CN107012122A - One kind immortalizes chicken embryo liver cell system and its production and use - Google Patents

One kind immortalizes chicken embryo liver cell system and its production and use Download PDF

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CN107012122A
CN107012122A CN201611140312.8A CN201611140312A CN107012122A CN 107012122 A CN107012122 A CN 107012122A CN 201611140312 A CN201611140312 A CN 201611140312A CN 107012122 A CN107012122 A CN 107012122A
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liver cell
hmrp18s
embryo liver
chicken embryo
cell
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冯磊
恽君雯
吴培培
陈丽
侯继波
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Jiangsu Academy of Agricultural Sciences
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Abstract

Chicken embryo liver cell system and its production and use is immortalized the invention discloses one kind.Immortalization chicken embryo liver cell system CEL hMRP18S 2 were deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 6th, 2016（Referred to as CGMCC）, deposit number is that CGMCC NO.12333. preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica.The immortalization chicken embryo liver cell obtained using the present invention remains the principal character and major function of primary chick embryo liver cell.And the cell can unconfined amplification cultivation in vitro, the cell line passed on for 100 generations in vitro still has preferable vigor, keeps division growth, does not occur aging and apoptosis.The preparation efficiency of viral antigen can be significantly improved by being applied to the propagation of I type aviadenovirus.

Description

One kind immortalizes chicken embryo liver cell system and its production and use
Technical field
Chicken embryo liver cell system and its production and use is immortalized the present invention relates to one kind, belongs to field of biology, In particular belong to bioengineering and veterinary biologicses technical field.
Background technology
Since 2013, I types aviadenovirus is largely propagated in fowl plant of China, has triggered serious epidemic situation, to cultivation Cause heavy economic losses in field.
For this virus, the main means of prevention that current China uses is to use aviadenovirus related vaccines.Existing skill We generally carry out aviadenovirus propagation to art using SPF chicken embryos or primary chicken embryo liver cell, so as to obtain related vaccines.
But, both modes all have certain defect.For example, the use of SPF chicken embryos can produce substantial amounts of virus dirt Dye production discarded object, and the acquisition of primary chicken embryo liver cell needs also exist for using SPF chicken embryos, and the preparation behaviour of primary cell Make extremely waste time and energy, easily pollute again.Therefore, existing aviadenovirus vaccine preparation technology falls behind, and prouctiveness is low, it is impossible to Demand is immunized in the reality for meeting fowl plant.
Cell immortality technology can cause the ability that target cell obtains the propagation of continuous passage in vitro.If can be right Primary chick embryo liver cell carries out immortalization transformation, being immortalized primary chick embryo liver cell system, it is possible to realize I types Aviadenovirus is bred in continuous cell line.So as to remove the troublesome operation for preparing primary cell every time from, disease is improved The preparation efficiency of malicious antigen.Solve the practical problem in current aviadenovirus related vaccines production.
What is more important avoids the use of SPF chicken embryos, it is to avoid pollution.
But, still not exclusively understand for the mechanism that primary cell is immortalized, it is considered that be that primary cell is passed some It there occurs the unstable rearrangement of cell chromosome after generation or genetic engineering operation and result in cell immortality, so as to possess body The ability of outer continuous passage propagation.At present, the remodeling method that primary cell is immortalized includes the domestication of spontaneous immortalization, exogenous virus Infection causes to immortalize, recombinantly express exogeneous viral protein cause immortalization these three.
The content of the invention
Present invention is primarily intended to provide a kind of immortalization chicken embryo cell line and preparation method thereof.The immortalization chicken embryo tire Cell line has the major function of primary chick embryo cell, and being capable of continuous passage in vitro.
In order to realize this purpose, a kind of immortalization chicken embryo liver cell system, the immortalization chicken embryonic liver are we disclosed Cell line CEL-hMRP18S-2 is deposited in the common micro- life of China Committee for Culture Collection of Microorganisms on December 7th, 2016 Thing center（Referred to as CGMCC）, deposit number is that CGMCC NO.13290. preservation address is BeiChen West Road, Chaoyang District, BeiJing City 1 Number No. 3 Institute of Microorganism, Academia Sinica of institute.
