed of changeing with per minute 3000, centrifugal 3 times of the speed that supernatant changes with per minute 3000 again, each centrifugal 30 minutes, it was standby-20 ℃ of preservations to collect supernatant.
On November 18th, 2003, take a morsel peritoneal fluid and culture fluid carried out determining the protein quantity with peritoneal fluid and culture fluid multigelation 3 times, and the protein content of peritoneal fluid is 30.67mg/ml as a result, and the protein content of cell culture fluid is 8.58mg/ml.With peritoneal fluid and the cell culture fluid mixed with 1: 8, mixed liquor is 3500ml, and its protein content is 10mg/ml.
On November 25th, 2003 is mixed liquor and the M206 adjuvant ratio mixing with 1: 1, emulsifying, and packing is Toxoplasma metabolic secretion antigen vaccine.
Embodiment 2:
On February 5th, 2004 is with 1 * 10^5 toxoplasma tachyzoite/only, 100 of abdominal cavity inoculation 20-25 gram body weight mices, February 9, mice occurred spiritual depressed, by the thick symptom such as disorderly of hair, white mice is taken off neck cause death, be soaked in and carry out the body surface sterilization in 75% ethanol.Every mice is washed abdomen with 2-3 milliliter PH7.2PBS buffer and collects polypide, collect peritoneal fluid 250ml altogether, centrifugal 20 minutes of the speed that collected peritoneal fluid is changeed with per minute 4000, collect supernatant, centrifugal 3 times of the speed that supernatant changes with per minute 4000 again, each centrifugal 20 minutes, collect supernatant, standby-15 ℃ of preservations.
While is the amount with 2 * 10^7 toxoplasma tachyzoite/bottle on February 5th, 2004, be inoculated in well-grown and do not contain in the vero cell of serum culture fluid original fluid is toppled over and add new 100ml, inoculate 28 bottles altogether, in incubator, continue to cultivate under 37 ℃ of conditions, after February 10, cell all came off, collect culture fluid, centrifugal 20 minutes of the speed of changeing with per minute 4000, centrifugal 3 times of the speed that supernatant changes with per minute 4000 again, each centrifugal 20 minutes, it was standby-15 ℃ of preservations to collect supernatant.
On February 18th, 2004, take a morsel peritoneal fluid and culture fluid carried out determining the protein quantity with peritoneal fluid and culture fluid multigelation 3 times, and the protein content of peritoneal fluid is 32.15mg/ml as a result, and the protein content of cell culture fluid is 7.24mg/ml.With peritoneal fluid and the cell culture fluid mixed with 1: 10, mixed liquor is 2750ml, and its protein content is 10mg/ml.
On February 25th, 2004 is with mixed liquor and the M206 adjuvant ratio mixing with 1: 1, emulsifying, packing.Be Toxoplasma metabolic secretion antigen vaccine.
Claims (6)
1. a Toxoplasma metabolic secretion antigen vaccine is characterized in that containing toxoplasma antigen, and described toxoplasma antigen adopts the following steps preparation:
(1) with 1 * 10^5 toxoplasma tachyzoite, abdominal cavity inoculation 20-25 gram body weight mice, through 72-96 hour, mice occur spirit depressed, during by the thick random symptom of hair, take off the neck white mice that causes death, be soaked in body surface sterilization in the 70-75% ethanol; Every mice is washed abdomen with 2-3 milliliter PH7.2PBS buffer and collects polypide; Collected peritoneal fluid is changeed with per minute 3000-4000 the centrifugal 20-30 of speed minute, collect supernatant, centrifugal 3-5 time of the speed that supernatant changes with per minute 3000-4000 again, each centrifugal 20-30 minute, the collection supernatant ,-15--20 ℃ preservation is standby;
(2) with 2 * 10^7 toxoplasma tachyzoite, be inoculated in the vero African green monkey kidney cell, in incubator, continue to cultivate under 37 ℃ of conditions, after treating that cell all comes off, collect culture fluid, change with per minute 3000-4000 the centrifugal 20-30 of speed minute, centrifugal 3 times of the speed that supernatant changes with per minute 3000-4000 again, each centrifugal 20-30 minute, collect supernatant-15--20 ℃ preservation is standby;
(3) with peritoneal fluid and culture fluid multigelation 3 times;
(4) with peritoneal fluid and cell culture fluid with 1: the mixed of 8-10 obtains toxoplasma antigen.
2. Toxoplasma metabolic secretion antigen vaccine according to claim 1 is characterized in that described antigen is Toxoplasma metabolic secretion antigen.
3. Toxoplasma metabolic secretion antigen vaccine according to claim 2 is characterized in that described metabolic secretion antigen is for inoculating the antigenic hybrid antigen of gained from cell culture and mouse peritoneal.
4. Toxoplasma metabolic secretion antigen vaccine according to claim 1 is characterized in that in the described antigen that from cell culture and the antigenic ratio of mouse peritoneal inoculation gained be 8-10: 1.
5. method for preparing Toxoplasma metabolic secretion antigen vaccine as claimed in claim 1 is characterized in that toxoplasma antigen is finished according to the following steps:
(1) toxoplasma antigen is carried out determining the protein quantity, protein content is 10mg/ml;
(2) with mixed liquor and M206 adjuvant ratio mixing with 1: 1, emulsifying, packing is Toxoplasma metabolic secretion antigen vaccine.
6. the preparation method of Toxoplasma metabolic secretion antigen vaccine as claimed in claim 5 is characterized in that antigen protein content wherein is 10mg/ml.
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| CN102198267A (en) * | 2011-03-22 | 2011-09-28 | 吉林大学 | Recombinant bacillus calmette guerin vaccine for toxoplamasis and preparation method thereof |
| CN111407886B (en) * | 2020-02-28 | 2022-08-26 | 南京农业大学 | Nano-material subunit vaccine for preventing Toxoplasma gondii infection and preparation method and application thereof |
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| CN1450087A (en) * | 2003-05-09 | 2003-10-22 | 李越希 | Recombinant Toxoplasma fusion protein antigen and preparation process and use thereof |
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| CN1450087A (en) * | 2003-05-09 | 2003-10-22 | 李越希 | Recombinant Toxoplasma fusion protein antigen and preparation process and use thereof |
Non-Patent Citations (3)
| Title |
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| 弓形虫不同组分抗原免疫保护性的研究. 黄炳成等.中国寄生虫病防治杂志，第16卷第6期. 2003 * |
| 弓形虫疫苗研究进展. 蒋华等.国外医学寄生虫病分册，第31卷第6期. 2004 * |
| 用弓形虫代谢分泌抗原制备间接血凝诊断试剂的研究. 张德林等.中国兽医科技，第33卷第9期. 2003 * |
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