CN101759774A - Method for preparing porcine circovirus disease resisting transfer factor - Google Patents
Method for preparing porcine circovirus disease resisting transfer factor Download PDFInfo
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- CN101759774A CN101759774A CN200810154346A CN200810154346A CN101759774A CN 101759774 A CN101759774 A CN 101759774A CN 200810154346 A CN200810154346 A CN 200810154346A CN 200810154346 A CN200810154346 A CN 200810154346A CN 101759774 A CN101759774 A CN 101759774A
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Abstract
The invention discloses a method for preparing a transfer factor naturally resisting porcine circovirus disease in high efficiency. The preparation method comprises the steps of: preparing viral antigens, extracting viral antigens, immunizing swine with the viral antigens, extracting the porcine circovirus disease resisting transfer factor from the spleen of the immune swine and the like. The transfer factor has the advantages of preventing porcine circovirus disease, protecting cells of a normal body against porcine circovirus disease, and lowering incidence; and simultaneously, when the transfer factor is used for treating the swine infected with porcine circovirus disease, the transfer factor can effectively improve clinical symptoms, increasing a curative ratio and lowering a death rate.
Description
Technical field
The present invention relates to medicine and formulation art thereof, more particularly, relate to a kind of preparation method of porcine circovirus disease resisting transfer factor.
Background technology
In recent years, the porcine circovirus disease is widely current in many countries as a kind of new virus disease.Porcine circovirus II type can cause wean pigling multisystemic exhaustion syndrome, pig interstitial pneumonia, the scorching nephrotic syndrome of pigskin) and the sow breeding difficulty etc., these diseases are generically and collectively referred to as the porcine circovirus disease.
Breeding difficulty porcine circovirus II type can cause sow to return the feelings rate increasing, and stillborn foetus, mummy tire and weakon etc. are produced in miscarriage, the preceding mortality ratio rising of the institute's pigling that produces wean.After boar infects PCV-II II type, can by mating infect to join sow, thereby cause its breeding difficulty.
Scorching and this disease of nephrotic syndrome of pigskin betides the pig in 8～18 all ages usually, and sick pig feed is for waste exhausted, fervescence to 41.5 ℃, subcutaneous dropsy.Red-purple pathology patch appears in skin, and is the most obvious at perineal position and four limbs, and these patches merge sometimes mutually.Dermatosis can disappear under few situation.Red-purple extravasated blood, petechiae or ecchymosis appear in the rear quarters position that the pathology that also has shows as pig.Yellow hydrothorax or pericardial effusion appear in visual superficial lymph knot enlargement.Kidney is glomerulonephritis and interstitial nephritis, the visible extravasated blood point in surface.Common sympton also has serious diarrhea and expiratory dyspnea, and disorderly thick by hair.
The pig in ages in interstitial pneumonia interstitial pneumonia main harm 6～14 week, sickness rate is 2%～30%, mortality ratio is 4%～10%.Observing pathology is the diffuse interstitial pneumonia, and color is bois de rose.Sometimes visible lung exists II type pneumonocyte hyperplasia district and the necrosis of bronchiole epithelium and contains the zone of non-viable non-apoptotic cell fragment, visible sometimes hyalin in the alveolar space.
Wean pigling multisystemic exhaustion syndrome be mainly in 6～8 age in week pigling, sickness rate is 20%～60%, mortality ratio is 5%～35%.Occur in 2～3 weeks of pigling wean back with cough, have difficulty in breathing, become thin gradually, mortality ratio and mortality all significantly rising be the disease of feature.Sick pig heating (generally being no more than 41 ℃), appetite stimulator occurs becoming thin,, ochrodermia thick disorderly by hair or symptoms such as jaundice, expiratory dyspnea then.Indivedual pig eyes have secretory product, diarrhoea, elbow joint and knee joint swelling.It cuts open inspection and is characterized as the lymph nodes of body as a whole enlargement and pericarditis, pleuropneumonia, peritonitis etc. take place, and uses multiple antibiotic therapy effect undesirable.
