CN105861444A - Anti-subgroup B avian leukosis virus strain cell line and application thereof - Google Patents
Anti-subgroup B avian leukosis virus strain cell line and application thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention provides an anti-subgroup B avian leukosis virus (ALV-B) strain cell line and application thereof. The ALV-B env gene is inserted into an expression vector, an established transfer vector is expressed in a chick embryo fibroblast cell line DF-1, and Zeocin drug screening is performed to obtain the Zeocin-resistant chick embryo fibroblast cell line DF-1/B with stably expressed ALV-B env gene; and the collection number of the cell line is CCTCC No.C201609. The detection and antivirus infection analysis on the DF-1/B cell line detect that the ALV-B env protein expressed in the DF-1 cell line has superinfection resistance to the ALV-B virus and can resist the virus infection replication quantity with the titer of 104TCID50. The DF-1/B cell line can be used for differential diagnosis on infection of different subgroup avian leukosis viruses, and has obvious clinical diagnosis value and wide application prospects.
Description
Technical field
The present invention relates to field of immunology, in particular it relates to a strain anti-B subgroup avian leucosis
The cell line of virus and application thereof.
Background technology
Avian leukosis virus (ALV) belongs to Retroviridae fowl α type retrovirus retrovirus group.
Avian leukosis virus is closely related with sarcoma virus, is often commonly referred to as avian leukosis/sarcoma virus.
According to cell infection scope, disturbance spectrum and envelope antigen feature, can be by avian leukosis/sarcosis
Poison group further discriminates between as 10 subgroups such as A to J, wherein A, B, C, D, E,
J subgroup sees chicken etc..A, B, J subgroup is common exogenous virus, and C, D are sub-
Group is the exogenous virus of few report, and wherein E subgroup is endogenous contaminating virus.Avian leukosis is sick
Poison envelope protein (the viral glycosylation albumen of env gene code) is the main table of this viroid
Face albumen, it determines the host range that virus infects, can induce generation neutralizing antibody.This egg
White with target cells specific receptor identification, combination, it is cell entry cell, duplication institute
Required.After infecting specific subgroup ALV, cell surface virus receptor can be produced by this virus
The viral glycosylation protein blocking of env gene code, show powerful superinfection resistance,
The subinfection again of corresponding virus can be limited.
At present avian leukosis is serious to China's aviculture harm, and occur in that myelocytome type and
The multiple pathological manifestations type such as angiomatous type, is also easy to and reticuloendothelium in clinical manifestation
The neoplastic disease such as hypertrophy disease, Marek are obscured mutually.This disease is mainly vertical transmission,
Controlling primary disease effective measures is to reduce the infection rate of breeder flock and set up without the white blood of exogenous fowl
The breeder flock that virus infects, its Main Means is exogenous by periodically carrying out chicken group
ALV detects, and eliminates band poison positive chicken in time, with the ALV purified and eliminate in chicken group.?
In exogenous ALV, chicken is had bright with J subgroup, A subgroup and B subgroup avian leucosis virus
Aobvious is pathogenic, and the harm to avian production is the biggest.
Both at home and abroad detection and authentication method for avian leukosis virus mainly have cell to cultivate,
ELISA, indirect immunofluorescence assay, PCR etc., wherein ELISA applies the most clinically
Extensively, its advantage is that this method operates relative ease, has the detection kit of commercialization to utilize,
Its weak point is to cannot distinguish between endogenous and exogenous ALV；Indirect immunofluorescence assay ratio
Relatively directly perceived, but generally require and use specific antibody (monoclonal antibody).Different ALV can be distinguished
PCR primer and the work system thereof of subgroup have had been reported that.
In the separation of exogenous avian leukosis virus, the most conventional cell has Embryo Gallus domesticus fibroblast
Dimension cell (CEF) and to come from 0 be the fibroblast DF-1 of chicken.U.S. ADOL is real
Test room based on DF1 cell line, establish the DF-1/J cell of J substock lymphoid leuoosis-resistant virus
System.Although U.S.'s ADOL laboratory has some specific chicken strains, its produce primary carefully
Born of the same parents can limit specific exogenous avian leukosis virus respectively and grow, but these Strains of Chickens
The rarity performance limiting its usefulness.There is presently no and can specifically limit B subgroup fowl
The relevant report of the cell line of leucovirus.
Summary of the invention
It is an object of the invention to provide a specific anti-B subgroup avian leucosis virus of strain
Cell line.
Another object of the present invention is to provide the cell of above-mentioned anti-B subgroup avian leucosis virus
The application of system.
