CN107937348A

CN107937348A - 15 cell lines of PK of pig PLIN2 genes overexpression and the method for expanding PCV2 viruses

Info

Publication number: CN107937348A
Application number: CN201711191623.1A
Authority: CN
Inventors: 高其双; 刘明; 彭霞; 程百炼; 邓兵; 谭隽珺; 田晨雪; 濮振宇; 谭纤茹
Original assignee: WUHAN ENGINEERING SCIENCE & TECHNOLOGY INSTITUTE; Wuhan Academy of Agricultural Sciences
Current assignee: WUHAN ENGINEERING SCIENCE & TECHNOLOGY INSTITUTE; Wuhan Academy of Agricultural Sciences
Priority date: 2017-11-24
Filing date: 2017-11-24
Publication date: 2018-04-20
Anticipated expiration: 2037-11-24
Also published as: CN107937348B

Abstract

The method that high-titer PCV2 viruses are produced the present invention provides 15 cell lines of PK of one plant of overexpression PLIN2, the preparation method of the cell and with the cell.The cell line of the present invention is susceptible to PCV2 viruses, is to prepare the viral high efficiency amplifying cells.

Description

The PK-15 cell lines of pig PLIN2 genes overexpression and the method for expanding PCV2 viruses

Field that the present invention belongs to

, specifically, can be significantly the present invention relates to one kind the invention belongs to cell engineering and Virus culture technical field Improve the recombinant cell strain of porcine circovirus 2 type (PCV2) culture potency, preparation method and culture high-titer PCV2 virus sides Method.

Technical background

Domestic pig circular ring virus vaccine currently in use mainly has 2 kinds at present ----subunit seedling and whole virus vaccine, it is sub- Though unit seedling security is preferable, production cost is high, and duration of immunity is short.Therefore most of the country pig farm is still with totivirus epidemic disease Based on seedling.But whole virus vaccine production also has its technical bottleneck：Virus culture potency is difficult to rise to perfect condition, to solve The problem, vaccine producers increase by one in its culture process and are handled with D- Glucosamines, make cellular retention in division II phase, ensures the program of virus amplification.But the program not only increases raw material input, and increases production link and people Power is put into, and is added waste discharge amount, production cost is at least increased by more than 1 times.Nonetheless, domestic many producer's lifes The circovirus vaccine of production, its virus titer is 10⁴Hereinafter, immune effect is poor.American-European countries takes screening to circovirus Extremely sensitive cell line, cultivates circovirus and carries out viral concentration, its viral level is 10⁶More than, immune effect compared with It is good, but its production cost is high, and selling price is often 3-5 times of domestic vaccine.Therefore the training of circovirus how is improved It is the main direction that circovirus vaccine competitiveness improves at present in domestic manufacturer to support potency and reduce production cost.

PLIN, that is, perilipin is distributed across the most perilipin of content on fat drips surface.Most early in epididymis Found in adipocyte, be the molecular switch in lipolysis regulation and control.PLIN2 genes are a member in PLIN families, pig PLIN2 genes participate in the lipid metabolism of animal or cell in vivo.But the influence on gene pairs circovirus propagation is so far There is not yet document report.

The technology contents of the present invention

It is an object of the invention to overcome the shortcomings of the prior art, there is provided and it is a kind of susceptible to PCV2 viruses, and can be big Width improves the transgenosis PK-15 cell lines of PCV2 culture potency.

It is also another object of the present invention to provide a kind of method for preparing PCV2 viruses.

It is a further object to provide application of the PCV2 viruses in PCV2 vaccines are prepared of amplification.

The invention discloses a kind of PK-15 cell lines for overexpressing pig PLIN2 genes, it is characterized in that：PK-15 cells contain There are the external source pig PLIN2 genes manually transfected, and PLIN2 genes have SEQ ID NO：Nucleotide sequence shown in 35, this is thin Born of the same parents are susceptible to PCV2 viruses, and can produce potency and reach 10^-7Virus above, which is pig kidney PK-15/PLIN2 cells Strain, is preserved in China typical culture collection center, deposit number is CCTCC NO:C2017251.

