CN104250636A - Porcine circovirus culture method and application - Google Patents
Porcine circovirus culture method and application Download PDFInfo
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Abstract
The invention provides a porcine circovirus culture method and application and a mycoplasma hyopneumoniae culture method. The porcine circovirus culture method can simultaneously culture porcine circovirus and mycoplasma hyopneumoniae, simultaneously proliferate the titer of the porcine circovirus and the mycoplasma hyopneumoniae, and obtain high titer porcine circovirus. A vaccine combination prepared by adding mycoplasma hyopneumoniae antigens into the porcine circovirus which is prepared by the porcine circovirus culture method can be used for the prevention and/or treatment of diseases caused by the porcine circovirus and the mycoplasma hyopneumoniae.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of cultural method and application thereof of pig circular ring virus, and the cultural method of mycoplasma hyopneumoniae.
Background technology
1974, German scholar Tischer etc. detected a kind of cell contamination thing from the porcine kidney cell line PK15 cell of continuous passage, were that one can not cause cytopathic virus like particle.Nineteen eighty-two, this scholar studies confirmation further: the genome of this virus is made up of the DNA of sub-thread ring-type, thus by its called after pig circular ring virus (Porcine Circovirus, PCV).
Pig circular ring virus belongs to PCV-II section Circovirus, is a kind of minimum animal virus found so far, diameter average out to 17 nm.Pathogenic according to pig circular ring virus, the difference of antigenicity and nucleotide sequence, PCV is divided into no pathogenicity PCV1 and pathogenic PCV2 two genotype, wherein PCV2 mainly causes pig multisystem asthenic syndrome (Pig Multisystemic Wasting Syndrome, PMWS), also can cause scorching nephrotic syndrome (the Porcine Dermatitis and Nephropathy Syndrome of pigskin, PDNS), PRDC (porcine respiratory disease complex) (Porcine Respiratory Disease Complex, PRDC), Hypertrophic necrotizing pneumonia (Proliferative and Necrotizing Pneumonia, PNP), congenital tremors (Congenital Tremors, the disease such as CT), be generically and collectively referred to as Porcine circovirus desease (Procine Circovirus Disease, PCVD).Clinically, PCV2 mainly encroaches on pulmonary alveolar macrophage and the lymphocyte of pig, main infection 5-12 week age piglet, the clinical symptom of sick pig is: gradually become thin or growth retardation, malnutrition, cough, expiratory dyspnea, jaundice, diarrhoea, ochrodermia, lymphadenectasis, the greyish white oedema of kidney etc., can become cad pig, severe patient can cause death.
PCV2 infects and can cause the immunosuppression of pig, thus makes body more other cause of diseases of easy infection, and this is also the pig circular ring virus reason relevant with the numerous disease polyinfection of pig.Modal polyinfection has mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp), porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, pig parvoviral, in superinfection or triple infection, the case fatality rate of disease pig is caused also will greatly to improve, reached at the 25%-40% had.
Mycoplasma hyopneumoniae can cause porcine mycoplasmal pneumonia (Mycoplasmal pneumonia of swine, MPS), this disease is also known as swine enzootic pneumonia (Li Yanming, Zhang Ying. mycoplasma hyopneumoniae Advances in Biological Study, animal medicine is in progress, 2003, 24 (3): 25-27) or pig endemic conditions pneumonia, it is a boar contact chronic respiratory tract disease, be widely distributed, with chronic, contact, hyperinfection, high incidence and low actual etc. are feature, main manifestations is cough and expiratory dyspnea, when dissected is based on pulmonary lesion, especially with two lung lobus cardiacuses, middle leaf and sharp leaf occur that pancreas sample becomes and carnification is its feature.University of Iowa Ross studies discovery: after Mhp infects, lymphocyte produces the ability decline of antibody, cellular immunity declines, pulmonary alveolar macrophage to cause of disease engulf and Scavenging activity also declines, the activity of suppressor T cell strengthens, cause respiratory immunity power to weaken, disease resistance declines, thus other pathogenic agent are more easily invaded, can secondary infection porcine reproductive and respiratory syndrome, pig annulus etc., thus cause multiple vaccine inoculation failure.
The biological characteristics of PCV2 is more special, although can exist with high titre for a long time in vivo, in cultivating in vitro, its viral titer is lower on the contrary and unstable, applies the most high-content that PK15 passage cell cultivates PCV2 in the world and is difficult to more than 10
3.5tCID
50/ ml; And the PK15 cell proportion that the display of immunofluorescent test result is infected by PCV2 only has 20%-30%, thus to produce malicious titre lower for PCV2, this and current extensive animal vaccine produce incompatible (Shen Lu. the foundation of porcine circovirus 2 type fluorescent quantitative PCR detection method and the Primary Study of culture process. Agricultural University Of Nanjing's master thesis).