This immortalization chicken embryo liver cell system is to primary chick embryo by carrier for expression of eukaryon pCAG-hMRP18S-2 Liver cell transfects hMRP18S-2 genes, and continuous passage is expanded and obtained after being screened with G418 medicines through two-wheeled.
Specifically, this preparation method can be described as：
1）Using the upstream and downstream primer with KpnI and NotI restriction enzyme sites from the pUC- containing hMRP18S-2 coded sequences The fragment is expanded on hMRP18S-2 plasmids, in the carrier for expression of eukaryon through double digestion rear clone to equal double digestion, is built into hMRP18S-2（Human mitochondrion robosomal protein 18 S-2）Carrier for expression of eukaryon pCAG-hMRP18S-2, for transfection procedure.
There is part chicken β-actin promoter sequence in the carrier for expression of eukaryon in CAG promoters, apply what is originated in fowl There is stronger startup transcriptional activity in cell.
2）PCAG-hMRP18S-2 is transfected to primary chick embryo liver cell, is screened, obtained pair with G418 medicines two-wheeled G418 medicines are resistant, can stablize the restructuring chicken embryo liver cell for expressing hMRP18S-2 albumen.
3）Collect restructuring chicken embryo liver cell and implement continuous passage amplification, obtain immortalizing chicken embryo liver cell system CEL- hMRP18S-2。
The present inventor divides for this generation of cell line continuous passage amplification cultivation 100, and according to different generations Other freeze-stored cell, carries out function and feature detection showed after 100 generations of passage, still is able to retain the main spy of jungle fowl fetal hepatocytes Seek peace major function.
Wherein it is preferred to, step 1）Middle human mitochondrion robosomal protein 18 S-2 nucleic acid coding sequence is close according to fowl source The recombinant nucleotide sequence that the inclined preferendum of numeral is optimized, as shown in SEQ ID NO.1.
Also, it is used as another preferred, step 2）2 wheel screenings of middle G418 medicines refer to that concentration is 200ug/ml respectively With 400ug/ml G418 drug solutions, and the step sizing of 20 generations is carried out under various concentrations.
The present invention further discloses this immortalization chicken embryo liver cell and tied up in I types aviadenovirus propagation simultaneously Using.
Specifically, its application mode is：
1）Continuous passage expands CEL-hMRP18S-2 cells and builds storehouse.
The cell has the characteristic of adherent growth, is placed under 37 DEG C, 5%CO2 culture environment, cell growth degree of converging reaches Aviadenovirus, which can be carried out, to 90% connects malicious operation.
2）According to MOI be 0.1 inoculation I types aviadenovirus in CEL-hMRP18S-2 cells, connect after poison 3~5 days cells i.e. It may occur in which obvious cytopathy.
3）In the continuous generation of blind passage I types aviadenovirus 20, can detect viral Effective multiplication in CEL-hMRP18S-2 cells, Virus multiplication potency can reach 108.5TCID50/ml。
And then, the present invention is also further claimed this immortalization chicken embryo liver cell and ties up to preparation I type aviadenovirus Application in disease vaccine.
The immortalization chicken embryo liver cell obtained using the present invention remains the principal character of primary chick embryo liver cell And major function.And the cell can unconfined amplification cultivation in vitro, the cell line passed on for 100 generations in vitro still to be had Preferable vigor, keeps division growth, does not occur aging and apoptosis.The propagation for being applied to I type aviadenovirus can be notable Improve the preparation efficiency of viral antigen.
Brief description of the drawings
Fig. 1 is the digestion qualification result figure of carrier；
M：Nucleic acid marker, 1:Digestion products of the pCAG-hMRP18S-2 through KpnI and NotI.
Fig. 2 is to identify testing result figure with RT-PCR；
M：Nucleic acid marker, 1:CEL-hMRP18S-2 cells, 2:Primary chick embryo liver cell.
Fig. 3 is that Western blot identify testing result figure；
M：Protein marker, 1:CEL-hMRP18S-2 cells, 2:Primary chick embryo liver cell.
Fig. 4 is CEL-hMRP18S-2 cell growth curve figures.
