CN102732486A - Porcine circovirus type2 strain and application thereof - Google Patents

Abstract

The invention provides a porcine circovirus type2 (PCV2) strain through separation and identification. The strain is named as a PCV2 SD strain, and has a strain collection number of CGMCC NO.5774. The strain PCV2 SD has relatively high titer. After 4 generations of blind passage, a virulence is 10<5.0>TCID50/mL. After passage, the virulence of the 20th generation is 10<6.0>TCID50/mL. The separation and identification of the strain provides a certain basis for further researches of PCV2 and the control of PCV2.

Description

One strain porcine circovirus 2 type strain and application thereof

Technical field

The invention belongs to mikrobe animal vaccine field, relate in particular to a strain porcine circovirus 2 type strain and a related application thereof.

Background technology

Pig circular ring virus is no cyst membrane sub-thread cyclic DNA virus, and the virus particle diameter is 17-20nm, is 20 body symmetries.In international virusology classification, pig circular ring virus, chicken anaemia virus and parrot beak ptilosis poison are divided into one newly, i.e. PCV-II section.Existing two genotype PCV1 of this virus and PCV2, genome contains 2 main reading frames, ORF1 gene encoding production relevant with rdrp virus (Rep) wherein, the ORF2 gene encoding production is formation viral capsid proteins (Cap) composition.PCV1 was found in the PK-15 cell culture by Tischer etc. in 1974 first, to the pig no pathogenicity.Porcine circovirus 2 type (porcine circovirus type2; PCV2) can cause relative diseases such as wean back piglet multisystemic exhaustion syndrome (PMWS), the scorching nephrotic syndrome (PDNS) of pigskin, breeding difficulty; Wherein serious with PMWS; PMWS finds in Canada first that in 1991 its cause of disease is that the PCV2 infection causes.This disease mainly encroach on 6～12 age in week piglet, cause and gradually become thin, have difficulty in breathing, suffer from diarrhoea and yellow subcutaneous ulcer etc., caused enormous economic loss for world's pig industry.

Domestic since calendar year 2001 report should disease, in various degree popular all arranged in each province and city.Clinical sample is being carried out on the basis that PCV2 detects the case of test positive has been carried out viral separation for this this research, because the PCV2 vitro culture does not produce cytopathy, the virus multiplication ability is brought certain difficulty to research work.In order to further investigate the characteristic of PCV2 in the cell in vitro multiplication by culture; This research separates the PCV2 strain from clinical PMWS pathological material of disease; Through the cell continuous passage; Obtain a strain cell culture and adapt to poison, methods such as (PCR), indirect immunofluorescence experiment, transmission electron microscope and nucleic acid sequence analysis identify, confirm that strain isolated is PCV2 through the polymerase chain reaction.Carry out the PCV2 correlative study in a deep going way for next step certain material and theoretical basis is provided.

Description of drawings

Fig. 1 PCV2SD virus PCR increasing product: 1.DNA standard DL 2000; 2-5 viral cultures PCR product; 6 positive controls, 7. negative controls

Fig. 2 IFA method detects PCV2 SD and infects PK-15 cell result: figure A is a cells infected, and figure B is the non-infected cells contrast.

Observed PCV2 SD particle photo under Fig. 3 Electronic Speculum: (140,000 *) scale=50nm.

Summary of the invention

The present invention has obtained a strain porcine circovirus 2 type strain (PCV2) through isolation identification; And called after PCV2 SD strain; This strain has been preserved in (address: village, Chaoyang District, Beijing City road, China Committee for Culture Collection of Microorganisms common micro-organisms center on February 8th, 2012; Institute of Microorganism, Academia Sinica), strain preserving number: CGMCC NO.5774, its called after of classifying: porcine circovirus 2 type.

The detection of PCV2 usually need be by methods such as immunofluorescence experiments, uses special viral antibody and could confirm virally in cell, to duplicate.This research adopts the IFA method to detect the antigen in the virus infected cell.Observe fluorescent dye and the sparse interior fluorescent dye of endochylema in the visible fine and close nucleus of PCV2 monoclonal anti body opening under the fluorescent microscope.PCV2 cultivates at cell in vitro and does not present cytopathy, and virus is also relatively poor at the multiplication capacity of cell.Maybe with the process of this virus replication in to depend on the host cell polysaccharase unduly relevant because this enzyme is mainly synthetic in the cell proliferation S cycle, has shortened viral nursery stage, thereby caused the virus multiplication ability drop.D-glucosamine on the one hand can strengthen the proteic expression of cell S phase, can promote viral DNA to advance people's nucleus on the other hand, thus the breeding of enhanced virus duplicate, it is reported to make PCV antigen-positive cell number improve 30%.In view of the above, we divide a bottle back 12h at cell, and cell is in the time standby D-glucosamine of logarithmic phase to be handled, thereby has more effectively promoted the breeding of PCV2 to duplicate.But when handling cell culture with D-glucosamine, note the period, treating processes is unsuitable long, otherwise will produce toxic action.