The cultivation difficulty of mycoplasma hyopneumoniae is larger, have impact on its research in hereditary, biochemical, immune etc. (Shao Guoqing. mycoplasma hyopneumoniae Progress on Molecular Biology. Chinese Amphixenosis's magazine, 1999,15 (2): 68-73).
Summary of the invention
The present inventor is surprised to find that in process of the test, and when pig circular ring virus and mycoplasma hyopneumoniae co-cultivation, the titre of pig circular ring virus has raising by a relatively large margin.By this cultural method, the pig circular ring virus of high titre can be obtained, and the pig circular ring virus utilizing this cultural method to prepare also can be applicable to the vaccine composition preparing pig circular ring virus and mycoplasma hyopneumoniae.
Thus, main purpose of the present invention is the cultural method providing a kind of pig circular ring virus, described cultural method comprises: (1) cultivates zooblast, (2) pig circular ring virus, mycoplasma hyopneumoniae are inoculated on zooblast respectively, to cultivate pig circular ring virus and mycoplasma hyopneumoniae simultaneously.
Preferably, in described cultural method, zooblast is the cell in porcine kidney cell system, African green monkey kidney cell line, monkey embryo kidney epithelial cell line and Pig testicular cell system in arbitrary clone.
Preferably, in described cultural method, zooblast is any one in PK15 cell, PKK cell, Vero cell, Marc145 cell and ST cell.
Further preferably, in described cultural method, zooblast is PK15 cell or PKK cell.
Term " PKK cell " refers to the high responsive cell of a strain PCV2 that Pulaike Biological Engineering Co., Ltd. clones from PK15 cell, this cell has been disclosed in Peng Wu equality (Peng Wuping, Wang Yanhui, Hu Dongbo etc. the cultivation of the cell adapted strain of porcine circovirus 2 type. the academic seminar collection of thesis of the 4th swine disease, 263-265) document in.
Preferably, in described cultural method, the training method of zooblast is monolayer adherence cultivation, suspension culture or Immobilized culture.
Further preferably, in described cultural method, the training method of zooblast is that monolayer adherence is cultivated.
Preferably, cultivate zooblast cell culture fluid in described step (1) and cultivate, described cell culture fluid is the substratum containing 5% (V/V) foetal calf serum; Described substratum is any one in MEM substratum, DMEM substratum, RPMR1640 substratum and 199 substratum.
Preferably, in described step (2), pig circular ring virus, mycoplasma hyopneumoniae are distinguished Simultaneous vaccination on cultured zooblast.
Preferably, in described step (2), pig circular ring virus, mycoplasma hyopneumoniae press 0.01-0.05MOI, 1%-10% (V/V) Simultaneous vaccination respectively on cultured zooblast.
Preferably, in described step (2), pig circular ring virus is porcine circovirus 2 type totivirus strain, and mycoplasma hyopneumoniae is the full bacteria strain of mycoplasma hyopneumoniae.
Preferably, in described step (2), pig circular ring virus is the arbitrary strain in SH strain, SD strain, D strain, WH strain, LG strain, ZJ/C strain, ZJ/H strain, DBN-SX07 strain and V201312 strain, and mycoplasma hyopneumoniae is the arbitrary bacterial strain in HN0613 strain, P strain, J strain, 168 strains, P-5722-3 strain, BQ14 strain, NJ strain, HDZK-Mhp57 strain, AN306 strain, DJ-166 strain and NM04/41259 strain.
The preserving number of term " SH strain " is CGMCC No.23890, and preservation date is on March 4th, 2008, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and this strain is open in Chinese patent CN101240264A.
The preserving number of term " SD strain " is CGMCC No.7707, and preservation date is on May 31st, 2013, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and this strain is open in Chinese patent CN103421748A.
The preserving number of term " D strain " is CGMCC No.7245, and preservation date is on April 24th, 2013, and depositary institution is China Microbiological preservation management committee's common micro-organisms center, and this strain is open in Chinese patent CN103275938A.
The preserving number of term " WH strain " is CCTCC No:V201333, and preservation date is on July 19th, 2013, and preservation address is China typical culture collection center, and this strain is open in Chinese patent CN103409374A.
Term " LG strain " produces porcine circovirus 2 type inactivated vaccine (LG strain) porcine circovirus type 2 strain used from Harbin Wei Ke biotechnology development company.
Term " ZJ/C strain " produces porcine circovirus 2 type inactivated vaccine (ZJ/C strain) porcine circovirus type 2 strain used from Zhejiang Nuobeiwei Biotechnology Co., Ltd..
The preserving number of term " ZJ/H strain " is CGMCC No.6391, and preservation date is on July 17th, 2012, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and this strain is open in Chinese patent CN102787100A.
