CN1970082A - Preparation process of autogenous high-immunity serum for treating post-weaning multi systemic wasting syndrome of baby pig - Google Patents

Preparation process of autogenous high-immunity serum for treating post-weaning multi systemic wasting syndrome of baby pig Download PDF

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CN1970082A
CN1970082A CN 200610116631 CN200610116631A CN1970082A CN 1970082 A CN1970082 A CN 1970082A CN 200610116631 CN200610116631 CN 200610116631 CN 200610116631 A CN200610116631 A CN 200610116631A CN 1970082 A CN1970082 A CN 1970082A
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primer
serum
immune
internal organs
hyper
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CN100406062C (en
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朱建国
华修国
张艳玲
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Shanghai Jiaotong University
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Abstract

The invention discloses a house hyper-immune serum making method to treat multi-system weaning exhaustion syndrome PMWS of pigling, which is characterized by the following: adopting multiple PCR technique to detect main pathogen PCV2 in the organ and the content of synergic infection PPV and PRRSV virus; selecting relative high-virus content organ as raw material to make house organ vaccine; proceeding multi-point injection strengthening immune through inactivated organ vaccine; detecting serum antibody level of immune pig; separating serum through high-antibody level pig; detecting the serum after biological safety as treating hyper-immune serum.

Description

Oneself hyper-immune serum preparation method of treatment weaned piglet multisystemic wasting syndrome
Technical field
The present invention relates to a kind of oneself hyper-immune serum preparation method for the treatment of the weaned piglet multisystemic wasting syndrome, the hyper-immune serum for preparing is used for the control of intensive pig production field weaned piglet multisystemic wasting syndrome (PMWS), to guarantee pig farm veterinary's public health security, reduce the M ﹠ M of pig farm PMWS.
Background technology
(the Post-Weaning multi systemic WastingSyndrome of multisystemic wasting syndrome behind the weaned piglet, PMWS) be that (Porcinecircovims 2 by porcine circovirus 2 type, PCV2) etc. weaned piglet that causes and a kind of new disease of the stage of growth can cause the high mortality rate of ablactational baby pig and fattening pig.This disease is worldwide distribution, and the sound development of pig industry in serious harm, more and more is subjected to the extensive concern of association area.In addition, because PCV2 is present in the pig body more widely, so in research or use pig organ transplantation through genetic modification in human body the time, might cause the human infection and cause human new infectious disease, for the safety of organ transplantation has proposed new requirement, need carry out risk assessment as a kind of potential Amphixenosis's cause of disease to PCV2.This disease is proved to be the ubiquity in China, enormous economic loss is caused in China large scale of pig farm field.Studies have shown that, just can not cause PMWS if there is PCV2 to exist, but PMWS excites PCV2 by some factors and causes.These motivating factors comprise virulence factor: pig parvoviral (PPV), pig syndrome of breeding and respiration disorders virus (PRRSV) etc., and non-virulence factor: poor environment, high density, all ages and classes pig mix raising etc.Basic demand by the anti-system of infectious disease, the sick pig of PMWS should all be eliminated, but because this disease sickness rate height, many clinically in addition these type of cases are difficult to make a definite diagnosis for the moment, be subjected to restrictions such as economic condition, many producers are difficult to accomplish all eliminate sick pig, in fact exist the problem that the sick pig of PMWS is treated.For now, this disease does not still have effective specificity vaccine, and its cause of disease belongs to virus in addition, and it is invalid using antibiotic therapy.In a single day this disease popular on the pig farm, constantly be difficult to eradicate with touching, and be one of main eqpidemic disease that endangers at present China's pig industry development.Therefore, development PMWS effectively prevents and treats preparation, has become China's intensive pig production already to produce urgent problem in the reality.