Fig. 5 is the PCR qualification result figures that I types aviadenovirus is continuously bred in CEL-hMRP18S-2 cells；
M：Nucleic acid marker, former poison：The former poison of I types aviadenovirus of SPF chicken embryos amplification,
P1~P20：I types aviadenovirus continuous passage sample in CEL-hMRP18S-2 cells.
Fig. 6 is cell growth form, the cytopathy that I types aviadenovirus is continuously bred in CEL-hMRP18S-2 cells Aspect graph；
A：Cytopathy of the I types aviadenovirus on CEL-hMRP18S-2 cells
B：The cellular morphology of malicious CEL-hMRP18S-2 cells is not connect
Embodiment
In order to be better understood from the present invention, below we in conjunction with specific embodiments to the present invention further explained State.
The structure of embodiment 1pCAG-hMRP18S-2 expression vectors
1.1 reagent
HMRP18S-2 protein code genetic fragments after codon optimization（SEQ ID NO.1）By Jin Sirui biotech companies Chemical synthesis, and be cloned into pUC57 carriers.PMD19-T carriers, various restriction enzymes, ExTaq enzymes, T4DNA ligases Equimolecular biologic material is purchased from TAKARA companies.Carrier for expression of eukaryon containing CAG promoter sequences is by national life for animals Tetramune Engineering Technical Research Centre voluntarily builds and preserved.Glue reclaim kit, plasmid are small to refer to that big extraction reagent kit is purchased from Qiagen companies.
1.2 primers are synthesized：Primer for expanding hMRP18S-2 albumen ORF sequences is closed by Jin Sirui biotech companies Into.
p1：GCCGGTACCGCCACCATGGCCGCG（It is KpnI restriction enzyme sites at line）
p2：GCCGCGGCCGCCTAGAGAGCACTCTG（It is NotI restriction enzyme sites at line）
The structure of 1.3 pCAG-hMRPS18-2 expression vectors and carry greatly
Using above-mentioned primer, using pUC-hMRP18S-2 as template, the complete ORF fragments for amplifying hMRP18S-2 are reacted through PCR, PCR reaction conditions：95 DEG C 3 minutes；95 DEG C 15 seconds, 62 DEG C 30 seconds, 72 DEG C 50 seconds, repeat 35 circulation；72 DEG C extend 10 points Clock.The purpose fragment and pCAG plasmids of PCR amplifications are handled using KpnI and NotI double digestions, by purpose fragment and pCAG plasmids Skeleton fragment carry out glue reclaim, it is quantitative after carry out nucleic acid attended operation, convert into DH5 α, select positive bacterium colony and largely expand Increase, digestion verification recombinant eukaryon expression vector pCAG-hMRP18S-2 after plasmid extraction.Reagent is carried greatly through Qiagen companies plasmid Box extracting obtains the higher transfection plasmid of quality.
1.4 result
Purpose fragment has been connected into carrier for expression of eukaryon by this experiment using two restriction enzyme sites, obtains pCAG-hMRP18S-2 Carrier for expression of eukaryon is used for follow-up transfection assay.It is aobvious using KpnI and NotI double digestion pCAG-hMRP18S-2 carriers qualification result Show that purpose fragment connection is correct（Such as Fig. 1）.Carried greatly through plasmid and obtain the higher plasmid of quality, OD260/OD280 value is 1.86, For the experiment of follow-up plasmid transfection.
The recombinant C EL-hMRP18S-2 of embodiment 2 structure
2.1 material：
Reagent：MEM, G418, Opti-MEM, transfection reagent are purchased from Invitrogen companies.The primary antibody of rabbit-anti hMRP18S-2 albumen Purchased from Santa Cruz biotech companies, goat anti-rabbit igg, the goat anti-rabbit igg of HRP marks of FITC marks are purchased from Hangzhou connection section Biotech company.RNA extracts kits, Reverse Transcriptase kit equimolecular biological reagent are purchased from Qiagen companies.
Biomaterial：Primary chick embryo liver cell by National Research Center of Veterinary Biologicals Engineering and Technology voluntarily from Separate and obtain in SPF chicken embryos, the primary cell is inoculated in standby in the MEM culture mediums containing 10% NBCS after preparing.
