CN107629996A - The construction method of one plant of grass carp pectoral fin cell line - Google Patents
The construction method of one plant of grass carp pectoral fin cell line Download PDFInfo
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Abstract
The invention discloses the construction method of one plant of grass carp pectoral fin cell line, it comprises the following steps：1) original cuiture：Grass carp pectoral fin tissue is taken, epidermis membrane tissue is removed, is shredded after PBS rinsed cleans；Digestive juice A is added, is well mixed；By the broken tissue block even spread of mixture slaking enzyme into blake bottle；Blake bottle is inverted in incubator, after being inverted 6~8h of dry doubling, complete culture solution is added and is just putting and continue 10~15h of culture, addition complete culture solution continues culture 5 10 days, obtains growing to 80%~90% primary cell converged；2) Secondary Culture：After primary pectoral fin cell confluent monolayers, old nutrient solution is removed, with twice of PBS, adds trypsin digestion cell, after cell rounding, complete culture solution is added, piping and druming, hang cell, progress Secondary Culture.The present invention establishes grass carp pectoral fin cell line and have detected sensitiveness of the cell to GCHV, and the research for exhaling long lonely virus for later stage grass carp is laid a good foundation.
Description
Technical field
The present invention relates to biological cell to build field, more particularly to a kind of side that cell line is established using grass carp pectoral fin cell
Method.
Background technology
Grass carp (Ctenopharyngodonidellus) is as cultivation product mostly important in China's cultured freshwater fish
Kind, there is extremely important value in aquaculture industry, for its yield in 2015 more than 5,000,000 tons, it is light that its yield accounts for China
More than the 20% of water cultivation total output.Current marine industry is just quickly to intensive and large-scale development so that high density, intensive
Change aquaculture model on a large scale to be carried out, so as to cause the outburst of aquatic animal disease, cause great economic loss, and
Development to the industry causes huge resistance.It is easy to make the diseases such as bacterium, virus and parasite under highdensity breeding environment
Former infection host, and amount reproduction, aquatic livestock is caused greatly to endanger, at the same time brings huge economic loss.
In all cause of diseases for infecting grass carp, GCRV (Grass carp reovims, GCRV), hemorrhagic disease of grass carp disease is commonly called as
Malicious (GCHV), hemorrhagic disease of grass carp harm is maximum caused by infection, turn into China's fresh water aquaculture the most distinct issues it
One.Disease preventing and treating is difficult, and period of disease length, infectiousness is strong, and the death rate is up to 90%, once outburst, makes to China's grass carp aquaculture
Into huge loss, according to incompletely statistics, China every year the economic loss as caused by hemorrhagic disease of grass carp more than 1,000,000,000.2008
Hemorrhagic disease of grass carp is defined as the class epidemic disease of aquatic animal two by the Ministry of Agriculture.
Because hemorrhagic disease of grass carp causes significant damage to grass carp cultivation, numerous domestic research institution is from cause of disease, host and ring
The each side in border deploys actively research to the anti-reovirus of grass carp, it is intended to seeks the effective ways for controlling this sick.To being at present
Only hemorrhagic disease of grass carp does not have special effect medicine therapeutic also, and immunoprophylaxis is the control maximally effective prevention and controls of hemorrhagic disease of grass carp.Establish
The sensitive cell line of virus is the first step for researching and developing vaccine, and studies viral route of infection, infection mechanism and develop virus
The important system of vaccine.Therefore, Grass Carp Cell system is established, is the separation and identification for carrying out GCRV, studies its cause
Anttdisease Mechanism, vaccine prepares and the important foundation of immunoprophylaxis technology.The establishing techniques of fish primary cell line be currently it is a kind of compared with
For the technology of maturation, but to obtain and still deposit very big contingency and difficulty for the sensitive cell line of virus, so building
It is large batch of primary cell strain to be prepared, to quality so as to sieve with quantity that vertical one plant, which needs the effective approach of fish cell system,
Select the cell line of sensitivity.