The construction method of the anti-B subgroup avian leucosis virus cell line of the present invention, including as follows
Step:
(1) utilize the primer of band KpnI and NotI restriction enzyme site from ALV-B (CD08 strain)
Genomic PCR amplification env fragment, with restricted enzyme KpnI and NotI respectively to expansion
Increase env gene and eukaryon expression plasmid pcDNA3.1/Zeo (+) carry out double digestion, point
The most after purification B subgroup avian leucosis virus env genetic fragment is connected to pcDNA3.1/Zeo
(+) eukaryon expression plasmid, is built into recombiant plasmid pcDNA-env-B；
(2) transfer vector pcDNA-env-B expresses in DF-1 chick embryo fibroblast system
On the basis of, repeatedly screened by Zeocin medicine, obtain there is Zeocin resistance, can stablize
Expressing the chick embryo fibroblast system DF-1/B of ALV-B env albumen, the most anti-B subgroup fowl is white
The cell line of disorders of blood virus.
The anti-B subgroup avian leucosis virus chick embryo fibroblast name that present invention screening obtains
For DF-1/B, on January 21st, 2016 China typical culture collection center (address:
Wuhan, China Wuhan University, China typical culture collection center, is called for short CCTCC, postcode
430072) preservation, deposit number CCTCC No.C201609.
The invention provides the anti-B white blood of subgroup fowl that deposit number is CCTCC No.C201609
The application in preparing B subgroup avian leucosis virus diagnostic reagent of the cell line of virus.
The invention provides the anti-B white blood of subgroup fowl that deposit number is CCTCC No.C201609
The application in preparing B subgroup avian leucosis virus antibody test reagent of the cell line of virus.
Deposit number of the present invention is that the cell line of CCTCC No.C201609 can be used for preparing
ALV-B monoclonal antibody.
Further, the present invention provides a kind of ALV-B monoclonal antibody, and it by deposit number is
The cell line of CCTCC No.C201609 prepares.The invention provides deposit number is
The cell line of the anti-B subgroup avian leucosis virus of CCTCC No.C201609 is in preparation B subgroup
Application in avian leukosis virus biological product.
Described biological product are vaccine.
The invention provides the anti-B white blood of subgroup fowl that deposit number is CCTCC No.C201609
The application in preparing anti-B subgroup avian leucosis transgenic chicken of the cell line of virus.
Present invention also offers a kind of reagent for detecting B subgroup avian leucosis virus, contain
The cell of the anti-B subgroup avian leucosis virus having deposit number to be CCTCC No.C201609
System.
It is white that the cell line of the anti-B subgroup avian leucosis virus of the present invention can be applicable to B subgroup fowl
The diagnosis of disorders of blood virus.
The invention provides the spy of a kind of cell line for detecting anti-B subgroup avian leucosis virus
Specific primer pair, its upstream and downstream primer sequence is:
Forward primer: CGGGGTACCGCCACCATGGAAGCCGTCATAAAGA
TGAGGCGAGCCCTCTTTTTGC；
Downstream primer: AAGGAAAAAAGCGGCCGCCTATACTGCTCTTTCGGG
CTGCTTA。
Target fragment size about 1845bp.
Test kit containing above-mentioned specific primer pair belongs to protection scope of the present invention.
The present invention is from env albumen superinfection resistance, by B subgroup avian leucosis virus env
Gene insert pcDNA3.1/Zeo (+) construction of eukaryotic expression vector becomes transfer vector
PcDNA-env-B, then transfects chick embryo fibroblast system by carrier pcDNA-env-B
DF-1, is screened by medicine Zeocin, it is thus achieved that anti-Zeocin, stably expression ALV-B env
The cell line of the anti-B subgroup avian leucosis virus of albumen.The obtained anti-B white blood of subgroup fowl
The cell line of virus can stably pass on, and preserves for a long time；Can resist titre is 104TCID50
Viral infection duplication amount, high specificity, the DF-1/B cell line of the present invention can be used for B
The diagnosis of subgroup avian leucosis virus.
Accompanying drawing explanation
Fig. 1 is the technology path of the cell line structure of anti-B subgroup avian leucosis virus
Fig. 2 is normal DF-1 cell state: threadiness, fusiformis, adherent growth.
Fig. 3 is env genetic results in PCR detection DF-1/B, with drawing of present invention design
Thing successfully be detected the env gene of 1845bp in DF-1/B, and does not has in DF-1.
Fig. 4 is that indirect immunofluorescence (IFA) detects env expression conditions result, wherein
Fig. 4 A is DF-1 cell (negative control) redgreen fluorescence, and Fig. 4 B is that DF-1/B cell has
Green fluorescence, illustrates external source env genetic fragment successful expression in DF-1/B.
Fig. 5 is the expression of results of Western-blot detection env gene, has env in DF-1/B
Protein expression, and DF-1 does not has, external source env fragment success table in DF-1/B is described
Reach.Actin is internal reference albumen, albumen for the purpose of env albumen.
Fig. 6 is anti-B subgroup avian leucosis virus (ALV-B) experimental result, and CD08 can be
The upper normal breeding of DF-1, but on DF-1/B, CD08 can not normally breed, only in disease
Poison titre is 105TCID50Time occur replicating, but virus replication is substantially suppressed, the lowest
In the duplication on DF-1.Result illustrates, DF-1/B has the strongest anti-ALV-B and infects
The ability of cell.