The screening of 2.2 G418 activities
The initial cell density for adjusting primary chick embryo liver cell is 5 × 105Cells/ml, carries out G418 works in 96 orifice plates With the screening of concentration.In nutrient solution add various concentrations G148, concentration be 100,200,300,400,500,600ug/ml, put In 37 DEG C, 5%CO2Incubator in, observe cell state daily, cultivate 6-12 days, determine that the minimum of G418 effectively works dense Degree.
2.3 cell transfecting
The inoculation initial cell density of primary chick embryo liver cell is 5 × 105Cells/ml, in 6 orifice plates after overnight incubation For transfecting.Culture medium is replaced by Opti-MEM culture mediums before transfection and is incubated 10 minutes in incubator.According to 25kDa branch The operation instruction configuration transfection composite of chain PEI transfection reagents, and be incubated 5 minutes at room temperature.It is after the completion of incubation that transfection is multiple Compound is added dropwise in primary chick embryo liver cell, is placed in incubator 6~8 hours.Afterwards will be multiple containing remaining transfection The culture supernatant of compound is removed, and is replaced with the normal incubation medium culture of the primary cell.
The screening of 2.4 CEL-hMRP18S-2 cell clones：
After transfection the primary chick embryo liver cell of 24~72 hours after digestion according to 1:3~1:5 ratio renewed vaccination is to 6 In orifice plate, in 37 DEG C, 5%CO2And enter recombinant cell screening in the nutrient solution containing 200ug/ml G418.One was changed according to 3 days The frequency of not good liquor carries out G418 and persistently screened, first round screening time substantially 2~3 weeks.Selection can have G418 screening pressures The recombinant C EL-hMRP18S-2 cell clones continued to multiply under power continue to cultivate.It is in second wheel screening that G418 screening operations is dense Degree, which is improved to 400ug/ml, continues screening of pressurizeing.Final obtain can stablize the restructuring of continued propagation in G418 screens resistance CEL-hMRP18S-2 cell clones.
2.5 RT-PCR, western blot are identified：
Will the CEL-hMRP18S-2 cell clones that screened digest after be collected by centrifugation, tried with being extracted after PBS 3 times by RNA Agent box illustrates the total serum IgE extracted.Vessel, pipette tips, water used in above-mentioned experiment etc. are whole using DEPC processing and high pressure Gloves are worn to be operated.The RNA samples extracted enter according to Reverse Transcriptase kit step explanation synthesis cDNA by masterplate of cDNA Row target sequence PCR is expanded, and is operated in design parameter be the same as Example one.
Through being collected by centrifugation after the CEL-hMRP18S-2 cell clones screened are digested, according to line after PBS 3 times The step of mitochondrial protein extraction agent box explanation extracting mitochondrial protein, for western blot detections.With 5% concentration glue Separation gel with 8% carries out SDS-PAGE protein electrophoresises, and albumen on glue is transferred on pvdf membrane using the operation of half dry type transferring film, After being closed through BSA, the goat-anti rabbit secondary antibody marked using the rabbit source primary antibody and HRP of hMRP18S-2 albumen, which is incubated, turns purposeful albumen Pvdf membrane, using chromogenic reagent identify destination protein expression.
Above-mentioned qualification test is reflected using primary chick embryo liver cell as blank control according to same operation step It is fixed.
2.6 experimental result
2.6.1 the screening of G418 working concentrations is determined
To kill the concentration of whole primary chick embryo liver cells in 5 days as G418 screening operation concentration.Determine 200ug/ml Working concentration during for normal screening, 400ug/ml is selected in pressurization screening.
2.6.2 the clone of recombinant cell, screening and identification
Cell after transfection passes through the screening of 3 weeks in 200ug/ml G418 screening environment, and obtaining being capable of normal continuous life Long recombinant cell strain.The cell line of survival is dispersed in the G418 screening and culturing mediums containing 400ug/ml and continues the sieve that pressurizes Choosing, it is final to obtain the CEL-hMRP18S-2 recombinant cell clones for being capable of continued propagation.