PCV-II II type can cause the immunizing power of infected pigs to reduce greatly, thereby has created condition for the invasion of other viruses or germ.As PCV-II and reproductive and respiratory syndrome virus synergy, when making pig secondary infection gold-coloured staphylococci, suis, thereby make the disease pig fervescence, congested fash, vomiting, diarrhoea, expiratory dyspnea etc. occur, because the keying action that these bacterium institute toxin producings are risen in this disease takes place can cause pig multiple organ dysfunction syndrome syndrome.
Although the clinical application of some anti-pig circular ring virus 2s is arranged in the market, striving property of existing medicament is not strong, and curative effect is limited, and mostly therapeutic preparation is what adopted the morbidity back, can not play the effect of prevention.Swinery for the easy infection pig circular ring virus 2.What every year was regular is the effective ways of prevention pig circular ring virus 2 to pig inoculation porcine circovirus vaccine, secondly should strengthen nutrition or drug intervention, improving autoimmunity, also is a kind of effective ways that prevent pig circular ring virus 2 by medicine raising Abwehrkraft des Koepers or immunizing power.
Transfer factor (TF, Transfer Factor) is a class low molecular peptide and a nucleotide complex that from lymphocyte, extracts, have premunition information, excite immunologic cellular activity, regulate effects such as immunologic function, enhancing body non-specific cell immunologic function, be described as the triggering agent of T cytoactive, the toughener of cellular immunization, cell immunomodulator and Interferon, rabbit produce and start agent.The transfer factor molecular weight is little, nontoxic, no antigen, do not play anaphylaxis, and antibody and can to surmount kind be advantage such as boundary application does not create antagonism.Since the famous immunology expert Lawrence of the U.S. found and studies transfer factor, many theoretical investigationes had not only been carried out in countries in the world, and have carried out a large amount of clinical application researchs, make transfer factor become present widely used enhancing immunity preparation.
The transfer factor great majority of the extraction of having reported at present all are the preparation methods about non-specific transfer factor.The present invention adopts the antigenic method of virus immunity to extract specific transfer factor, and extracting method is improved on the traditional method basis of having reported, shortens the production time, simplifies production technique.
Adopt special virus strain immunizing antigen to extract the transfer factor of anti-porcine circovirus, have important theory and realistic meaning.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, and a kind of transfer factor of anti-porcine circovirus of efficiency natural is provided.
First purpose of the present invention provides a kind of preparation method of simple and effective anti-pig circular ring virus 2 transfer factor, and step comprises:
(1) cleans the pig spleen of crossing with the porcine circovirus antigen immune with water for injection earlier, remove tissues such as its surperficial manadesma, fat, use normal saline flushing again;
(2) pig spleen of cleaning is shredded, add 1～2 times of cold 10mmol PBS damping fluid, smash to pieces 3～5 times with the 1000～3000rpm of high-speed tissue mashing machine, each 3～5min makes pig spleen homogenate;
(3) pig spleen homogenate is handled 5～10min with ultrasonic wave (4KW, 3KHz);
(4) homogenate that obtains in the step (3) is transferred pH5.0～6.0 or added 0.1～0.3% citric acid with HCl, 4 ℃, the centrifugal 10～30min of 3000～8000rpm collects supernatant liquor;
(5) supernatant liquor is with 0.2 μ m hollow fiber column micro-filtration, and filtrate is 6000 daltonian hollow fiber column ultrafilters with molecular weight cut-off again, collects filtrate;
(6) filtrate that ultrafiltration is obtained is carried out sterile filtration with 0.22 μ m filter membrane, promptly obtains porcine circovirus disease resisting transfer factor stoste, 4 ℃ of preservations.