Theory and practice proves that all for the eqpidemic disease of still not having effective vaccine and medical treatment, adopting hyper-immune serum to carry out mass treatment is feasible way.With regard to present China intensive pig production field present situation, the eqpidemic disease that swinery is taken place in most cases has not been by due to the single pathogen infection, and it has been universal phenomenon that a plurality of pathogen are mixed coinfection.The healthy serum of prior art for preparing pick up under the natural infection state pig only, be under the situation of indeterminate cause of disease, to collect mostly, the pathological material of disease cause of disease that relates to is single, and the healthy serum antibody titer that is obtained is not high, can not satisfy the control needs to intensive pig production field PMWS.Studies show that, utilize oneself the PMWS pig internal organs of dying of illness to prepare vaccine, this perforated gesture boar is carried out immunity according to certain procedure, develop hyper-immune serum, under the isolated condition of strictness, the sick pig of this class of the whole audience is treated, can receive excellent curative.Have not yet to see the public technology report relevant with this research.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art and actual demand, a kind of oneself hyper-immune serum preparation method for the treatment of the weaned piglet multisystemic wasting syndrome is provided, the hyper-immune serum that makes can be under the prerequisite of indiffusion cause of disease, PMWS pig to this generation is treated, reduce M ﹠ M, reduce economic loss.
For achieving the above object, the present invention upgrades traditional serum preparation method, under the situation of making a definite diagnosis weaned piglet multisystemic wasting syndrome PMWS epidemic situation through immunological method, adopt main pathogens PCV2 (porcine circovirus 2 type) and coinfection cause of disease PPV (pig parvoviral) and PRRSV (pig syndrome of breeding and respiration disorders virus) viral level in multiplex PCR (polymerase chain reaction) the technology for detection internal organs, select the high relatively internal organs of viral level to make oneself internal organs Seedling of feedstock production, again according to certain immune programme for children, with deactivation internal organs Seedling oneself perforated gesture boar is carried out the multi-point injection reinforced immunological, detect the serum antibody level of immune swine with agar diffusion test, take the high person's of antibody horizontal blood separation serum, after biological safe check, can obtain being used for the treatment of oneself hyper-immune serum of weaned piglet multisystemic wasting syndrome.
The inventive method specifically comprises the steps:
1, the preparation of oneself internal organs inactivated vaccine:
Choose positive and typical PMWS pathological changes occurs and cut open the sick pig internal organs that kill through the PCV2 antibody test, after according to conventional method tested sick pig internal organs being extracted DNA, utilize multiple PCR method to carry out PCV2, PPV, three kinds of virus detections of PRRSV, choose the raw material of viral relative amount the higher person as preparation internal organs Seedling.Wherein, the primer of design detection viral gene is respectively:
PCV2 primer 5 '-primer:5 '-GCGGATCCATGCCAAATAACAAC-3 ',
3’-primer:5’-ATCTCGAGTCATGCTGAGGGTGA-3’;
PPV primer 5 '-primer:5 '-CAGGATCCGCAACCTCACCACC-3 ',
3’-primer:5’-GCCTCGAGTGCATGTTAGATTTCCC-3’;
PRRSV primer: 5 '-primer:5 '-GCGGATCCATGCCAAATAACAAC-3 ',
3’-primer:5’-ATCTCGAGTCATGCTGAGGGTGA-3’
With Potter-Elvehjem Tissue Grinders the internal organs Seedling raw material of choosing is carried out homogenate, filter with sterile gauze then, add 1% formaldehyde in the filtrate in order to deactivation, add the oils adjuvant stirring and emulsifying with filtrate equivalent again, it is standby to make 4 ℃ of preservations of oneself internal organs inactivated vaccine.
2, the preparation of hyper-immune serum: utilize above-mentioned oneself the internal organs inactivated vaccine that makes, the healthy boar of the perforated gesture of this 3 monthly age size is carried out the multi-point injection reinforced immunological, after 2~3 immunity, expand the antibody that detects anti-PCV2 in the immune swine serum with fine jade.To expand through fine jade and detect antibody titer and cut open blood-letting extremely at the immune swine 1: 32 or more, separation of serum adds each 2000 μ g/1000ml of penicillin and streptomycin, after aseptic detection and biological safe detect, obtains hyper-immune serum and packs in the container 4 ℃ of preservations by every 10ml branch into.