Detected through RT-PCR find CEL-hMRP18S-2 recombinant cell clones it is amplifiable go out 795bp specific band, it is and former Amplification for chicken fetal liver cells is feminine gender（As shown in Figure 2）.Western blot detections find recombinant C EL- HMRP18S-2 cell clones express about 29kDa destination protein, and primary chick embryo liver cell is expressed without destination protein （As shown in Figure 3）.
The continuous growth performance of the CEL-hMRP18S-2 cells of embodiment 3 is determined
3.1 materials and methods
Primary chick embryo liver cell is voluntarily prepared by National Research Center of Veterinary Biologicals Engineering and Technology.
Recombinant C EL-hMRP18S-2 cells screen acquisition as previously described, and it is to preserve to build.
Culture medium：MEM culture mediums containing 10% NBCS.
The recombinant C EL-hMRP18S-2 cells of primary chick embryo liver cell generation different with continuous passage with 3 × 105Cells/ml initial density is inoculated in T150 culture square vase, is positioned over 37 DEG C, 5%CO2Incubator in cultivate.Often Cell density and survival rate is measured by sampling within 24 hours, draws the cell growth curve of different growth generations.
3.2 experimental result
The growth curve of recombinant C EL-hMRP18S-2 cells is as shown in Figure 4.Compared with primary chick embryo liver cell, recombinated The growth rate of CEL-hMRP18S-2 cells is apparently higher than primary chick embryo liver cell.Particularly possessing continuous passage energy After power, with the generation increase of passage, the cell cultivate in T150 bottle cell acquisition maximum cell density can reach 2.6 × 106Cells/ml, and primary chick embryo liver cell cell propagation efficiency in first culture is poor, breeds unobvious.Explanation The continuous passage that the recombinant cell obtained after foreign gene transfection and screening operation possesses not available for primary cell increases Grow ability.
The CEL-hMRP18S-2 cells of embodiment 4 breed aviadenovirus
4.1 test material
Primary chick embryo liver cell is voluntarily prepared by National Research Center of Veterinary Biologicals Engineering and Technology.
Recombinant C EL-hMRP18S-2 cells screen acquisition as previously described, and it is to preserve to build.
Cell culture medium：MEM culture mediums containing 10% NBCS.
Virus multiplication maintaining liquid：MEM culture mediums containing 2% NBCS.
I type aviadenovirus：Separated, identified by National Research Center of Veterinary Biologicals Engineering and Technology, preservation.
4.2 test method
Recombinant C EL-hMRP18S-2 cells and primary chick embryo liver cell are inoculated in 6 orifice plates.Treat that cell confluency degree reaches More than 90%, culture supernatant is removed, after PBS 3 times, 200ul aviadenovirus is accessed（It is 0.1 to calculate MOI）, in 37 DEG C of trainings Support and virus liquid is removed after being incubated 1 hour in case, change virus multiplication maintaining liquid and continue to cultivate 3~5 days, observe cytopathy, receive Obtain virus multiplication sample, -40 DEG C freeze it is to be measured.In continuous blind passage viral 20 generation, detect virus multiplication potency.
Virus multiplication titration.Sample after virus multiplication is subjected to 10 times of serial dilutions, is inoculated in containing having reached 90% In the orifice plate of CEL-hMRP18S-2 cells 96 for degree of converging, each 8 holes of dilution factor, 37 DEG C, 50%CO2Cultivated 3 days in incubator After observe cytopathy, according to Reed-Muench methods calculate virus cell median infective dose（TCID50/ml）.
Virus PCR is detected.Detection primer, F：CAACTACATCGGGTTCAGGGATAACTTC, R： CCAGTTTCTGTGGTGGTTGAAGGGGTT.It is 766bp or so to expand purpose fragment.PCR amplification conditions are：Step one：98℃ 30 seconds；Step 2：98 DEG C 15 seconds, 55 DEG C 30 seconds, 72 DEG C 20 seconds, 30 circulation；Step 3：72 DEG C 10 minutes.