The invention also discloses the method for preparing PK-15/PLIN2 cell lines, this method comprises the following steps:

1) clone of PLIN2 genes：Overlapping PCR primers are designed according to PLIN2 gene orders, amount to 34, adjacent primer Between have that 17bp reverse complementals are overlapping, there is the non-complementary series of 25bp in centre, and 34 primers are spliced into the total length of PLIN2 genes, will By annealing, the 17bp sequence complementary pairings of adjacent primer after 34 primer mixing.Middle unpaired 25bp sequences, Sequence forms the full length sequence of complete double-strand PLIN2 genes by polishing under the action of high-fidelity enzyme, by 25 circulations Afterwards, PLIN2 genes are expanded, and then will expand the fragment obtained through electrophoresis detection；

2) using plastic recovery kit will expand obtain PLN2 genetic fragments recycling, then with by the fragment with PCDNA3.1-EGFP carriers are passed through through NheI and AflII digestion with restriction enzyme, electrophoresis detection after recycling purpose fragment respectively T4 ligases are connected in pCDNA3.1-EGFP carriers, are built into pCDNA3.1-EGFP-PLIN2 plasmids, and test through sequencing Card；

3) transfection and screening：Transfected plasmids PLIN2-pCDNA3.1-EGFP is to PK-15 cell lines, individual cells cloning Cultivation filters out the PK-15/PLIN2 cell lines stablized and replicate external source PLIN2.

The present invention further discloses the method for preparing PCV2, this method comprises the following steps:

1) prepared by cell：PK-15/PLIN2 cell lines are thawed, are incubated in Kolle flask, are passaged to requirement；

2) virus inoculation：PCV2 virus inoculations on PK-15/PLIN2 cell lines, are continued culture 24 by synchronous inocalation method Hour；

3) Virus culture：24 it is small when after, culture medium is changed to the maintaining liquid containing 2% hyclone, and to continue culture 72 small When；

4) harvest virus：The culture supernatant in step (3) is harvested, and carries out PCV2 poison valency measures, virus titer can reach To 10^-7More than.

The specific purpose of the present invention is achieved through the following technical solutions：

1st, the preparation of the PK-15 cell lines containing pig PLIN2 genes

1.1 design of primers and synthesis

According to target gene PLIN2 sequences and over-lap PCR principle design primer, sequence is following (being shown in Table 1), and primer sequence is special Levy and be：Numbering is the primer and PLIN2 gene orders (5'- of odd numbers>3') direction is consistent, and numbering is the primer and PLIN2 of even numbers Gene order (5'->3') reverse complemental, it is in addition overlapping there are 17bp between the sequence of adjacent numbering.Before first sequence 20bp is that the homologous sequence of carrier is consistent with carrier sequence.The rear 20bp of the last item sequence is the homologous sequence and load of carrier Body sequence is consistent.

1 list of primers of table

50pmol/ μ l are diluted to deionized water after primer synthesis, appropriate centrifugation is uniformly mixed.

1.2 gene magnification

Reaction system is prepared pressing table 2 on ice, PCR instrument is put into after mixing, has 17bp between the adjacent primer of 34 primers Reverse complemental, has the non-complementary series of 25bp (18-42bp positions) among every primer, 34 primers cover PLIN2 genes Total length.By making annealing treatment (55 DEG C, 1min) after 34 primers are mixed, the 17bp sequence complementary pairings of adjacent primer.It is middle Unpaired 25bp sequences, sequence is by polishing under the action of high-fidelity enzyme, so as to form complete double-strand PLIN2 genes Full length sequence.Pass through 25 cyclic amplifications in PCR instrument, PLIN2 genes are expanded, and PCR amplification program is shown in Table 3.

2 pcr amplification reaction system of table

3 pcr amplification reaction condition of table

The electrophoresis detection of 1.3 amplified fragments and recycling

1.5% agarose electrophoresis 30min is utilized after PCR amplification, then according to DNAmarker sizes, is returned in the UV lamp Purpose band is received, then recycles target gene PLIN2 bands using plastic recovery kit.