The preserving number of term " DBN-SX07 strain " is CGMCC No.3064, and preservation date is on May 12nd, 2009, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and this strain is open in Chinese patent CN101549155A.
The preserving number of term " V201312 strain " is CCTCC No:V201312, and preservation date is on May 28th, 2013, and depositary institution is China typical culture collection center, and this strain is open in Chinese patent CN103436498A.
The preserving number of term " HN0613 strain " is CCTCC No.M2012230, and preservation date is on June 13rd, 2012, and depositary institution is China typical culture collection center, and this bacterial strain is open in patent application CN103083655A.
Term " P strain " produces porcine mycoplasmal pneumonia inactivated vaccine (P strain) mycoplasma hyopneumoniae bacterial strain used from Protatek International Inc. of the U.S..
Term " J strain " from American Type culture center ATCC, and specifies ATCC registration number 25934 and 27715, and this bacterial strain is open in Chinese patent CN101883581A.
The mycoplasma hyopneumoniae bacterial strain that the porcine mycoplasmal pneumonia living vaccine (168 strain) produced from Nanjing Tianbang Bio-industry Co., Ltd. of term " 168 strain " is used.
Term " P-5722-3 strain " produces porcine mycoplasmal pneumonia inactivated vaccine (P-5722-3 strain) mycoplasma hyopneumoniae bacterial strain used from Lincoln factory of Pfizer Inc..
Term " BQ14 strain " produces porcine mycoplasmal pneumonia inactivated vaccine (BQ14 strain) mycoplasma hyopneumoniae bacterial strain used from factory of France of Merial Limited.
The preserving number of term " NJ strain " is CCTCC No:M2012286, and preservation date is on July 16th, 2012, and depositary institution is China typical culture collection center, and this strain is open in Chinese patent CN103585622A.
The preserving number of term " HDZK-Mhp57 strain " is CGMCC No.8096, preservation date is on August 15th, 2013, depositary institution is the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and this bacterial strain is open in Chinese patent CN103484414A.
The preserving number of term " AN306 strain " is CCTCC No.M2012431, and preservation date is on October 25th, 2012, and depositary institution is China typical culture collection center, and this strain is open in Chinese patent CN103740625A.
The preserving number of term " DJ-166 strain " is CGMCC No.4545, and depositary institution is the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and this strain is open in Chinese patent CN103184171A.
The preserving number of term " NM04/41259 strain " is NM04/41259 strain, and depositary institution is national measurement research institute (Australia), and this strain is open in Chinese patent CN102458462A.
Preferably, described step (2) cultivates porcine circovirus 2 type and mycoplasma hyopneumoniae cell maintenance medium is cultivated, described cell maintenance medium is the substratum containing 4% (V/V) calf serum and 2mmol/l D-glucosamine hydrochloric acid, or contains the substratum of 1.5% (V/V) foetal calf serum; Described substratum is any one in MEM substratum, DMEM substratum, RPMR1640 substratum and 199 substratum.
Another object of the present invention is to the cultural method providing a kind of mycoplasma hyopneumoniae, described cultural method comprises: (1) cultivates zooblast, and mycoplasma hyopneumoniae is inoculated on zooblast by (2), to cultivate mycoplasma hyopneumoniae.
Another object of the present invention is to provide a kind of vaccine composition, pig circular ring virus antigen prepared by the described pig circular ring virus cultural method that described vaccine composition comprises immunity amount, the mycoplasma hyopneumoniae antigen of immunity amount and/or veterinarily acceptable adjuvant.
Preferably, the content of described pig circular ring virus antigen is 9.0 × 10
7.5tCID
50/ ml, the content of described mycoplasma hyopneumoniae antigen is 2.7 × 10
9.0mHCDE/ml.
Term " veterinarily acceptable adjuvant " can comprise aluminium glue adjuvant; Saponin(e (saponin), as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); Water-in-oil emulsion; Oil-in-water emulsion; Water-in-oil-in-water emulsion; The polymkeric substance of acrylic or methacrylic acid; The compound that the multipolymer of MALEIC ANHYDRIDE and alkenyl (alkenyl) derivative is selected.