In infection GCRV ill grass carp, clinical symptoms are frequently found grass carp pectoral fin and its bleeding of substrate position, take portion
Divide pectoral fin detection, detection has GCRV infection, therefore speculates that grass carp pectoral fin cell can exhale grass carp long orphan's virus sensitive.Present invention grass
The pectoral fin tissue of fish carries out the condition of culture that cell culture has primarily determined that grass carp pectoral fin cell, establishes grass carp pectoral fin cell line
(being named as CPF, Ctenopharyngodonidellus Pectoral Fin) simultaneously have detected cell to GCHV
(GCRV) sensitiveness, the research for exhaling long lonely virus for later stage grass carp are laid a good foundation.
The content of the invention
It is an object of the invention to disclose a kind of method for establishing cell line using grass carp pectoral fin cell.
The technical solution used in the present invention is：A kind of construction method of grass carp pectoral fin cell line, it comprises the following steps：
1) original cuiture：Grass carp pectoral fin tissue is taken, epidermis membrane tissue is removed, is shredded after PBS rinsed cleans；Digestive juice A is added, mixing is equal
It is even；By the broken tissue block even spread of mixture slaking enzyme into blake bottle；Blake bottle is inverted in incubator, inversion dry doubling 6~
After 8h, add complete culture solution and just putting and continue 10~15h of culture, addition complete culture solution continues culture 5-10 days, is grown
The primary cell converged to 80%~90%；2) Secondary Culture：After primary pectoral fin cell confluent monolayers, old nutrient solution is removed, is used
Twice of PBS, trypsin digestion cell is added, after cell rounding, add complete culture solution, piping and druming, hanged cell, passed
It is commissioned to train foster.
Preferably, digestive juice A contains Collagenase I and bovine serum albumin(BSA).
Preferably, digestive juice A composition is that 0.1% Collagenase I adds 5% bovine serum albumin(BSA).
Preferably, complete culture solution is containing hyclone, Grass Carp Serum, basic fibroblast like growth factor, epidermis life
The long factor, penicillin, streptomysin, amphotericin B, M199 nutrient solutions.
It is further preferred that complete culture solution is containing 20% hyclone, 0.5%~1% Grass Carp Serum, 20ng/L alkali
Property is into fiber like growth factor, 1ng/mL EGF, 100U/mL penicillin, 100 μ g/mL streptomysins, 0.25 μ g/
ML amphotericin Bs, pH value are 7.2-7.4 M199 nutrient solutions.
The preparation method of Grass Carp Serum is：The health and live grass carp in 1~2 age is taken, from the tail vein blood of fish, at room temperature
Half an hour is placed, after blood clotting contraction, then is transferred in 4 DEG C of refrigerator, is stood overnight, clot is further shunk and is separated out
Serum；Rotating speed 5000-6000rpm/min centrifuges 5~10min, takes supernatant filter membrane at 4 DEG C within second day, and -20 DEG C of preservations are standby
With.
Preferably, the cultural method of original cuiture is：Take body to grow 10 ± 0.5cm fresh and alive grass carp to soak in 75% alcohol
30~60 seconds, be placed in it is sterile in super-clean bench remove pectoral fin tissue, gently wipe epidermis membrane tissue off with scalpel, after PBS rinsed cleans,
It is placed in 5mL sterile penicillin bottle, PBS containing 200ul in bottle, is shredded with operating scissors；Add the digestion of 2 times of volumes
Liquid A, it is well mixed；With by the broken tissue block even spread of mixture slaking enzyme in advance with the coated blake bottle of 0.1% gelatin；
Blake bottle is inverted into 27 DEG C of incubators of saturated humidity, after being inverted 6~8h of dry doubling, is slowly added to 2mL complete mediums
Just putting and continuing 10~15h of culture, addition culture medium continues culture 5~10 days to 5mL, obtains growing to 80%~90% and converges
Primary cell.
Preferably, the cultural method of Secondary Culture is：After primary pectoral fin cell confluent monolayers, old culture is removed with suction pipe
Liquid, with twice of PBS, 0.25% pancreatin 1mL vitellophags are added, after Microscopic observation cell rounding, it is complete to add 10mL
Nutrient solution, cell has been hanged in piping and druming, with 1:2 are passed on, and are added complete culture solution to 5mL/ bottles, are put into 27 DEG C of incubators and carry out
Secondary Culture；Passage in about 3~5 days later is once；When reaching for 10 generation, hyclone content is kept to cumulative volume in cell culture fluid
10%, no longer add Grass Carp Serum, Basic Fibroblast Growth Factor and EGF in nutrient solution.