PCV2 infects the tremendous economic loss that has caused world's pig industry, and the PCV2 vaccine is also stepping up development.But because the cultivation titre of PCV2 on cell is very low, a lot of production of vaccine producers are too high based on cost, abandoned the plan of exploitation PCV2 vaccine, and therefore, how improving the titre of PCV2 on cell should also be one of focus of studying from now on.The isolating PCV2 SD of this institute strain, malicious price ratio is higher, after 20 generations went down to posterity, is 10 ^6.0TCID50/ml has the value as vaccine research.In addition, the malicious price differential of different strains different whether with its pathogenic relevant further research of also treating.Isolated strain increases progressively with the passage number of times, and virus titer also significantly raises, and the PCV2 multiplication capacity is except that the influence that receives the synthetic external source polysaccharase of host cell, and is also relevant with the flexibility of cell in vitro cultivation.Virus isolated strain is cultivated domestication through cell in vitro, can effectively change the affinity relation of virus and cell surface receptor.

PCV2 can duplicate and produce lesion tissue at BALB/c mouse and kunming mice.In view of the above, this research adopts kunming mice as laboratory animal, and the virus inoculation culture cuts open inspection and finds that 60% connects malicious mouse submandibular lymph nodes obvious hyperemia is arranged; PCR successfully detects the PCV2SD strain at the mouse proliferation in vivo.

The pathogenic spectrum of PCV2 is in continuous expansion; The relative disease that causes for PCV2 does not still have proper prophylactic methods; Rapidly effective diagnosis, infected pigs is removed and good breed operative technique standard is to eliminate the optimal path that PCV2 infects loss at present; Also have the scholar to propose to tackle PMWS " 20 plans ", in eliminating the PMWS process, also produced positive effect.Therefore the research work of carrying out PCV2 has important economic implications.This research successfully is separated to the PCV2 SD strain that a strain has higher titre, for next step carries out the PCV2 correlative study in a deep going way and control provides certain basis.

The porcine circovirus 2 type strain PCV2 SD that isolation identification obtains, and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, strain preserving number: CGMCC NO.5774.

Specific embodiment

Embodiment

1. pathological material of disease, cell, laboratory animal

Pathological material of disease is from the Shandong large-scale pig farm, mainly shows as fervescence before the sick pig death, the expiratory dyspnea etc. of becoming thin, through PCR detect for PCV2 positive.PK-15 cell (no PCV pollutes) is for the control of Shandong Province's livestock and poultry pestilence and breed a laboratory preservation.SPF level kunming mice is available from Shandong Province's Experimental Animal Center.

2. main agents

The PCV2 positive serum is preserved for this laboratory; The PCV2 monoclonal antibody is so kind as to give by Harbin Veterinary Medicine Inst., China Academy of Agriculture; The anti-pig fluorescence two anti-D-glucosamine that reach of the rabbit of FITC mark are available from Sigma company; Pancreatin, DMEM and foetal calf serum are U.S. GIBCO company and produce; E.coli DH5 α bacterial classification is by the control of Shandong Province's livestock and poultry pestilence and breed key lab's preservation; PMD18-T carrier, dna ligation kit, PCR test kit, DL-2000 Marker etc. are available from the precious biotinylated biomolecule in Dalian Engineering Co., Ltd; Plasmid extraction kit, gel reclaim test kit, penbritin (Amp) etc. available from Jin Bo Bioisystech Co., Ltd, and other reagent is import or homemade analytical pure article.

3. the virus separation is gone down to posterity with virus

The pathological material of disease (spleen and lymphoglandula) of clinical trouble " PMWS " is ground ,-20 ℃ of multigelations 3 times, the centrifugal 5min of 5000rpm removes deposition, and the aperture is 0.2 μ m filter filtration sterilization.

Covered with the PK-15 cell of individual layer and used 0.05% trysinization, the pathological material of disease supernatant of the above-mentioned preparation of inoculation 1mL discards nutrient solution behind the 12h, use 300mmoldm ^-3D-glucosamine is handled 30min, after the PBS washing, changes the fresh DMEM substratum that contains 3% serum and continues to cultivate 48h.Continuously 4 generations of blind passage, each digestion divides a bottle back 12h, all handles 30min with D-glucosamine.If normally do not connect the contrast of pathological material of disease cell.Therefrom separate and obtain virus strain called after PCV2 SD strain.

4.PCR identify and nucleotide sequencing

Primer: according to the PCV2 complete genome sequence that GenBank delivers, adopt Primer software design primer, give birth to worker's biotechnology Services Co., Ltd by Shanghai and synthesize.Amplification PCV2 full-length gene primer sequence is P1:5 '-gaaccgcgggctggctgaacttttgaaagt-3 ', P2:5 '-gcaccgcggaaatttctgacaaacgttaca-3 '.Above-mentioned 4 generation the PK-15 cell extraction genome that obtains of blind passage; Use above-mentioned primer to carry out pcr amplification; The PCR product cloning that pcr amplification is therefrom obtained is served the sea and is given birth to the order-checking of worker's biotechnology Services Co., Ltd to the pMD18-T carrier behind the purifying, adopt DNAStar software to carry out sequential analysis.