Term " emulsion " can especially based on light liquid paraffin oil (European Pharmacopea type); Because of the isoprenoid oil (isoprenoid oil) that olefin oligomerisation produces, as squalane (squalane) or squalene oil (squalene oil), especially iso-butylene or certain herbaceous plants with big flowers alkene; The ester containing linear alkyl of acid or alcohol, more specifically vegetables oil, ethyl oleate, propylene glycol two-(octanoate/certain herbaceous plants with big flowers acid esters), glycerine three-(octanoate/certain herbaceous plants with big flowers acid esters) or Rikemal PO 200; The ester of branched chain fatty acid or alcohol, especially isostearate.Oil and emulsifier combination use to form emulsion.Emulsifying agent preferred nonionic surfactants, especially the ester of the ester of the ester of the ester (as anhydrous N.F,USP MANNITOL oleic acid ester) of the ester of sorbitanic, mannide (mannide), aliphatic dihydroxy alcohol (glycol), the ester of Polyglycerine (polyglycerol), the ester of propylene glycol and oleic acid, the ester of Unimac 5680, the ester of ricinolic acid or oxystearic acid, their optional ethoxylations, also has Pluronic L121, especially Pluronic product, particularly L121.See " The theory and practical application of adjuvants " (Ed.by DES Stewart-Tull that Hunter etc. writes, John Wiley and Sons, New York, " Vaccine " (1997,15:564-570) of 1995:51-94) writing with Todd etc.Such as, " the Vaccine design that Powell M and Newman M writes can be used, the Subunit and adiuvant approach " (Plenum Press, 1995) the 147th page describe SPT emulsion and the 183rd page describe MF59 emulsion.
Term " polymkeric substance of acrylic or methacrylic acid " is preferably crosslinked acrylic or methacrylic acid polymer, especially be cross-linked with the sugar poly alkenyl ether of (sugar) or polyalcohols, these compounds are known is called as carbomer (Carbomer, trade(brand)name Carbopol) (Phameuropa, 1996,8 (2)).Those skilled in the art also can see US Patent No. 2909462, which depict this kind of acrylate copolymer, itself and poly-hydroxylated compound crosslink, described compound has at least 3 hydroxyls, preferably more than 8, wherein the hydrogen atom of at least 3 hydroxyls is had the unsaturated lipid alkyl of at least 2 carbon atoms (aliphatic radical) replacement.Preferred group is that those contain the group of 2-4 carbon atom, such as vinyl, allyl group and other ethylenically unsaturated group (ethylenically unsaturated group).Described unsaturated group self can comprise other substituting group, as methyl.These products are sold with the name of carbopol, and (BF Goodrich, Ohio, USA) is suitable especially.They and allyl sucrose or be cross-linked with Allyl pentaerythritol (allyl pentaerythritol).This wherein can mention carbopol 974P, 934P and 971P, most preferably uses carbopol 971P.
The multipolymer EMA (Monsanto) of MALEIC ANHYDRIDE and ethene also can be considered in term " multipolymer of MALEIC ANHYDRIDE and alkenyl derivative ", these polymkeric substance dissolve and produce acidic solution in water, through neutralization, preferably be neutralized to physiological pH, to produce assist agent solution, immunogenicity, immunogenicity or vaccinal composition itself can be mixed wherein.
Term " adjuvant " also comprises, but be not limited to, RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A), Avridine lipid-amine adjuvant, E.coli LT (restructuring or other), Toxins,exo-, cholera, IMS 1314, Muramyl dipeptide, Gel adjuvant etc.
Preferably, described adjuvant is one or more in the multipolymer of aluminium glue adjuvant, saponin(e, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, the polymkeric substance of acrylic or methacrylic acid, MALEIC ANHYDRIDE and alkenyl (alkenyl) derivative, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, E.coli LT, Toxins,exo-, cholera, IMS 1314, Muramyl dipeptide or Gel adjuvant; Preferably, described adjuvant is Gel adjuvant.
Preferably, the volume ratio of described adjuvant in described vaccine composition is 5%-30%.
Term used herein " pig " refers to any animal belonging to Suidae (Suidae) member, as pig.
The present invention has following outstanding advantage:
(1) cultural method of described pig circular ring virus can breed the titre of pig circular ring virus and mycoplasma hyopneumoniae simultaneously;
(2) cultural method of described pig circular ring virus once can cultivate preparation 2 kinds of antigens, thus lowers vaccine cost.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
In the embodiment of the present invention to adopt mycoplasma hyopneumoniae to attack poison bacterial strain be CVCC354 strain, purchased from China Veterinery Drug Inspection Office.
The animal cell culture mode adopted in the embodiment of the present invention is set forth for the rolling bottle cell cultures in monolayer adherence cultivation, but no matter this embodiment does not under any circumstance all form limitation of the invention.
The embodiment of the present invention is using the Carbopol in the polymkeric substance of acrylic or methacrylic acid as adjuvant, and be the adjuvant of 10% with volume ratio, preparing vaccine composition is that example is set forth, but no matter this embodiment does not under any circumstance all form limitation of the invention.
In the embodiment of the present invention, used diluent is aseptic phosphate buffered saline buffer, i.e. the PBS of pH7.4, and its 1L volume formula is: NaCl 8.0g, KCl 0.2g, Na
2hPO
412H
2o 2.9g, KH
2pO
40.24g, after adding the dissolving of appropriate distilled water, then is settled to 1L with distilled water, adjust pH to 7.4, autoclave sterilization sterilizing 112Kpa, 20min, after cooling, is stored in 4 DEG C of refrigerators for subsequent use.