The digestion of 1.4 carriers and target gene

4 digestion system of table

The PLIN2 genetic fragments of recycling and pCDNA3.1-EGFP carriers are prepared into reaction system by above-mentioned table 4 respectively, so Afterwards in 37 DEG C of digestion 1-2h；Digestion products are detected using 1.5% agarose electrophoresis and utilize plastic recovery kit to recycle enzyme Genetic fragment and carrier segments after cutting.

The connection of 1.5 carriers and target gene：Carrier segments after digestion and PLIN2 genetic fragments are pressed into following system Reaction system table 5 is prepared, and is put in 16 DEG C of connection 1h.

5 linked system of table

1.6 conversion

(50 μ l competence will be added in the pipe equipped with TOP10 competent cells through the product after the connection of T4 ligases Cell needs 25ng DNA), volume should be no more than the 5% of competent cell, gently rotate and mix content several times, ice bath 30min。

Centrifuge tube mixture is put into and is heated up in 42 DEG C of recirculated water, heat shock 90s, not shake pipe.Quickly pipe is turned Move on in ice bath, cell is cooled down 1~2min.

Often pipe adds 200 μ l SOC fluid nutrient mediums, and culture medium is heated up to 37 DEG C with water-bath, is then transferred to pipe It is arranged on 37 DEG C of shaking table, 220rpm culture 45min, makes the resistant maker gene of cell recovery and expression plasmid coding.

The competent cell that proper volume (each 90mm tablets up to 200 μ l) has converted is transferred to containing corresponding antibiosis On the LB culture mediums of element.Tablet is inverted, when culture 12~16 is small in 37 DEG C of incubators.

1.7 bacterium colony PCR are verified

Treat to grow bacterium colony on tablet, random picking several, carry out bacterium colony PCR verifications, detect transformant, preliminary screening Bacterium colony (positive colony) containing target gene.

1.8 sequence verifications, send positive colony to sequencing company sequence verification.

1.9 transfection plasmid amplifications and extraction：Using endotoxin-free plasmid extraction kit, plasmid is extracted, is contained The plasmid of PLIN2 genetic fragments is PLIN2-pCDNA3.1-EGFP.

1.10 transfections are cultivated with screening：The plasmid vector that will be obtained using liposomal transfection teclmiques in above-mentioned steps 1.9 PLIN2-pcDNA3.1-EGFP transfects PK-15 cell lines, and is screened using G418.Fluorescence microscopy Microscopic observation, when positive thin Intracellular growth into clone cell roll into a ball when, positive cell is chosen into independent culture with fine glass needle tubing, and finally obtain PK-15/ PLIN2 cell lines, on November 22nd, 2017 PK-15/PLIN2 cell lines be preserved in China typical culture collection center, protect It is CCTCC NO to hide numbering:C2017251.China typical culture collection center abbreviation CCTCC, is that Patent Office of the People's Republic of China specifies Preservation mechanism, positioned at Wuhan City, Hubei Province Wuhan University in the school, postcode 430072, network address：www.cctcc.org, phone： 027-68752319, Email:cctcc@whu.edu.cn.After the present invention discloses, nonprofit researcher can This microorganism fungus kind is asked for collection reproduce the present invention in accordance with the law.

Application of the cell line disclosed by the invention in PCV2 viruses are prepared.

Application of the PCV2 viruses in PCV2 viral vaccines are prepared caused by the present invention.

Plasmid vector pCDNA3.1-EGFP in the present invention can be obtained by commercially available commercial biological company, such as Thremo The companies such as Fisher Scientific inc., PK-15 cells, PCV2 viruses can pass through commercially available commercial biological company or strain Preservation mechanism obtains, and as China typical culture collection center preservation has the cell and virus, researcher can be to the axial cable Take.

Advantages of the present invention

Compared with prior art, the invention has the advantages that；

(1) virus titer of culture increases substantially, on the PCV2 with the gene-recombinated cell strain culture of invention Clear liquid virus immunity fluorescence TCID50 methods measure potency reaches 10^-7More than, the virus liquid effect of more conventional PK-15 cell culture Valency improves more than 100 times.