The present invention selects to have typical PMWS pathological changes, measure the high internal organs of PCV2, PPV, PRRSV viral level as raw material through PCR, and the former content height of oneself internal organs seedling diseases of preparation can improve immune effect greatly.Utilize the healthy boar reinforced immunological of perforated gesture of this no commodity value, can improve the hyper-immune serum quality.The cause of disease that the present invention is directed to is known, making a definite diagnosis under the prerequisite of epidemic situation, prepare hyper-immune serum with oneself oneself boar of internal organs Seedling immunity and treat oneself sick pig, but whole development process all is in monitor state, can avoid because of cause introducing the probability of new cause of disease with external source serum.
Oneself hyper-immune serum of the present invention preparation, can be under the prerequisite of indiffusion cause of disease, PMWS pig to this generation is treated, make pig farm PMWS epidemic situation obtain stable control, reduce M ﹠ M greatly, reduce economic loss, can save a large amount of medicine spendings simultaneously, have remarkable economic efficiency, be suitable for the production reality of China intensive pig production field.
The specific embodiment
Below in conjunction with specific embodiment technical scheme of the present invention is further described.
Embodiment 1
The intensification in somewhere ten thousand popular for successive years doubtful weaned piglet multisystemic wasting syndromes in pig farm (PMWS), sickness rate reaches 30%-50%, causes this piglet growth to be obstructed, and death increases year by year, suffers heavy losses.The present invention selects this for project implementation base, at first a pig farm PMWS suspected case is carried out the PCV2 antibody test: with the PCV2 strain of PK-15 cell culture, behind the density gradient centrifugation purification as envelope antigen; The content bag of pressing the 10ug/ hole is by 96 hole elisa plates, and 37 ℃, 2h, PBS (phosphate buffer) washing; The sick porcine blood serum of taking the doubtful symptom of PMWS is as sample to be checked; Serum to be checked is pressed doubling dilution, add respectively ELISA bag by in the plate carrying out antigen antibody reaction, 37 ℃, 2h, PBS washing; The goat-anti pig IgG that adds the HRP labelling of working concentration (description regulation) is two anti-ly to react equally, 37 ℃, and 2h, PBS washs; Add substrate benzidine (OPD), 37 ℃, 20min develops the color; On enzyme mark reading apparatus, read every hole count value.Be judged to the positive with OD value sample well/negative control hole ratio greater than 2, have only the male case of PCV2 testing result just can carry out next step and cut open the inspection diagnosis.
The positive pig of PCV2 antibody test cutd open kill, cut open the inspection program according to routine pathology then and carry out pathological observation, great majority are suffered from pigs and are presented many organs popularity pathology damage, visible lymphadenitis, and with superficial inguinal lymph nodes and mesenteric lymph node for very.Superficial inguinal lymph nodes enlargement, many kitchen ranges property interstitial pneumonia, tonsillitis, hepatitis interstitialis chronica and gastric ulcer, enteritis etc.Kidney cortex and medullary substance have and are dispersed in white downright bad point not of uniform size, renomegaly.Granuloma can appear in liver, lung, pancreas, adrenal gland, salivary gland and lymphoid tissue.Have only and detect positive person and carry out comprehensive diagnos, just can be diagnosed as the PMWS pig above-mentioned typical PMWS pathological changes and PCV2 occurring.
Oneself the preparation method step of hyper-immune serum of the present invention is as follows:
1, the preparation of oneself internal organs inactivated vaccine:
Choose positive and typical PMWS pathological changes occurs and cut open the sick pig internal organs that kill through the PCV2 antibody test, according to conventional method tested sick pig internal organs are extracted DNA after, utilize multiple PCR method to carry out PCV2, PPV, three kinds of viruses of PRRSV detect.Wherein, the primer of design detection viral gene is respectively:
PCV2 primer 5 '-primer:5 '-GCGGATCCATGCCAAATAACAAC-3 ',
3’-primer:5’-ATCTCGAGTCATGCTGAGGGTGA-3’;
PPV primer 5 '-primer:5 '-CAGGATCCGCAACCTCACCACC-3 ',
3’-primer:5’-GCCTCGAGTGCATGTTAGATTTCCC-3’;
PRRSV primer: 5 '-primer:5 '-GCGGATCCATGCCAAATAACAAC-3 ',