Chemical reagent used in the present invention is analytical pure, purchased from traditional Chinese medicines group.
In the embodiment of the present invention, the morbidity standard of pig circular ring virus is attacked malicious method for piglet immunological and is carried out, and needs the condition met after attacking poison: attack malicious control group should at least 4 hairs sick, immune group should at least 4 head protections.Judge according to body temperature, relatively day weight gain and the morbidity of virus antigen assay to pig circular ring virus, Details as Follows:
(1) body temperature standard, fervescence (>=40 DEG C) (morning about 10 every day measures piglet body temperature once), at least continues 3;
(2) relative day weight gain standard, the individual relative day weight gain according to every pig calculates the average relative day weight gain often organizing pig, and the average relative day weight gain of each group and the average relative day weight gain of blank group is compared.When average relative day weight gain is significantly lower than blank group (P<0.05), judge that all pigs of this group have clinical symptom, wherein, according to following formulae discovery individual relative day weight gain:
The calculating of P value can adopt relevant biometric software (as SPSS) to carry out;
(3) virus antigen detection, detects lymph node tissue with ImmunohistochemistryMethods Methods, PCV2 antigen should be detected.
Meet above any two, can morbidity be judged to.
In the embodiment of the present invention, mycoplasma hyopneumoniae method for testing efficacy is passed judgment on pulmonary lesion according to 28 point-scores.Wherein, described 28 point-scores observe pathology according to the typical lung of porcine mycoplasmal pneumonia, carries out quantification judge occurring degree, comprises the essence sample pathology of apex pulmonis leaf, lobus cardiacus, lobus diaphragmaticus, the appearance of middle leaf, as " carnification ", " change of pancreas sample ".Pneumonia pathology standards of grading are: 2 sharp leaves, 2 lobus cardiacuses, 2 lobus diaphragmaticus, 1 middle leaf amount to 7 lobes of the lung, and each lobe of the lung full marks are 4 points, amount to 28 points.Shared by the lobe of the lung area of generation essence pathology, the ratio of this leaf, gives a mark to each lobe of the lung respectively.0 point is designated as without pneumonia pathology; Lesion area ratio is 1%-25%, is designated as 1 point; Lesion area ratio 26%-50%, is designated as 2 points; Lesion area ratio is 51%-75%, is designated as 3 points; Lesion area ratio is 76%-100%, is designated as 4 points.As lobe of the lung tow sides all have pathology, score with the one side that lesion area is large.The pneumonia disease that each lobe of the lung marking summation is this morbidity strain becomes point.After respectively immune group pig and control group pig being scored, the lobe of the lung pathology decrement according to following formulae discovery immune group pig:
According to the tuberculosis varying index of biometric method statistic 7 lobes of the lung, determine lesion degree, carry out statistical analysis.
For making the present invention easier to understand, below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for the present invention to limit the scope of the invention.Experimental technique of the present invention, if without specified otherwise, is ordinary method; Described biomaterial, if without specified otherwise, all can obtain from commercial channels.
The cultivation of embodiment 1 different components and the mensuration of content
The cultivation of 1.1 different componentss
1.1.1 PCV2 single culture
Cultivate PK15 cell, the individual layer PK15 cell covering with 80%-100% is removed cell culture fluid.
Poison is planted on PCV2 SH strain basis and is inoculated in PK15 cell by 0.01MOI, 37 DEG C adsorb 30 minutes, add in the cell maintenance medium containing the MEM medium preparing of 4% (V/V) calf serum and 2mmol/l D-glucosamine hydrochloric acid, cultivate 5-7 day for 37 DEG C,-40 DEG C of freeze thawing 2-3 time, results virus, as culture 1.
1.1.2 the Dual culture of PCV2 and Mhp
Cultivate PK15 cell, the individual layer PK15 cell covering with 80%-100% is removed cell culture fluid.
PCV2 SH strain, mycoplasma hyopneumoniae HN0613 strain basis are planted poison/bacterial classification and pressed PCV2SH strain 0.01MOI, mycoplasma hyopneumoniae HN0613 strain 1% (V/V) Simultaneous vaccination respectively in PK15 cell, 37 DEG C adsorb 30 minutes, add in the cell maintenance medium containing the MEM medium preparing of 4% (V/V) calf serum and 2mmol/l D-glucosamine hydrochloric acid, juxtaposition 37 DEG C cultivates 5-7 day.-40 DEG C of freeze thawing 2-3 time, results culture, as culture 2.
1.1.3 mycoplasma hyopneumoniae single culture
During mycoplasma hyopneumoniae single culture, mycoplasma hyopneumoniae HN0613 strain basis bacterial classification is inoculated in separately PK15 cell according to the cultural method of embodiment 1.1.2, in contrast.And culture mycoplasma hyopneumoniae single culture gathered in the crops is as culture 3.