The beneficial effects of the invention are as follows：The pectoral fin tissue of grass carp carries out cell culture and has primarily determined that grass carp pectoral fin cell
Condition of culture, establish grass carp pectoral fin cell line and have detected sensitiveness of the cell to GCHV, be later stage grass carp
The research of long lonely virus is exhaled to lay a good foundation.
Brief description of the drawings
Fig. 1 observes for grass carp pectoral fin cellular morphology, and wherein A is primary cell；B is 15 generation cells；C is 50 generation cells；D is
100 generation cells.
Fig. 2 is the adherent observation of grass carp pectoral fin cell primary culture, and wherein A is that tissue block method's 15d cells are moved out situation；B is
Trypsase is digested method culture 13d cell attachment situations；C is that clostridiopetidase A tissue block Combined culture method 3d cells are moved out situation.
Fig. 3 is the growth curve of the 60th generation grass carp pectoral fin cell.
Fig. 4 is GCRV- I agarose gel electrophoresis figure piece.M:Marker 1,2：Purpose band
Fig. 5 is that grass carp pectoral fin cell line infects GCRV GCRV pictures, and wherein A is control；B is cell infection
Viral GCRV is after 2 days；C, D is virus infection GCRV cell electron microscopic section figure.
Embodiment
Embodiment
1. prepare cell culture fluid：
GIBCO companies M199 culture mediums are taken, add the hyclone for accounting for cumulative volume 10~20% used, 0.5~1% grass carp
Serum, 20ng/mL Basic Fibroblast Growth Factor (bFGF), 1ng/mL EGF (EGF), 4 DEG C of storages are standby
With；
2 original cuitures:
Take body to grow 10 ± 0.5cm fresh and alive grass carp to soak 30~60 seconds in 75% alcohol, be placed in sterile in super-clean bench take
Lower pectoral fin tissue, gently wipes epidermis membrane tissue off with scalpel, after PBS rinsed cleans, is placed in 5mL sterile penicillin bottle
(PBS containing 200ul), is shredded with operating scissors；The digestive juice A of about 2 times of volumes is added, is well mixed.With the stainless steel of sterilizing
Spatula is by the broken tissue block even spread of mixture slaking enzyme in advance with the coated 25cm of 0.1% gelatin2In blake bottle；Will training
Foster bottle is inverted into 27 DEG C of incubators of saturated humidity, after being inverted 6~8h of dry doubling, is slowly added to 2mL complete mediums and is just put
Continue 10~15h of culture, addition culture medium to 5mL, continue culture 5-10 days, obtain growing to 80%~90% converge it is primary
Cell.Wherein, digestive juice A composition is that 0.1% Collagenase I adds 5% bovine serum albumin(BSA).
This method has been contrast experiment with trypsinization and tissue block method, as a result such as table 1, Fig. 2.
1 grass carp pectoral fin cell of table, three kinds of primary culture method contrasts
3. Secondary Culture:
After primary pectoral fin cell confluent monolayers, old nutrient solution is removed with suction pipe, with twice of PBS, adds 0.25% pancreas
Enzyme-EDTA (1:1) 1mL vitellophags, after Microscopic observation cell rounding, 10mL complete culture solutions is added, piping and druming, have been hanged thin
Born of the same parents, with 1:2 are passed on, and are added complete culture solution to 5mL/ bottles, are put into 27 DEG C of incubators and carry out Secondary Culture；Later about 3
Passage in~5 days is once；When reaching for 10 generation, hyclone content is kept to the 10% of cumulative volume in cell culture fluid, in nutrient solution
No longer add Grass Carp Serum, Basic Fibroblast Growth Factor (bFGF) and EGF (EGF).
Complete medium formula is that described culture medium prescription is containing 20% hyclone, 0.5%~1% grass carp blood
Clearly, 20ng/L basic fibroblasts like growth factor (bFGF), 1ng/mL EGF (EGF), 100U/mL penicillin,
100 μ g/mL streptomysins, 0.25 μ g/mL amphotericin Bs, pH value are 7.2-7.4 M199 nutrient solutions.