3’-primer:5’-ATCTCGAGTCATGCTGAGGGTGA-3’
The PCR reaction system is 50 μ L, consists of: 10 * PCR buffer, 5 μ L, dNTPs (2.5mM) 4 μ L, 3 couples of each 1 μ L of primer, template DNA (eDNA) 10 μ L, rTaq enzyme 0.5 μ L (5U/ μ L), add sterilization ultra-pure water to 50 μ L, mix homogeneously adds 30 μ L sterilization paraffin oil.Behind 95 ℃ of degeneration 10min of mixture, by 94 ℃ of degeneration 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 1.5min, carry out 30 circulations altogether, and last 72 ℃ are extended 10min.The PCR product carries out electrophoresis with 1% agarose gel, and uviol lamp is observed the amplified fragments size down.Positive with the sample that three specific bands occur.Choose the relative the higher person of viral level, the material of conduct preparation internal organs Seedling after the aseptic taking-up.
With Potter-Elvehjem Tissue Grinders the high internal organs of choosing of viral level are carried out homogenate, filter with sterile gauze then, add 1% formaldehyde in the filtrate and do in order to deactivation, the oils adjuvants such as pitch that add again with filtrate equivalent carry out emulsifying with motor stirrer, and it is standby to make 4 ℃ of preservations of oneself internal organs inactivated vaccine.
2, the preparation of hyper-immune serum:
Picking is originally seen healthy active outside the venue, the perforated gesture boar pig of 3 monthly age sizes, utilize above-mentioned oneself the internal organs inactivated vaccine that makes respectively to select carry out intramuscular injection at 5 along boar pig spinal column both sides, every some 1ml, carry out reinforced immunological, exempt from the back headed by the immune programme for children and carry out 23 weeks and exempt from, 2 exempt from the back carries out 33 weeks and exempts from, and 3 exempt from the back expands the antibody that detects anti-PCV2 in the immune swine serum with fine jade.
To expand the detection antibody titer through fine jade and cut open blood-letting extremely at the immune swine more than 1: 32, separation of serum adds each 2000 μ g/1000ml of penicillin and streptomycin, obtains hyper-immune serum.Get the 10ml hyper-immune serum and pack in the container, cultivate down at 37 ℃, to carry out aseptic detection; By every part 10ml intramuscular injection 31 monthly ages piglets, observe its health status simultaneously, detect to carry out biological safe.It is qualified to detect, and then divides in the container of packing into 4 ℃ of preservations by every 10ml with the hyper-immune serum that obtains.
The application of oneself hyper-immune serum that the present invention prepares:
Cut open inspection, serological test etc. and be diagnosed as sick and weak piglet (about 2 monthly ages) in the PMWS swinery through epidemiology, the clinical shape, pathology of examining, under the isolated condition of strictness, treated 3 days continuously with antibiotic, sulfanilamide etc. earlier, do not see and take a favorable turn, oneself hyper-immune serum of the each intramuscular injection 10ml of every then pig the present invention preparation, once a day, for three days on end.Afterwards, consider that PMWS has the situation of secondary bacterial infection, be aided with treatments such as gentamycin, penicillin and streptomycin, fluid infusion salt and stomachic 3 days.Test carries out twice altogether, and the first time is totally 338 sick pigs, and wherein 39 have obvious systemic bleeding speckle, and dead 37, the whole rehabilitations of the not obvious person in all the other petechias, cure rate reaches 89%.247 sick pigs have 61 of obvious systemic bleeding speckle persons for the second time, and dead 46, all the other whole rehabilitations, cure rate reaches 81%.Oneself hyper-immune serum of the present invention simultaneously, is aided with anti-microbial type medicine and some symptomatic treatment measures to treating then works very well of the sick pig of early stage or chronic PMWS, can receive even more ideal therapeutic effect.
After oneself hyper-immune serum that self-application the present invention developed, this pig farm PMWS epidemic situation obtains stable control.At present, the pig mortality rate drops to present about 1% by about 30% before 3 years.What utilize is the test pig of this perforated gesture boar that has no commercial value as the preparation hyper-immune serum, not only do not increase cost, saved the spending of a large amount of medicines on the contrary, this Accounting Department's cost accounting shows that this new hyper-immune serum has brought tangible economic benefit.