1.1.4 healthy cell
Meanwhile, cultivate PK15 cell, as healthy cell contrast, as culture 4.
The mensuration of PCV2 and Mhp content in 1.2 different cultures.
1.2.1 the mensuration of PCV2 content
Use indirect immunofluorescence assay (IFA) to measure the content of PCV2 virus in culture 1, culture 2, culture 4 respectively, operation steps is: each culture MEM maintenance medium of results is done 10 times of serial dilutions, gets 10
-5, 10
-6, 10
-7three extent of dilution, each extent of dilution inoculates 96 well culture plate PK15 cell monolayer 4 holes respectively, and every hole 0.1ml, sets up negative control simultaneously.When measuring the content of PCV2 in PCV2 and Mhp coculture, while healthy cell contrast is set, Mhp single culture thing is inoculated, in contrast.Culture plate is contained 5%CO at 37 DEG C
2incubator in cultivate 24 hours, change the MEM maintenance medium of the D-glucosamine hydrochloric acid containing 2mM, continue cultivation 24 hours; Use cold acetone fixed cell, measure with IFA the hole count that each extent of dilution contains PCV2 positive cell (in green), while contrast is without non-specific fluorescence, calculate viral TCID according to KarberShi method
50.
Measurement result shows: during PCV2 single culture, PCV2 viral level is 10
6.5tCID
50during/ml, PCV2 and Mhp Dual culture, PCV2 viral level is 10
8.5tCID
50/ ml, and in healthy cell, all there is not fluorescence (details are in table 1).
1.2.2 the mensuration of Mhp content
By PCR method, each culture is counted.By 10 times of cultures that doubling dilution is examined, be then PCR, the minimum limitation that PCR detects is 3 × 10
-3μ g (be equivalent to about 1000 thalline), by the multiple of dilution calculate thalline number (Shen Changchun, Tan Qingsong, Wang Qin wait .PCR method to measure Mhp culture bacterium number. Chinese Preventive Veterinary Medicine report, 2006,28 (1): 55-57).The content of culture 2, culture 3 is respectively 1 × 10
6, 1 × 10
7mHDCE/ml (1MHDCE=mycoplasma hyopneumoniae DNA cell equivalents, namely 1MHDCE is equivalent to 1 mycoplasma hyopneumoniae).The measurement result of Mhp content is in table 1.
Measurement result after the cultivation of table 1 different components
Note :-represent and do not do.
The Dual culture of embodiment 2 PCV2 and Mhp
The Dual culture of 2.1 PCV2 and Mhp
The coculture of PCV2 and Mhp is prepared according to embodiment 1.1.2, wherein PCV2 strain is respectively SD strain, WH strain, namely the coculture of PCV2 SD strain and mycoplasma hyopneumoniae HN0613 strain is cultivated respectively, and the coculture of PCV2 WH strain and mycoplasma hyopneumoniae HN0613 strain, successively by its called after culture 5, culture 6.
The mensuration of PCV2 and Mhp content in 2.2 cultures 5, culture 6
Measure the mensuration of PCV2 and Mhp content in culture 5, culture 6 respectively according to the method in embodiment 1.2, measurement result is in table 2.
Measurement result after the cultivation of table 2 different components
The preparation of the mono-seedling of embodiment 3 PCV2 and PCV2-Mhp vaccine composition
3.1 deactivations and inspection
The culture 1 prepared in embodiment 1-2, culture 2, culture 5 and culture 6 are directly added 10% formaldehyde solution deactivation by 0.1% (V/V) respectively, 37 DEG C of deactivations 24 hours, stirred once every 4 hours.Culture 1 after deactivation, culture 2 sample respectively and carry out aseptic and deactivation inspection, put 4 DEG C of preservations using the culture 1 be up to the standards, culture 2, culture 5 and culture 6 as antigen liquid.
The preparation of 3.2 mycoplasma hyopneumoniae list seedlings
According to Chinese patent CN103127497A (Zhang Xu section, Sun Jinzhong, Bai Chaoyong. porcine circovirus 2 type, mycoplasma pneumoniae bivalent inactivated vaccine and preparation method thereof. Chinese patent CN103127497A) described in the method preparation of mycoplasma hyopneumoniae, obtained mycoplasma hyopneumoniae enriched material is 3 × 10
10mHDCE/ml.
3.3 join seedling
By mycoplasma hyopneumoniae list seedling prepared by culture prepared in embodiment 2.1, embodiment 3.2, use the dilution of pH7.4 PBS solution, antigen liquid after dilution is added Carbopol adjuvant respectively in 10% (V/V) ratio, the final contained component of each vaccine composition and ratio are mixed by table 3, after stirring, sampling carries out steriling test respectively, it is anticorrosion that the sample that steriling test is qualified adds 0.02% (V/V) Thiomersalate, make its final concentration be no more than ten thousand/, abundant vibration mixing, after packing, 2-8 DEG C saves backup.