(2) production technology significantly simplifies.Current art handles the PK-15 after infection PCV2 with D- Glucosamines Cell, makes cultivating system Toxin producing C quickly improve.There is certain shadow since D- Glucosamines subsequently produce cultivating system poison Ring, therefore must be added in its flow and discard D- Glucosamines, then several inferior programs of cell are washed with PBS solution, it is real in production In border, often increase a program, will imply that the costs such as raw material, manpower are scaling up, often increase a program and also mean Increase and once open cultivating system entrance, this will greatly increase the possibility of pollution, this is also to cause current PCV2 epidemic diseases The main reason for seedling production cost is high, and the market price is higher, directly adds D- Glucosamines although also having in production at present Enter culture production poison cell in maintaining liquid, save washing process, but the more classical program of PCV2 value-added effects is greatly lowered in it, The product quality of production is also greatly lowered.The streptococcus training that can increase substantially PCV2 culture potency prepared by the present invention Thing preparation is supported, then can directly be incorporated the maintaining liquid of culture virus, the programs such as culture medium, washing are replaced without experience, therefore will be big Width reduces production cost.

(3) the purity higher of culture：Cell need to be frozen together with virus when poison is received after ordinary cells culture circovirus Melt, contained cell fragment is more in the virus liquid of freeze thawing harvest, influences viral purity and follow up vaccine quality.It is and special with this Need to only collect supernatant during profit culture PCV2 can obtain required exclusive virus, and cell fragment is compared with cell freeze thawing in supernatant Liquid much less, therefore virus liquid is more pure.

Brief description of the drawings：

Fig. 1 is PLIN2-pCDNA3.1-EGPF vector construction schematic diagrames

Fig. 2 is the fluorescence micrograph of PK-15/PLIN2 cell lines

Fig. 3 is target gene electrophoretogram after carrier digestion

Embodiment

With reference to specific embodiment, the present invention is further explained.It should be appreciated that these examples are merely to illustrate The present invention, and cannot limit the scope of the invention.

The preparation of the PK-15 gene-recombinated cell strains of 1 pig PLIN2 genes of embodiment overexpression

1st, material and method

1.1 inoculums and main agents：

PK-15 cells, PCV2 viruses：Purchased from CCTCC

PBS pulvis：Market purchase, homemade goods

Hyclone：It is domestic.

DMEM culture mediums：Buy Hyclone Products.

Trypsase：Purchased from sigma Products.

Carrier：PcDNA3.1-EGFP is purchased from Biofeng companies

Ligase, restriction endonuclease：T4 ligases are purchased from NEB purchased from Takara companies, NheI and AflII restriction enzymes Company.

1.2 main operational steps and method：

1.2.1 design of primers and synthesis

Target-gene sequence and clonal fashion the design primer sequence such as table 1 provided according to client：

It is 50pmol/ μ l to dilute synthetic primer to concentration with deionized water, and appropriate centrifugation is uniformly mixed.

1.2.2 gene magnification

Reaction system is prepared pressing table 6 on ice, is put into PCR instrument after mixing, program carries out PCR amplifications as set by table 7.

6 pcr amplification reaction system of table

7 pcr amplification reaction condition of table

1.2.3 the electrophoresis detection of amplified fragments and recycling

1.2.4 the digestion of carrier and target gene

8 digestion system of table

The PLIN2 genetic fragments of recycling and pCDNA3.1-EGFP carriers are pressed into above-mentioned table 8 respectively, prepare reaction system, Then in 37 DEG C of digestion 1-2h；Digestion products are detected using 1.5% agarose electrophoresis and utilize plastic recovery kit to recycle Genetic fragment and carrier segments after digestion.

1.2.5 the connection of carrier and target gene：Carrier segments after digestion and PLIN2 genetic fragments are pressed into such as lower body It is that table 9 prepares reaction system, and is put in 16 DEG C of connection 1h.