Claims (2)

1, a kind of oneself hyper-immune serum preparation method for the treatment of the weaned piglet multisystemic wasting syndrome is characterized in that comprising the steps:
1) preparation of oneself internal organs inactivated vaccine: choose positive and typical weaned piglet multisystemic wasting syndrome pathological changes occurs and cut open the sick pig internal organs that kill through the PCV2 antibody test, behind tested sick pig internal organs extraction DNA, utilize multiple PCR method to carry out PCV2, PPV, three kinds of virus detections of PRRSV, choose the raw material of viral relative amount the higher person as preparation internal organs Seedling; Wherein, the primer of design detection viral gene is respectively:
PCV2 primer 5 '-primer:5 '-GCGGATCCATGCCAAATAACAAC-3 ',
3’-primer:5’-ATCTCGAGTCATGCTGAGGGTGA-3’;
PPV primer 5 '-primer:5 '-CAGGATCCGCAACCTCACCACC-3 ',
3’-primer:5’-GCCTCGAGTGCATGTTAGATTTCCC-3’;
PRRSV primer: 5 '-primer:5 '-GCGGATCCATGCCAAATAACAAC-3 ',
3’-primer:5’-ATCTCGAGTCATGCTGAGGGTGA-3’;
With Potter-Elvehjem Tissue Grinders the internal organs Seedling raw material of choosing is carried out homogenate, filter with sterile gauze then, add 1% formaldehyde in the filtrate in order to deactivation, add the oils adjuvant stirring and emulsifying with filtrate equivalent again, make oneself internal organs inactivated vaccine, 4 ℃ of preservations are standby;
2) preparation of hyper-immune serum: utilize above-mentioned oneself the internal organs inactivated vaccine that makes, the healthy boar of the perforated gesture of this 3 monthly age size is carried out the multi-point injection reinforced immunological, after 2~3 immunity, expand the antibody that detects anti-PCV2 in the immune swine serum with fine jade, to expand the immune swine of detection antibody titer more than 1:32 through fine jade and cut open blood-letting extremely, separation of serum, add penicillin, each 2000 μ g/1000ml of streptomycin, after aseptic detection and biological safe detection, obtain hyper-immune serum and pack in the container 4 ℃ of preservations by every branch of every 10ml/ into.
2, according to oneself hyper-immune serum preparation method of the treatment weaned piglet multisystemic wasting syndrome of claim 1, it is characterized in that carrying out 23 weeks after described multi-point injection reinforced immunological is exempted from according to head exempts from, 2 exempt from the back carries out 3 immune programme for children of exempting from 3 weeks, respectively selects carry out intramuscular injection at 5 along immune swine spinal column both sides.
CNB2006101166315A 2006-09-28 2006-09-28 Preparation process of autogenous high-immunity serum for treating post-weaning multi systemic wasting syndrome of baby pig Expired - Fee Related CN100406062C (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102382900A (en) * 2010-08-31 2012-03-21 上海市农业科学院 Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus type 2
CN104288760A (en) * 2013-07-18 2015-01-21 普莱柯生物工程股份有限公司 Vaccine composition, and preparation method and application thereof
CN110025781A (en) * 2019-04-25 2019-07-19 杭州洪扬生物工程有限公司 A kind of vaccine composition and preparation method thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1800375A (en) * 2005-12-23 2006-07-12 南京农业大学 Recombinant adenovirus of porcine reproductive and respirator syndrome virus and porcine Circovirus, and vaccine

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102382900A (en) * 2010-08-31 2012-03-21 上海市农业科学院 Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus type 2
CN102382900B (en) * 2010-08-31 2013-05-08 上海市农业科学院 Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus type 2
CN104288760A (en) * 2013-07-18 2015-01-21 普莱柯生物工程股份有限公司 Vaccine composition, and preparation method and application thereof
CN110025781A (en) * 2019-04-25 2019-07-19 杭州洪扬生物工程有限公司 A kind of vaccine composition and preparation method thereof
CN110025781B (en) * 2019-04-25 2022-09-20 杭州牧达智能科技有限公司 Vaccine composition and preparation method thereof

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