The each vaccine composition ingredient of table 3 and content
The mono-seedling of embodiment 4 PCV2 and PCV2-Mhp vaccine composition immune protective effect are evaluated
4.1 immunity and attack poison
Choose 14-21 age in days PRRSV, CSFV, PCV2 antigen and antibody and be negative healthy susceptible weanling pig 50, be divided into 4 groups.Wherein, the 1st group of 5 piglet mono-seedling of musculi colli injection 1ml PCV2 respectively, carried out second time inoculation by identical approach and dosage after 2 weeks; Each 10 piglets of 2-4 group through musculi colli inoculation 1ml PCV2-Mhp vaccine composition 1-3, carry out second time inoculation by identical approach and dosage after 2 weeks respectively; 5th group of 10 piglets attack poison contrast as nonimmune; 6th group of 5 piglet are as blank (nonimmune, non-attack poison).
Two exempt from latter 21 days, weigh to all pigs, carry out that PCV2 attacks poison, Mhp attacks poison afterwards respectively simultaneously.
Wherein, the 1st group of 5 piglets, and 2-5 group is respectively got at random 5 piglets and is all carried out PCV2 and attack poison, with PCV2 SH strain (containing 10
6.0tCID
50/ ml) collunarium 1ml, intramuscular injection 2ml attack poison, respectively organizes isolated rearing afterwards; 6th group of 5 piglets do not attack poison as blank, raise in independent room.Attack poison latter 4th, 7 day, respectively the oxter, both sides of every pig and both sides buttocks 4 totally o'clock to 25 piglet inoculations of 1-5 group keyhole hemocyanin (KLH/ICFA with Freund's incomplete adjuvant emulsification, 0.5mg/ml), each inoculation 1ml (4ml/ head), intraperitoneal inoculation thioglycollate medium simultaneously, 10ml/ head.Attack malicious latter 11st, 19 intraperitoneal inoculation thioglycollate mediums again, 10ml/ head.Attack the rear Continuous Observation of poison 25, slaughter after weighing on 25th and cut open inspection.
Meanwhile, each remaining 5 piglets of 2-5 group all carry out Mhp and attack poison, with tracheae injection Mhp Jinan system virulent strain CVCC354 strain 100MID
50/ head carries out attacking poison, observes and dissects after 18 days, and observe pulmonary lesion, mark according to 28 point-scores to pulmonary lesion.
4.2 PCV2 challenge test results
PCV2 attacks the test-results after poison in table 4.
Table 4 PCV2 attacks the Efficacy evaluation after poison
Note :+represent positive ,-represent negative.
Associative list 4 analyze PCV2 attack poison after test effect: 2-4 group (immune group of PCV2-Mhp vaccine composition), between each group, effect is suitable, and difference is not significantly (P>0.05); Relative to the 1st group for (immune group of the mono-seedling of PCV2), its average daily gain higher (P<0.05); (nonimmunely malicious control group is attacked relative to the 5th group, wherein there are 4 piglet body temperature >=40 DEG C number of days more than 3 days, there is the PCV2 Viral diagnosis of 4 piglets for positive), its piglet body temperature >=40 DEG C number of days is not all higher than 1 day, and all PCV2 not detected, average daily gain is higher (P<0.05) obviously; For the 6th group (blank group), its average daily gain is a little less than the 6th group (P>0.05).
4.3 Mhp challenge test results
The challenge test of Mhp the results are shown in Table 5.
Table 5 Mhp attacks the Efficacy evaluation after poison
Associative list 5 analyze Mhp attack poison after test effect: the pneumonia disease of each group piglet of 2-4 group (immune group of PCV2-Mhp vaccine composition) becomes point all between 7-11, average is respectively 6.00,6.20,5.80, pneumonia disease variability is all greater than 60%, namely all protection is obtained, and effect is suitable, difference is not significantly (P>0.05); Relative to the 5th group (nonimmunely attack malicious control group, pneumonia disease becomes point at 14-18, and average is 15.60), pneumonia disease becomes average higher (P<0.05).
The above is only the preferred embodiments of the present invention, not any pro forma restriction is done to the present invention, although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention, any those skilled in the art, not departing from the scope of technical solution of the present invention, make a little change when the technology contents of above-mentioned announcement can be utilized or be modified to the Equivalent embodiments of equivalent variations, in every case be the content not departing from technical solution of the present invention, according to any simple modification that technical spirit of the present invention is done above embodiment, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
Claims (10)
1. the cultural method of a pig circular ring virus, it is characterized in that, described cultural method comprises: (1) cultivates zooblast, (2) pig circular ring virus, mycoplasma hyopneumoniae are inoculated on cultured zooblast respectively, to cultivate pig circular ring virus and mycoplasma hyopneumoniae simultaneously.