The preparation of Grass Carp Serum：The health and live grass carp in 1~2 age is taken, the tail vein blood with syringe from fish, is gently squeezed
1.5mL centrifuge tube is pressed into, places half an hour at room temperature, after blood clotting contraction, then is transferred in 4 DEG C of refrigerator, stands
Overnight, clot is further shunk and separate out serum, be not easy to form haemolysis during centrifugation.Second day at 4 DEG C, rotating speed
5000-6000rpm/min, centrifuge 5-10min.Supernatant is taken, successively crosses 0.45 μm and 0.22 μm of filter membrane, -20 DEG C save backup.
From inoculation tissue so far, cell passed on for 100 generations, and cell line, which is built, is tied to form work(.
4 cells freeze and recovered
4.1 cells freeze：With trypsin digestion collect 2 bottles be in exponential phase grass carp pectoral fin cells, 1000g from
Supernatant discarding after heart 5min, frozen with 1mL after protecting liquid (the M199 nutrient solutions containing 20%FBS and 10%DMSO) that cell is resuspended
It is placed in cryopreservation tube, cryopreservation tube is placed in program temperature reduction box in -80 DEG C overnight, finally puts into liquid nitrogen (- 196 DEG C) medium-term and long-term guarantor
Deposit, and make a record.
The recovery of 4.2 cells：During recovery cell, cryopreservation tube is taken out from liquid nitrogen, is thawed rapidly in 37 DEG C of water-baths,
1000g centrifugations 5min is inoculated in 25cm after collecting cell2Blake bottle in, be positioned in incubator 27 DEG C of cultures, treat that cell pastes
Supernatant is abandoned after wall, nutrient solution is changed, continues to cultivate.
Cell after recovery is subjected to Trypan Blue, the survival rate ＞ 95% of cell is determined with cell counter.
5 viral infection experiments:
Sensitiveness of the grass carp pectoral fin cell to GCHV (GCRV)：
Cell is inoculated in 25cm2When cell monolayer reaches 80%~90% in blake bottle, with 2 additions of PBS cell
GCRV (GCRV-873) suspension 1mL, standing adsorption 1h, suction are abandoned viral suspension and cleaned carefully with the culture medium of serum-free
Cellular surface 2 times, add the culture medium containing 3%FBS and be placed in 27 DEG C of incubator cultures, cell is observed under inverted microscope daily becomes
Change and photograph to record.When CPE (cytopathic effect) occur be used as viral susceptibility index, show cell to the virus sensitivity
(Fig. 5 B).Take 200 μ L cell liquid to extract RNA, and enter performing PCR with GCRV- I primer and expand, obtain 532bp purpose band
(Fig. 4).The virion (Fig. 5 C, D) that GCRV is in lattice-like arrangement can be observed under Electronic Speculum.
Claims (8)
1. a kind of construction method of grass carp pectoral fin cell line, it comprises the following steps：
1) original cuiture：Grass carp pectoral fin tissue is taken, epidermis membrane tissue is removed, is shredded after PBS rinsed cleans；Digestive juice A is added, is mixed
Close uniform；By the broken tissue block even spread of mixture slaking enzyme into blake bottle；Blake bottle is inverted in incubator, is inverted dry doubling
After 6~8h, add complete culture solution and just putting and continue 10~15h of culture, addition complete culture solution continues culture 5-10 days, is given birth to
Grow to 80%~90% primary cell converged；
2) Secondary Culture：After primary pectoral fin cell confluent monolayers, old nutrient solution is removed, with twice of PBS, adds pancreatin digestion
Cell, after cell rounding, complete culture solution is added, piping and druming, has hanged cell, carries out Secondary Culture.
2. construction method according to claim 1, it is characterised in that the digestive juice A contains Collagenase I and ox blood is pure
Albumen.
3. construction method according to claim 1, it is characterised in that the composition of the digestive juice A is 0.1% clostridiopetidase A
I adds 5% bovine serum albumin(BSA).
4. construction method according to claim 1, it is characterised in that the complete culture solution is containing hyclone, grass
Fish serum, basic fibroblast like growth factor, EGF, penicillin, streptomysin, amphotericin B, M199 nutrient solutions.
5. construction method according to claim 1, it is characterised in that the complete culture solution is containing 20% tire ox blood
Clearly, 0.5%~1% Grass Carp Serum, 20ng/L basic fibroblasts like growth factor, 1ng/mL EGF, 100U/mL
Penicillin, 100 μ g/mL streptomysins, 0.25 μ g/mL amphotericin Bs, pH value are 7.2-7.4 M199 nutrient solutions.