In specific embodiments, to implement before the above-mentioned steps, step also comprises the preparation virus antigen, extracts the step of virus antigen, virus antigen immune swine.These steps can select for use routine techniques to implement.Wherein, described preparation virus antigen step, preferred: as porcine circovirus to be inoculated in 9～11 age in days chick embryo allantoic liquids to cultivate 24～72h for 30～40 ℃.Get chick embryo allantoic liquid ,-20 ℃ of multigelations 4～8 times, the centrifugal 10～30min of 3000～8000rpm discards precipitation and collects supernatant liquor.Wherein, porcine circovirus is selected good strains in the field for seed from the PCV-II of the applicant's preservation (Genbank accession NO.AF027217 or Genbank accession NO.AF201897).
Described extraction virus antigen step, preferably: supernatant liquor extracts virus with the sucrose density gradient centrifugation purifying, sucrose concentration is 10%～80%, centrifugal 2～4 hours of 150000～200000rpm, determine the position of porcine circovirus place centrifuge tube according to the porcine circovirus molecular weight, sucking-off is standby;
Described virus antigen immune swine step, preferred: select healthy immune swine, with the subcutaneous multi-point injection immune swine of the virus antigen of said extracted, immunity is 3～5 times altogether.Each immunity is a week at interval, and last immunity was got porcine vein after 7～10 days, detects the specific antibody titres of above-mentioned virus with immunofluorescence technique or ELISA method, the virus-specific immunizing potency appears and after, slaughter pig, get spleen in-20 ℃ of preservations, standby.
Second purpose of the present invention is to provide by the prepared transfer factor of aforesaid method.
In a preferred embodiment, the porcine circovirus of aforesaid method use is selected good strains in the field for seed from the PCV-II of the applicant's preservation (Genbank accession NO.AF027217 or Genbank accession NO.AF201897).
Because transfer factor is a class low molecular peptide and a nucleotide complex that extracts from lymphocyte, do not have clear and definite component or structure at present, the difference of preparation method or used virus strain, the transfer factor of gained also can difference, therefore is in process of production usually to come clear and definite transfer factor by limiting preparation method and used virus strain.In the present invention, owing to define concrete preparation method and special virus strain, therefore resulting transfer factor has characteristics such as component is stable, purity is high, activity is good, has good application prospects.
Technique effect
The preparation method of a kind of porcine circovirus disease resisting transfer factor of the present invention has the following advantages:
1, pig spleen belongs to the tankage in slaughterhouse, and raw material sources are easy;
2, tissue adopts powerful ultrasonic equipment to handle after tissue mashing machine smashs to pieces, shortens the multigelation time, and when having avoided because of the high pressure homogenizer broken wall treatment because high pressure homogenizer is stopped up in the existence of manadesma;
3, regulate pH with dilute hydrochloric acid before centrifugal, make most of albumen flocculation, make the clarity of the supernatant liquor after centrifugal significantly better than former method; And have and utilize follow-up micro-filtration, ultrafiltration;
4, in homogenate, add 0.1～0.3% citric acid and also play the effect that makes the albumen flocculation, simplify the centrifugal treating of back;
5, the present invention both can be based on the conventional virus antigen immunity step in early stage, again can preferred immune step of the present invention, have flexible and efficient advantage.
6, the extracting method of described transfer factor has shortened the production time significantly, has simplified production technique.
7, extract is through proofs such as external biological activity test and pharmacology, toxicity tests, have the purity height, immunocompetence is rapid-action and characteristics such as it is long to hold time, easily absorption, no any untoward reaction, its determined curative effect, safe and reliable, not only can treat but also can strengthen resistance against diseases.Formulations such as that this extract can be made is oral, injection, spraying, easy to use.
8, the used virus strain of the present invention, be selected from the PCV-II (Genbank accession NO.AF027217 or Genbank accession NO.AF201897) of the applicant's preservation, compare with conventional PCV-II strain, has higher immunogenicity, thereby the transfer factor extracted of the method that adopts this virus strain immunizing antigen, comprise specificity is good and purity is high low molecular peptide and nucleotide complex, its activity level obviously is better than ordinary method and the prepared transfer factor of conventional virus strain.