9 linked system of table

1.2.6 conversion

1.2.7 bacterium colony PCR is verified

Treat to grow bacterium colony on tablet, several bacterium colonies of random picking, carry out bacterium colony PCR verifications, detect transformant.

1.2.8 sequence verification

Positive colony is sent to sequencing company sequence verification, while parallel sample carries out restriction enzyme digestion and electrophoresis verification.

1.2.9 transfection plasmid amplification and extraction：Using endotoxin-free plasmid extraction kit, plasmid is extracted, is contained at this time The plasmid for having PLIN2 genetic fragments is named as PLIN2-pCDNA3.1-EGFP.

1.2.10 transfection is cultivated with screening：The plasmid vector that will be obtained using liposomal transfection teclmiques in above-mentioned steps 1.9 PLIN2-pCDNA3.1-EGFP transfects PK-15 cell lines, and is screened using G418.Fluorescence microscopy Microscopic observation, when positive thin Intracellular growth into clone cell roll into a ball when, positive cell is chosen into independent culture with fine glass needle tubing, and finally obtain PK-15/ PLIN2 cell lines.

1.2.11 positive cell overexpression product measure：WB measures pure positive cell culture supernatant and intracellular SFN contains Amount, and compared with the PK-15 cells of untransfected.

2nd, result：

The sequencing result SEQ ID NO of 2.1 gene chemical synthesis and conversion plasmid enzyme restriction target product：35.

2.2 transfection positive cells purify:The transfection positive cells of acquisition are subjected to monoclonal by cloning plate, by one Cell progressively expands, and makees Seed storage.

Embodiment 2：With PK-15/PLIN2 cell lines culture high-titer PCV2 and prepare PCV2 vaccines

2nd, material and method

2.1 inoculums and main agents

PK-15/PLIN2 cell lines：CCTCC patent depositories.

PK-15 cells, PCV2 kinds poison：Purchased from CCTCC

Hyclone, PBS pulvis, D- Glucosamines, the special PCR detection kits of PCV2：Market purchase, domestic production Product

DMEM culture mediums：Buy Hyclone Products.

Trypsase：Purchased from sigma Products

2.2 main operational steps and method

2.2.1 prepared by host cell：Conventionally at the same time by common PK-15 cells and PK-15/PLIN2 cell lines Thaw, be incubated at respectively in Kolle flask, be passaged to requirement.

2.2.2 prepared by virus kind poison：

With PBS dilute PCV2 freeze dried powders, by synchronous inocalation method by diluted virus inoculation in PK-15 cells, continuously More than generation, immunofluorescence TCID50 monitors its potency and reaches 10 squamous subculture 3^-5More than, harvest virus is as the art of this patent poison Kind.

2.2.3 virus inoculation liquid is prepared and is inoculated with：With 10 times of dilution seeds culture of viruses of DMEM culture mediums containing 10% hyclone Afterwards, according to conventional synchronous inoculation method by PCV2 virus inoculations in 2 kinds of cell suspensions of above-mentioned culture, it is 24 small to continue culture When.

2.2.4 Virus culture：After when virus inoculation 24 is small, nutrient solution is changed to the DMEM containing 2% hyclone and is maintained Liquid, continue culture 72 it is small when.

2.2.5 harvest virus：By above-mentioned steps 4. in the culture supernatants of 2 kinds of cells harvest respectively, carry out mark, and PCV2 poison valency measures are carried out respectively, and record is put on record, for vaccine formulation or other follow-up uses.

2.2.6PCV2 bioactivity：It is sub-packed in after healthy PK-15 cell dissociations are dispelled in 1.5mlEP pipes, often pipe point 0.9ml cell suspensions are filled, numbers in order and lines up 2 row, 10 EP pipes of each column, 2 kinds of cells for taking above-mentioned steps 5. to harvest respectively The virus liquid 0.1ml of culture is added in the first solencyte suspension of different lines, then according to 10^-1~10^-9Carry out gradient dilution, Then the cell suspension of each dilution gradient is inoculated with 6 holes of 96 well culture plates respectively, per hole 100ul, 24 it is small when after use The D- Glucosamines of 3mmol/L concentration maintain fluid exchange culture medium, continue culture 72 it is small when, culture plate is taken out, according to PCV2 Immunofluorescence TCID50 methods measure the virus-positive hole count of each dilution factor, calculate virus titer.