6. the construction method according to claim 4 or 5, it is characterised in that the preparation method of Grass Carp Serum is：Took for 1~2 age
Health and live grass carp, from the tail vein blood of fish, place half an hour at room temperature, after blood clotting contraction, then be transferred to 4 DEG C
Refrigerator in, stand overnight, clot is further shunk and separate out serum；Second day at 4 DEG C, rotating speed 5000-6000rpm/
Min centrifuges 5~10min, takes supernatant filter membrane, -20 DEG C save backup.
7. construction method according to claim 1, it is characterised in that the cultural method of original cuiture is：Take body length 10 ±
0.5cm fresh and alive grass carp is soaked 30~60 seconds in 75% alcohol, be placed in it is sterile in super-clean bench remove pectoral fin tissue, use scalpel
It is light to wipe epidermis membrane tissue off, after PBS rinsed cleans, it is placed in 5mL sterile penicillin bottle, PBS containing 200ul in bottle, uses hand
Art is shredded；The digestive juice A of 2 times of volumes is added, is well mixed；Arrived with by the broken tissue block even spread of mixture slaking enzyme
In advance with the coated blake bottle of 0.1% gelatin；Blake bottle is inverted into 27 DEG C of incubators of saturated humidity, is inverted dry doubling
After 6~8h, be slowly added to 2mL complete mediums and just putting continue 10~15h of culture, addition culture medium to 5mL, continue culture 5~
10 days, obtain growing to 80%~90% primary cell converged.
8. construction method according to claim 1, it is characterised in that the cultural method of Secondary Culture is：Primary pectoral fin is thin
After born of the same parents' confluent monolayers, old nutrient solution is removed with suction pipe, with twice of PBS, 0.25% pancreatin 1mL vitellophags is added, treats mirror
After lower observation cell rounding, 10mL complete culture solutions are added, cell has been hanged in piping and druming, with 1:2 are passed on, and add complete culture solution
To 5mL/ bottles, it is put into 27 DEG C of incubators and carries out Secondary Culture；Passage in about 3~5 days later is once；When reaching for 10 generation, cell
Hyclone content is kept to the 10% of cumulative volume in nutrient solution, and Grass Carp Serum, basic fibroblast growth are no longer added in nutrient solution
The factor and EGF.
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Cited By (4)
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| CN108148803A (en) * | 2018-02-01 | 2018-06-12 | 中国水产科学研究院珠江水产研究所 | One plant of grass carp musculature cell line and application |
| CN113234660A (en) * | 2021-04-02 | 2021-08-10 | 肇庆大华农生物药品有限公司 | Grass carp ureter tissue cell line and application thereof |
| CN113249298A (en) * | 2021-04-02 | 2021-08-13 | 肇庆大华农生物药品有限公司 | Grass carp arterial ball tissue cell line and application thereof |
| CN113278580A (en) * | 2021-04-02 | 2021-08-20 | 肇庆大华农生物药品有限公司 | Grass carp skin tissue cell line and application thereof |
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| CN108148803A (en) * | 2018-02-01 | 2018-06-12 | 中国水产科学研究院珠江水产研究所 | One plant of grass carp musculature cell line and application |
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| CN113234660A (en) * | 2021-04-02 | 2021-08-10 | 肇庆大华农生物药品有限公司 | Grass carp ureter tissue cell line and application thereof |
| CN113249298A (en) * | 2021-04-02 | 2021-08-13 | 肇庆大华农生物药品有限公司 | Grass carp arterial ball tissue cell line and application thereof |
| CN113278580A (en) * | 2021-04-02 | 2021-08-20 | 肇庆大华农生物药品有限公司 | Grass carp skin tissue cell line and application thereof |
| CN113278580B (en) * | 2021-04-02 | 2024-03-29 | 肇庆大华农生物药品有限公司 | A kind of grass carp skin tissue cell line and its application |
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Inventor after: Wang Yingying Inventor after: Zeng Weiwei Inventor after: Wang Qing Inventor after: Li Yingying Inventor after: Shi Cunbin Inventor before: Wang Yingying |