Embodiment
The present invention will be further described below in conjunction with embodiment.
Embodiment 1:
The preparation of porcine circovirus disease resisting transfer factor
1, porcine circovirus is inoculated in 9～11 age in days chick embryo allantoic liquids and cultivates 48h for 37 ℃.Get chick embryo allantoic liquid ,-20 ℃ of multigelations 5 times, the centrifugal 30min of 5000rpm discards precipitation and collects supernatant liquor;
2, get supernatant liquor, supernatant liquor extracts virus with the sucrose density gradient centrifugation purifying, sucrose concentration is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, centrifugal 4 hours of 150000rpm, determine the position of porcine circovirus place centrifuge tube according to the porcine circovirus molecular weight, sucking-off is standby;
3, the porcine circovirus antigen immune pig that obtains selects healthy immune swine, and with the subcutaneous multi-point injection immune swine of the virus antigen of said extracted, immunity is 4 times altogether.Each immunity is a week at interval, and last immunity was got porcine vein after 7 days, detects the specific antibody titres of above-mentioned virus with immunofluorescence technique or ELISA method, the virus-specific immunizing potency appears and after, slaughter pig, get spleen in-20 ℃ of preservations, standby;
4, extract porcine circovirus disease resisting transfer factor from the pig spleen that obtains:
(1) cleans spleen with water for injection earlier, remove tissues such as its surperficial manadesma, fat, use normal saline flushing again;
(2) pig spleen of cleaning is shredded, add 2 times of cold 10mmol PBS damping fluids, smash to pieces 3 times with the 2000rpm of high-speed tissue mashing machine, each 5min makes pig spleen homogenate;
(3) pig spleen homogenate is handled 5min with ultrasonic wave (4KW, 3KHz);
(4) homogenate that obtains in (3) is transferred pH5.0 with HCl, 4 ℃, the centrifugal 30min of 5000rpm collects supernatant liquor;
(5) supernatant liquor is collected filtrate with 0.2 μ m hollow fiber column micro-filtration;
(6) filtrate is 6000 daltonian hollow fiber column ultrafilters with molecular weight cut-off again, collects filtrate;
(7) filtrate that ultrafiltration is obtained is carried out sterile filtration with 0.22 μ m filter membrane;
(8) packing, 4 ℃ of preservations promptly obtain porcine circovirus disease resisting transfer factor stoste.
Embodiment 2:
The preparation of porcine circovirus disease resisting transfer factor
1, porcine circovirus is inoculated in 9～11 age in days chick embryo allantoic liquids and cultivates 72h for 37 ℃.Get chick embryo allantoic liquid ,-20 ℃ of multigelations 5 times, the centrifugal 30min of 5000rpm discards precipitation and collects supernatant liquor;
2, get supernatant liquor, supernatant liquor extracts virus with the sucrose density gradient centrifugation purifying, and sucrose concentration is 15%, 30%, 45%, 60%, 75%, centrifugal 3 hours of 180000rpm, determine the position of porcine circovirus place centrifuge tube according to the porcine circovirus molecular weight, sucking-off is standby;
3, the porcine circovirus antigen immune pig that obtains selects healthy immune swine, and with the subcutaneous multi-point injection immune swine of the virus antigen of said extracted, immunity is 4 times altogether.Each immunity is a week at interval, and last immunity was got porcine vein after 10 days, detects the specific antibody titres of above-mentioned virus with immunofluorescence technique or ELISA method, the virus-specific immunizing potency appears and after, slaughter pig, get spleen in-20 ℃ of preservations, standby;
4, extract porcine circovirus disease resisting transfer factor from the pig spleen that obtains:
(1) cleans spleen with water for injection earlier, remove tissues such as its surperficial manadesma, fat, use normal saline flushing again;
(2) pig spleen of cleaning is shredded, add 2 times of cold 10mmol PBS damping fluids, smash to pieces 3 times with the 1500rpm of high-speed tissue mashing machine, each 5min makes pig spleen homogenate;
(3) pig spleen homogenate is handled 10min with ultrasonic wave (4KW, 3KHz);
(4) with the homogenate that obtains in (3) adding 0.1% citric acid, mixing, 4 ℃, the centrifugal 30min of 6000rpm collects supernatant liquor;
(5) supernatant liquor is collected filtrate with 0.2 μ m hollow fiber column micro-filtration;
(6) filtrate is 6000 daltonian hollow fiber column ultrafilters with molecular weight cut-off again, collects filtrate;
(7) filtrate that ultrafiltration is obtained is carried out sterile filtration with 0.22 μ m filter membrane;
(8) packing, 4 ℃ of preservations promptly obtain porcine circovirus disease resisting transfer factor stoste.