2. cultural method according to claim 1, it is characterized in that, in described cultural method, zooblast is the cell in porcine kidney cell system, African green monkey kidney cell line, monkey embryo kidney epithelial cell line and Pig testicular cell system in arbitrary clone.
3. cultural method according to claim 2, it is characterized in that, in described cultural method, zooblast is any one in PK15 cell, PKK cell, Vero cell, Marc145 cell and ST cell, is preferably PK15 cell or PKK cell.
4. cultural method according to claim 1, is characterized in that, in described cultural method the training method of zooblast be that monolayer adherence is cultivated, suspension culture or Immobilized culture, be preferably monolayer adherence and cultivate.
5. cultural method according to claim 1, is characterized in that, cultivates zooblast cell culture fluid and cultivate in described step (1), and described cell culture fluid be the substratum containing 5% (V/V) foetal calf serum; Described substratum is any one in MEM substratum, DMEM substratum, RPMR1640 substratum and 199 substratum.
6. cultural method according to claim 1, is characterized in that, in described step (2) by pig circular ring virus, mycoplasma hyopneumoniae respectively Simultaneous vaccination on cultured zooblast; Preferably, in described step (2), pig circular ring virus, mycoplasma hyopneumoniae press 0.01-0.05MOI, 1%-10% (V/V) Simultaneous vaccination respectively on cultured zooblast.
7. cultural method according to claim 1, is characterized in that, in described step (2), pig circular ring virus is porcine circovirus 2 type totivirus strain, and mycoplasma hyopneumoniae is the full bacteria strain of mycoplasma hyopneumoniae; Preferably, in described step (2), pig circular ring virus is the arbitrary strain in SH strain, SD strain, D strain, WH strain, LG strain, ZJ/C strain, ZJ/H strain, DBN-SX07 strain and V201312 strain, and mycoplasma hyopneumoniae is the arbitrary bacterial strain in HN0613 strain, P strain, J strain, 168 strains, P-5722-3 strain, BQ14 strain, NJ strain, HDZK-Mhp57 strain, AN306 strain, DJ-166 strain and NM04/41259 strain.
8. cultural method according to claim 1, it is characterized in that, in described step (2), cultivation porcine circovirus 2 type and mycoplasma hyopneumoniae cell maintenance medium are cultivated, described cell maintenance medium is the substratum containing 4% (V/V) calf serum and 2mmol/l D-glucosamine hydrochloric acid, or contains the substratum of 1.5% (V/V) foetal calf serum; Described substratum is any one in MEM substratum, DMEM substratum, RPMR1640 substratum and 199 substratum.
9. a cultural method for mycoplasma hyopneumoniae, is characterized in that, described cultural method comprises: (1) cultivates zooblast, and mycoplasma hyopneumoniae is inoculated on zooblast by (2), to cultivate mycoplasma hyopneumoniae.
10. a vaccine composition, described vaccine composition comprise immunity amount any one of claim 1-8 described in cultural method prepare pig circular ring virus antigen, immunity amount mycoplasma hyopneumoniae antigen and/or veterinarily acceptable adjuvant.
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| CN109200284A (en) * | 2018-09-30 | 2019-01-15 | 江苏省农业科学院 | A kind of porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants, preparation method and applications |
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| CN104771754A (en) * | 2015-04-02 | 2015-07-15 | 武汉科前生物股份有限公司 | Water-based adjuvant of porcine circovirus type 2 inactivated vaccine and application thereof |
| CN104771754B (en) * | 2015-04-02 | 2018-02-13 | 武汉科前生物股份有限公司 | A kind of porcine circovirus 2 type inactivated vaccine aqueous adjuvants and application |
| CN107312746A (en) * | 2017-06-22 | 2017-11-03 | 中国农业科学院哈尔滨兽医研究所 | A kind of extensive full suspension culture method of porcine circovirus 2 type |
| CN107312746B (en) * | 2017-06-22 | 2020-10-16 | 中国农业科学院哈尔滨兽医研究所 | Large-scale full-suspension culture method for porcine circovirus type 2 |
| CN108795863A (en) * | 2018-06-06 | 2018-11-13 | 广西大学 | The method that Infection in Vitro porcine circovirus 2 type establishes porcine alveolar macrophage inflammatory model |
| CN109200284A (en) * | 2018-09-30 | 2019-01-15 | 江苏省农业科学院 | A kind of porcine mycoplasmal pneumonia live vaccine Mucosal Adjuvants, preparation method and applications |
| CN109200284B (en) * | 2018-09-30 | 2022-01-11 | 江苏省农业科学院 | Mycoplasma hyopneumoniae live vaccine mucosal immune adjuvant, and preparation method and application thereof |
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