4.3 result of the test
CEL-hMRP18S-2 cells can support I types aviadenovirus to carry out the blind passage amplification in continuous 20 generation, and virus multiplication is normal. As shown in figure 5, virus can clearly detect the viral nucleic acid fragment in the cell after the generation of blind passage 20.As shown in fig. 6, viral It can breed in the cell, connect cell after poison and show typically in CPs such as pencil, aggregation bunchiness, and not connect poison Compareing CEL-hMRP18S-2 cells, then cell face is smooth, and cellular morphology is normal.As shown in table 1, I types aviadenovirus is in CEL- Propagation potency ratio in hMRP18S-2 cells and primary chick embryo liver cell is compared with the immortalization CEL- in the present invention The virus multiplication titre of hMRP18S-2 cells is significantly higher than the viral titer of primary cell, reaches 8.5lgTCID50/ml。
Breeding ratio of the I types aviadenovirus of table 1. in CEL-hMRP18S-2 cells and primary chick embryo liver cell compared with
Unit：lgTCID50/ml。
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>One kind immortalizes chicken embryo liver cell system and its production and use
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 777
<212> DNA
<213>It is artificial synthesized
<400> 1
atggccgcgt ccgtactcaa caccgtactg cggaggctgc cgatgttgag tctgtttagg 60
ggttcacatc gcgtccaggt tcctttgcaa acgctgtgta ctaaagctcc ttccgaggaa 120
gatagtctga gctcagtacc tataagtccc tacaaggacg agccgtggaa gtaccttgaa 180
agtgaggagt accaggaaag gtacggcagt agacctgttt gggccgacta tcggcggaac 240
cataaagggg gggtcccacc tcaacggaca agaaagacct gtatacggcg caacaaagtg 300
gtcggcaatc cgtgccctat atgtcgggac cataagttgc atgtggattt tcggaatgtt 360
aagcttttgg aacaatttgt ttgtgctcac acagggatca tattttacgc cccgtacacg 420
ggggtgtgtg taaaacaaca taaacgcctt acccaagcta tccaaaaagc acgggatcac 480
ggcctgctca tttaccatat tcctcaagtt gaaccgaggg acctggattt ttctaccagc 540
cacggtgccg tctccgcaac cccgccggcc cctacattgg tcagtgggga cccatggtat 600
ccatggtata attggaagca gccgcccgaa cgcgagttga gcagactgcg gcggctctat 660
caaggacatc tccaggagga gagcggtcct ccacctgagt ccatgcccaa aatgccaccc 720
aggacaccgg ctgaagcaag ttcaaccggg caaacgggcc cgcagagtgc tctctag 777

Claims (5)

1. one kind immortalizes chicken embryo liver cell system, it is characterised in that：Immortalization chicken embryo liver cell system CEL-hMRP18S- 2 were deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 7th, 2016（Referred to as CGMCC）, deposit number is CGMCC NO.13290, and preservation address is the Chinese section of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of microbiology of institute.
2. the preparation method for immortalizing chicken embryo liver cell system described in claim 1, it is characterised in that：It is by eukaryotic expression Carrier pCAG-hMRP18S-2 transfects hMRP18S-2 genes to primary chick embryo liver cell, after being screened with G418 medicines through two-wheeled Continuous passage is expanded and obtained.
3. the preparation method according to claim 2 for immortalizing chicken embryo liver cell system, it is characterised in that：hMRP18S-2 Nucleic acid coding sequence as shown in SEQ ID NO.1.
4. the application that the immortalization chicken embryo liver cell described in claim 1 is tied up in I types aviadenovirus propagation.
5. the immortalization chicken embryo liver cell described in claim 1 ties up to the application prepared in I type aviadenovirus disease vaccines.
CN201611140312.8A 2016-12-12 2016-12-12 One kind immortalizes chicken embryo liver cell system and its production and use Pending CN107012122A (en)

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CN106075427A (en) * 2016-06-30 2016-11-09 武汉中博生物股份有限公司 1 type aviadenovirus vaccine, the preparation method of yolk antibody

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CN1934243A (en) * 2003-11-03 2007-03-21 普罗拜奥根股份公司 Immortalized avian cell lines for virus production
CN104293832A (en) * 2014-10-09 2015-01-21 河南农业大学 Eukaryotic recombinant micro-ring expression vector for cell immortalization
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