Embodiment 3:
Detection to the porcine circovirus disease resisting transfer factor of prepared of the present invention
Porcine circovirus disease resisting transfer factor to embodiment 1,2 described prepared carries out following detection:
1, ultraviolet spectrophotometry: above-mentioned gained sample has an absorption peak at 252 ± 2nm place, and ABS
260/ ABS
280＞2.0.
2, reference liquid is faint yellow, and the pH value is between 6.0～8.0.
3, bacteriological analysis: no aerobic, anaerobism, saprophytic microorganism and fungi exist in the above-mentioned gained sample.
4,20% sulphosalicylic acid detects: above-mentioned gained sample does not have muddiness and deposited phenomenon, illustrates that albumen test all becomes negative, and it does not contain macro-molecular protein.
5, nucleic acid content is measured: above-mentioned gained sample is measured nucleic acid content through orcin method and is respectively 603.14 μ g/mL, 594.55 μ g/mL.
6, determining content of peptides: above-mentioned gained sample is measured content of peptides through the Lowry method and is respectively: 7.08mg/mL, 6.88mg/mL.
7, transfer factor determination of activity: can make its function of receptors of thymus gland T cellular-restoring that takes off behind the E acceptor according to transfer factor, thus the biological activity of reaction transfer factor.The percentage that above-mentioned gained sample forms the E rosette is respectively 30.5%, 28.4%, and comparison increases by 18.9%, 16.8% (P≤0.01) respectively according to group.
8, supersensitivity, toxicity test: get 10 of healthy mices, male and female half and half, oral this product concentrated solution, be equivalent to 100 times of normal oral dosage, observe the variation of the viability of small white mouse, the result: do not have any allergy, toxic reaction, also do not have any dead appearance, illustrate that this product is safe, does not have any toxicity and side effect.
9, do skin test with porcine circovirus antigen and detect, weak positive reaction is arranged, illustrate that this product has good transfer activity and specificity.
By the foregoing description, we find that the anti-pig circular ring virus 2 transfer factor of being extracted can suppress porcine circovirus comprehensively, can protect the normal body cell to avoid the infection of porcine circovirus.Therefore use transfer factor of the present invention can play the prevention pig circular ring virus 2, protect the normal body cell to avoid the effect of porcine circovirus infringement, thereby reduce sickness rate.Use this transfer factor to treat for the pig that is subjected to the porcine circovirus infection simultaneously, can effectively improve clinical symptom, improve curative ratio, reduce mortality ratio.
Claims (7)
1. the preparation method of the transfer factor of an anti-porcine circovirus, its step comprises:
(1) cleans the pig spleen of crossing with the porcine circovirus antigen immune with water for injection earlier, remove tissues such as its surperficial manadesma, fat, use normal saline flushing again;
(2) pig spleen of cleaning is shredded, add 1～2 times of cold 10mmol PBS damping fluid, smash to pieces 3～5 times with the 1000～3000rpm of high-speed tissue mashing machine, each 3～5min makes pig spleen homogenate;
(3) pig spleen homogenate is handled 5～10min with ultrasonic wave (4KW, 3KHz);
(4) homogenate that obtains in the step (3) is transferred pH5.0～6.0 or added 0.1～0.3% citric acid with HCl, 4 ℃, the centrifugal 10～30min of 3000～8000rpm collects supernatant liquor;
(5) supernatant liquor is with 0.2 μ m hollow fiber column micro-filtration, and filtrate is 6000 daltonian hollow fiber column ultrafilters with molecular weight cut-off again, collects filtrate;
(6) filtrate that ultrafiltration is obtained is carried out sterile filtration with 0.22 μ m filter membrane, promptly obtains porcine circovirus disease resisting transfer factor stoste, 4 ℃ of preservations.