2.2.7 inactivation of virus and vaccine formulation：The disease of cell culture in take 5. required amount of above-mentioned steps harvest 2 Venom is inactivated and is configured to vaccine according to a conventional method respectively.

2.2.8 immune effect of vaccine detects：The PCV2 feminine genders pig 9 of weight about 30KG is chosen, is divided into 3 groups, the 1st group every 7. vaccine 5ml that head injection above-mentioned steps are prepared with the virus of PLIN2 overexpressing cells cultures, the 2nd group of every above-mentioned step of injection Suddenly the vaccine 5ml 7. prepared with the virus of common PK-15 cell culture, the 3rd group is set to control group, and each group blood is measured after 21 days PCV2 antibody levels in clear.

2.3rd, result：

2.3.1 PLIN2 genes overexpressing cells are contrasted with control cell culture PCV2 potency, 11 are shown in Table

11 potency contrast table of table

2.3.2PK-15/PLIN2 antibody test knot after the vaccine immunity that cell line is prepared with control cell culture PCV2 Fruit, is shown in Table 12

12 antibody test table of table

Sequence table

<110>Academy of Agricultural Sciences of Wuhan City

Engineering Scientific Technology Research Inst., Wuhan

<120>The PK-15 cell lines of pig PLIN2 genes overexpression and the method for expanding PCV2 viruses

<160> 35

<170> SIPOSequenceListing 1.0

<210> 1

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 1

ggagacccaa gctggctagc atggcatccg ttgctgttga atcacaaccg agtgtggtg 59

<210> 2

<211> 55

<212> DNA

<213>Pig (Sus scrofa)

<400> 2

tacgtggagc tcaccaaggg taggttggcc acccttgtca ccacactcgg ttgtg 55

<210> 3

<211> 55

<212> DNA

<213>Pig (Sus scrofa)

<400> 3

ttggtgagct ccacgtatga ccttgtctcc tcggcttata tcagtacaaa ggatc 55

<210> 4

<211> 55

<212> DNA

<213>Pig (Sus scrofa)

<400> 4

ctctgccatc tcacacagag acttcaagta gggatactga tcctttgtac tgata 55

<210> 5

<211> 55

<212> DNA

<213>Pig (Sus scrofa)

<400> 5

tgtgtgagat ggcagagaag ggcgtcaaga ccatcacctc cgtggccatg tccgg 55

<210> 6

<211> 55

<212> DNA

<213>Pig (Sus scrofa)

<400> 6

caatctgagg ctctagcttc tggatgatag ggagagcacc ggacatggcc acgga 55

<210> 7

<211> 55

<212> DNA

<213>Pig (Sus scrofa)

<400> 7

gctagagcct cagattgcca ttgccaacac ttacgcctgt aagggactag acaga 55

<210> 8

<211> 55

<212> DNA

<213>Pig (Sus scrofa)