Extracting cellular genome carries out the PCV2 specific band about 1700bp occurring in the cell of pcr amplification discovery inoculation pathological material of disease; Do not amplify any band (referring to Fig. 1) in the normal cell; The PCR positive plasmid is served the sea gives birth to the biological ltd of worker and checks order and identify that sequencing result carries out sequence alignment and finds that its full genome and known strain highest homology property are 95%.

5.IFA detect

The PK-15 cell that 4 generations of blind passage obtain in the step 3 is used 0.05% trysinization with quadrat method, packing 96 orifice plates (2 * 10 ⁴The every hole of cell), behind the cultivation 12h, use 300mmoldm ^-3D-glucosamine is handled 30min, and the washing back is added the fresh DMEM nutrient solution that contains 3% serum and continued to cultivate 48h.Discard nutrient solution,, clap and do with PBS (pH7.2) washing; With the fixing 5-10min of v (acetone): v (absolute ethyl alcohol)=6: 4, discard stationary liquid, dry naturally; With the PBS washing, clap and do; Add people's 100 μ L Standard PC V2 monoclonal antibodies (dilution in 1: 200), 37 ℃ of effect 1h, PBS washing 5 times; The anti-pig fluorescence two of the rabbit of Dropwise 50 μ L FITC mark anti-(dilution in 1: 200), 37 ℃ of effect 45min, same washing back is clapped and is done fluorescence microscope.If inoculating cell negative control not.

Under fluorescent microscope, observe and find, fluorescent dye and sparse cytoplasm fluorescent dye in the visible fine and close nuclear in the PK-15 cell (Fig. 2 A) of infection PCV2 SD.And the normally not no fluorescence appearance of inoculating cell (Fig. 2 B).

6. viral TCID ₅₀Measure

With 4 generations of blind passage and 20 generation PCV2 SD male PK-15 cell-20 ℃ multigelations 3 times, the PK-15 cell that grows fine (no PCV pollutes) use 0.05% trysinization, packing 96 orifice plates, the every hole of 90 μ L adds the DMEM substratum 10 that usefulness contains 10% serum then ^-1-10 ^-12The viral liquid of dilution.CO is put in each 6 hole of extent of dilution inoculation behind the mixing ₂Continue in the incubator to cultivate.Discard nutrient solution behind the 12h, use 300mmoldm ^-3D-glucosamine is handled 30min, and PBS washing back adds the DMEM nutrient solution that contains 2% serum and continues to cultivate 48h; Discard nutrient solution, 5 make indirect immunofluorescence assay (IFA) result of determination set by step.Press the TCID that the Reed-Muench method is calculated virus strain ₅₀

Isolating PCV2 SD blind passage after 4 generations malicious valency be 10 ^5.0TCID ₅₀/ mL, being passaged to the malicious valency of the 20th generation virus is 10 ^6.0TCID ₅₀/ mL.

7. electron microscopic observation

With the 20th generation cytopathy venom multigelation 3 times, the centrifugal 30min of 8000rpm discards deposition, with supernatant 40000rpm ultracentrifugation 3h, abandons supernatant, with resuspended deposition of PBS and collection, whether transmission electron microscope observing exists virus.

It is rounded to observe virus particle under the transmission electron microscope, and the about 17nm of diameter sees Fig. 3.

8. experimentation on animals

10 3 week SPF level kunming mices in age (female mouse) are divided into two groups at random, and 5 every group, virocyte culture (PCV2 SD, 10 of every abdominal cavity inoculation of control group 0.2mL ^6.0TCID ₅₀/ mL), control group is inoculated PBS with quadrat method.Cut open after one week and kill, from solid tissue organs such as blood, lymph, spleen, detect virus respectively.All detect virus in 5 inoculation culture thing mouse as a result, and control group mice does not all detect virus.The result: attack that 3 mouse lymphoodi mandibulares have obvious hyperemia in the poison group, control group is normal; The PCR detected result detects PCV2 SD 4 parenchymal tissues that connect malicious mouse; Control group does not all detect.

Claims (4)

1. porcine circovirus 2 type strain PCV2 SD, it is characterized in that: its deposit number is: CGMCC NO.5774.

2. the application of the described porcine circovirus 2 type strain of claim 1 PCV2 SD in the medicine that preparation treatment 2 porcine circovirus infects.

3. the described application of claim 2 is characterized in that said medicine is a vaccine.

4. the application of the described porcine circovirus 2 type strain of claim 4 PCV2 SD in treatment 2 porcine circovirus infected animals.

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