2. the described method of claim 1 is wherein stated before the step on the implementation, also comprises the preparation virus antigen, extracts steps such as virus antigen, virus antigen immune swine.
3. the described method of claim 2, wherein said preparation virus antigen step comprises: porcine circovirus is inoculated in 9～11 age in days chick embryo allantoic liquids cultivates 24～72h for 30～40 ℃.Get chick embryo allantoic liquid ,-20 ℃ of multigelations 4～8 times, the centrifugal 10～30min of 3000～8000rpm discards precipitation and collects supernatant liquor.
4. the described method of claim 2, wherein said extraction virus antigen step, comprise: supernatant liquor extracts virus with the sucrose density gradient centrifugation purifying, sucrose concentration is 10%～80%, centrifugal 2～4 hours of 150000～200000rpm, determine the position of porcine circovirus place centrifuge tube according to the porcine circovirus molecular weight, sucking-off is standby.
5. the described method of claim 2, wherein said virus antigen immune swine step comprises: select healthy immune swine, with the subcutaneous multi-point injection immune swine of the virus antigen of said extracted, immunity is 3～5 times altogether.Each immunity is a week at interval, and last immunity was got porcine vein after 7～10 days, detects the specific antibody titres of above-mentioned virus with immunofluorescence technique or ELISA method, the virus-specific immunizing potency appears and after, slaughter pig, get spleen in-20 ℃ of preservations, standby.
6. claim 1 or 2 described methods, wherein porcine circovirus is selected good strains in the field for seed from the PCV-II of the applicant's preservation (Genbankaccession NO.AF027217 or Genbank accession NO.AF201897).
7. the prepared transfer factor of the method for claim 1 to 6.
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| CN200810154346A CN101759774A (en) | 2008-12-23 | 2008-12-23 | Method for preparing porcine circovirus disease resisting transfer factor |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103724419A (en) * | 2013-12-30 | 2014-04-16 | 天津瑞普生物技术股份有限公司 | Pig spleen transfer factor purification method |
| CN103724420A (en) * | 2013-12-30 | 2014-04-16 | 天津瑞普生物技术股份有限公司 | Pig spleen transfer factor extracting method |
| CN104388509A (en) * | 2014-10-27 | 2015-03-04 | 合肥平光制药有限公司 | Preparation method of hepatocyte growth-promoting factors employing ultrasonic treatment |
-
2008
- 2008-12-23 CN CN200810154346A patent/CN101759774A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103724419A (en) * | 2013-12-30 | 2014-04-16 | 天津瑞普生物技术股份有限公司 | Pig spleen transfer factor purification method |
| CN103724420A (en) * | 2013-12-30 | 2014-04-16 | 天津瑞普生物技术股份有限公司 | Pig spleen transfer factor extracting method |
| CN103724419B (en) * | 2013-12-30 | 2015-11-18 | 天津瑞普生物技术股份有限公司 | A kind of Pig spleen transfer factor purification method |
| CN103724420B (en) * | 2013-12-30 | 2016-02-24 | 天津瑞普生物技术股份有限公司 | A kind of Swine spleen transfer factor extracting method |
| CN104388509A (en) * | 2014-10-27 | 2015-03-04 | 合肥平光制药有限公司 | Preparation method of hepatocyte growth-promoting factors employing ultrasonic treatment |
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Open date: 20100630 |