<400> 8

tttgttggct gattcagaat aggcagcttc tcctcaattc tgtctagtcc cttac 55

<210> 9

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 9

ctgaatcagc caacaaacca ggttgtagcc aatgctaaag gggctgtgac tggggcaaa 59

<210> 10

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 10

acacaatcct tggccccagt cacagtagtc gtcatagcat cttttgcccc agtcacagc 59

<210> 11

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 11

ggggccaagg attgtgtggc cagcacgatc actgaggtgg tggacaagac caaagaagc 59

<210> 12

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 12

ccattaacca cagacttggt cttttccaca cttccggtca ctgcttcttt ggtcttgtc 59

<210> 13

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 13

aagtctgtgg ttaatggaag cattaacact gtcctgggaa gtcggatgat gcagctggt 59

<210> 14

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 14

agcagctctg atttggtgaa tgctttttct actccactgc tcaccagctg catcatccg 59

<210> 15

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 15

accaaatcag agctgctggt agaccagtac ctccctctca ctgaagaaga actagaaaa 59

<210> 16

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 16

ggcttctgaa ccatatcaaa tccttccact tttttggctt ctttttctag ttcttcttc 59

<210> 17

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 17

gatatggttc agaagccaag ttattatgtt agactgggat ccctgtccac caagctccg 59

<210> 18

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 18

acttctttaa ccctggtgag ggcctgctgg taggcccgtg agcggagctt ggtggacag 59

<210> 19

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 19

accagggtta aagaagtcaa gcaaaaaagc caggagacca tttctcagct ccattccac 59

<210> 20

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 20

gcattatgca cattcttcct ggcaaattca atcaggttga cagtggaatg gagctgaga 59

<210> 21

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 21

aagaatgtgc ataatgccaa ccagaaaatt caaggcactc aggataagct ctatctgtc 59

<210> 22

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 22

tctgtgtcat catagccaat gcttctcttc cattccaccc aggacagata gagcttatc 59

<210> 23

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 23

ggctatgatg acacagatga atcccactgt gctgagcata tagagtcacg tactctggc 59

<210> 24

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 24

tggcacgtgg tctggagctg ctgagtcagg ttgcgggcaa ttgccagagt acgtgactc 59

<210> 25

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 25

ctccagacca cgtgccacac cctcgtgtcc aacatccaag ggttaccaca gaacatcca 59

<210> 26

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 26

tagatgtcgc cagccatcac ccccaagtgg ttggcctgat cgtggatgtt ctgtggtaa 59

<210> 27

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 27

atggctggcg acatctactc agtgtttcac aatgcttcct cctttaagga aatgtctga 59

<210> 28

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 28

ttcattttct gcagctgccc cttgctggaa ctgaggaggc catcagacat ttccttaaa 59

<210> 29

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 29

cagctgcaga aaatgaagga gtctttagat gatgtgatgg attatcttgt taacaacac 59

<210> 30

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 30

gtcagttgag gataaaaggg acctaccagc cagttgaggg gcgtgttgtt aacaagata 59

<210> 31

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 31

ttttatcctc aactgaccga gtctcaggat gctcagtccc ggggtgcaga gaacacaac 59

<210> 32

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 32

ggtttaatgc gttttgtctc aggttgctgg gtctctgggc tcgttgtgtt ctctgcacc 59

<210> 33

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 33

acaaaacgca ttaaacctgc ccctgccagc agtgcatggg gcagccagtc aggtgacac 59

<210> 34

<211> 59

<212> DNA

<213>Pig (Sus scrofa)

<400> 34

accatggtac caagcttaag gcaggttgca acagtacagg acgtgtcacc tgactggct 59

<210> 35

<211> 1377

<212> DNA

<213>Pig (Sus scrofa)

<400> 35

atggcatccg ttgctgttga atcacaaccg agtgtggtga caagggtggc caacctaccc 60

ttggtgagct ccacgtatga ccttgtctcc tcggcttata tcagtacaaa ggatcagtat 120

ccctacttga agtctctgtg tgagatggca gagaagggcg tcaagaccat cacctccgtg 180

gccatgtccg gtgctctccc tatcatccag aagctagagc ctcagattgc cattgccaac 240

acttacgcct gtaagggact agacagaatt gaggagaagc tgcctattct gaatcagcca 300

acaaaccagg ttgtagccaa tgctaaaggg gctgtgactg gggcaaaaga tgctatgacg 360

actactgtga ctggggccaa ggattgtgtg gccagcacga tcactgaggt ggtggacaag 420

accaaagaag cagtgaccgg aagtgtggaa aagaccaagt ctgtggttaa tggaagcatt 480

aacactgtcc tgggaagtcg gatgatgcag ctggtgagca gtggagtaga aaaagcattc 540

accaaatcag agctgctggt agaccagtac ctccctctca ctgaagaaga actagaaaaa 600

gaagccaaaa aagtggaagg atttgatatg gttcagaagc caagttatta tgttagactg 660

ggatccctgt ccaccaagct ccgctcacgg gcctaccagc aggccctcac cagggttaaa 720

gaagtcaagc aaaaaagcca ggagaccatt tctcagctcc attccactgt caacctgatt 780

gaatttgcca ggaagaatgt gcataatgcc aaccagaaaa ttcaaggcac tcaggataag 840

ctctatctgt cctgggtgga atggaagaga agcattggct atgatgacac agatgaatcc 900

cactgtgctg agcatataga gtcacgtact ctggcaattg cccgcaacct gactcagcag 960

ctccagacca cgtgccacac cctcgtgtcc aacatccaag ggttaccaca gaacatccac 1020

gatcaggcca accacttggg ggtgatggct ggcgacatct actcagtgtt tcacaatgct 1080

tcctccttta aggaaatgtc tgatggcctc ctcagttcca gcaaggggca gctgcagaaa 1140

atgaaggagt ctttagatga tgtgatggat tatcttgtta acaacacgcc cctcaactgg 1200

ctggtaggtc ccttttatcc tcaactgacc gagtctcagg atgctcagtc ccggggtgca 1260

gagaacacaa cgagcccaga gacccagcaa cctgagacaa aacgcattaa acctgcccct 1320

gccagcagtg catggggcag ccagtcaggt gacacgtcct gtactgttgc aacctgc 1377

Claims

1. the PK-15 cell lines of pig PLIN2 genes overexpression, it is characterized in that：PK-15 cells contain the external source pig manually transfected PLIN2 genes, and PLIN2 genes have SEQ ID NO：Nucleotide sequence shown in 35, the cell are susceptible to PCV2 viruses, and Potency can be produced and reach 10^-7Virus above, which is pig kidney PK-15/PLIN2 cell lines, is preserved in Chinese Typical Representative culture Thing collection, deposit number are CCTCC NO:C2017251.

2. preparing the method for PK-15/PLIN2 cell lines in claim 1, this method comprises the following steps:

1）The clone of PLIN2 genes：According toPLIN2Gene order designs overlapping PCR primers, 34 altogether, between adjacent primer Have that 17bp reverse complementals are overlapping, there is the non-complementary series of 25bp in centre, and 34 primers are spliced into the total length of PLIN2 genes, by 34 By annealing after primer mixing, the 17bp sequence complementary pairings of adjacent primer, middle unpaired 25bp sequences, are protected in height Sequence forms complete double-strand by polishing under the action of true enzymePLIN2The full length sequence of gene, after 25 circulations,PLIN2 Gene is expanded, and then will expand the fragment obtained through electrophoresis detection；

2）It will expand what is obtained using plastic recovery kitPLN2Genetic fragment recycles, then with by the fragment and pCDNA3.1- EGFP carriers pass through respectivelyNheI andAflII digestion with restriction enzyme, electrophoresis detection connect after recycling purpose fragment through T4 ligases It is connected in pCDNA3.1-EGFP carriers, is built into pCDNA3.1-EGFP-PLIN2 plasmids, and through sequence verification；

3）Transfection and screening：Transfected plasmids PLIN2-pCDNA3.1-EGFP is to PK-15 cell lines, individual cells colonized culture Method filters out the PK-15/PLIN2 cell lines stablized and replicate external source PLIN2.

3. preparing the method for PCV2, this method comprises the following steps:

1）It is prepared by cell：PK-15/PLIN2 cell lines are thawed, are incubated in Kolle flask, are passaged to requirement；

2）Virus inoculation：Synchronous inocalation method by PCV2 virus inoculations on PK-15/PLIN2 cell lines, continue culture 24 it is small when；

3）Virus culture：24 it is small when after, by culture medium be changed to the maintaining liquid containing 2% hyclone continue culture 72 it is small when；

4）Harvest virus：Harvest step（3）In culture supernatant, and carry out PCV2 poison valency measures, virus titer can reach 10^-7 More than.

4. application of the cell line in PCV2 viruses are prepared described in claim 1.

5. application of the caused PCV2 viruses in vaccine is prepared described in claim 3.