Fig. 7 is J substock lymphoid leuoosis-resistant virus (ALV-J CHN06) experimental result, ALV-J
Can normally breed on DF-1/B and DF-1, breeding situation does not has marked difference, explanation
DF-1/B does not has resistance to ALV-J.
Fig. 8 hives off diagnosis example result for clinical virus, and the S/P value of DF-1/B is substantially less than
DF-1, illustrates that DF-1/B has resistance to this ALV.
Detailed description of the invention
Following example further illustrate present disclosure, but should not be construed as the present invention
Restriction.Without departing from the spirit and substance of the case in the present invention, to the inventive method, step
Amendment that rapid or condition is made or replacement, belong to the scope of the present invention.
The DF-1 cell line of the present invention is 0 to be chick embryo fibroblast, and CD08 is B subgroup
Avian leukosis virus, is all from relying on Ministry of Agriculture's live vaccine initiative weight of Agricultural University Of South China
Point laboratory.Restricted enzyme Kpn I and Not I used by the present invention are public purchased from NEB
Department.PMD18-T carrier purchased from TaKaRa company, eukaryon expression plasmid pcDNA3.1/Zeo (+)
Purchased from Invitrogen company.ALV-A/B monoclonal antibody specific, U.S. ADOL is real
Test room present.The goat anti-mouse IgG antibodies of FITC labelling and the mountain sheep anti mouse of HRP labelling
IgG antibody is purchased from Simga company.SQ Tissue tissue DNA extracts test kit, glue
Reclaim test kit DNA Gel Extraction Kit, remove endotoxin plasmid extraction test kit
Plasmid Miniprep Kit is OMEGA Products.ALV antigen detection kit
(Avian Leukosis Virus Antigen Test Kit) is American I DEXX Products.High sugar
DMEM medium powder, 0.25% pancreatin (containing EDTA), hyclone are U.S. GIBCO
Products；Dimethyl sulfoxide (DMSO) is Sigma Products.
POLOdeliverer3000 transfection reagent box is purchased from Shanghai Rui Sai Bioisystech Co., Ltd.
The foundation of embodiment 1 anti-B group avian leukosis virus (ALV-B) cell line DF-1/B
The present invention expands the primer of ALV-B env gene by inventor's designed, designed:
Forward primer: CGGGGTACCGCCACCATGGAAGCCGTCATAAAGA
TGAGGCGAGCCCTCTTTTTGC
Downstream primer: AAGGAAAAAAGCGGCCGCCTATACTGCTCTTTCGGG
CTGCTTA
Reaction system (50 μ L): each 1 μ L, the ddH of LA Taq 25 μ L, upstream and downstream primer2O
21 μ L, template 2 μ L.
Response procedures: 94 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 55 DEG C of annealing 1min,
72 DEG C extend 2min, 30 circulations；8min is extended after 72 DEG C.
From ALV-B (CD08 strain) genome, env is amplified with the above-mentioned primer of designed, designed
Fragment, reclaims test kit DNA Gel Extraction Kit description according to glue and reclaims；Press
According to NEB company restricted enzyme KpnI, NotI description respectively to eukaryon expression plasmid
PcDNA3.1/Zeo (+) and pMD18-T-env fragment carry out double digestion；It is separately recovered enzyme action to produce
Thing, and be attached reclaiming product according to NEB company's T 4 ligase description, build weight
Group plasmid pcDNA-env-B；Transformed competence colibacillus cell DH5 α, goes according to OMEGA company
Endotoxin plasmid extraction test kit description, carries out recombiant plasmid pcDNA-env-B extracting.Matter
Grain send English Weihe River victory base (Invitrogen) company in Guangzhou to check order.
Check order correct plasmid according to Shanghai Rui Sai Bioisystech Co., Ltd
POLOdeliverer3000 transfection reagent box description transfects.
The DF-1 cell of digestion early-stage preparations, spreads six porocyte culture plates, 10%FBS, 5%CO2、
37 DEG C of cultivations.Transfect in 24h, Corning company six porocyte culture plate every hole transfection body
System is: 2 μ g pcDNA-env-B plasmids and 10 μ L liposomees are used respectively
Dilute and hatch 5min, then remixing, jointly hatching 15min, by 200 μ L plasmids-lipid
Nanocrystal composition joins in ready six porocyte culture dishs.After transfection, 5h changes cell culture fluid
(DMEM+10%FBS), after 24h, the cell dissociation in a hole of six orifice plates is got off, paving
24 orifice plates, every hole 500 μ L cell culture fluid (DMEM+15%FBS+200 μ g/mL zeocin),
Changed once the cell culture fluid of same system every 3-4 days, 10-15d forms single cell colonies,
About three weeks, single cell colonies was grown up, and digests to be placed in 6 orifice plates and cultivates, and adherent covered with
Proceed to 25cm afterwards2Cultivating in cell bottle, now cell culture is
(DMEM+10%FBS+200 μ g/mL zeocin).With medicine zeocin to cell step sizing
In 30 generations, from nucleic acid and protein level, intracellular env is detected, and it is real to carry out antiviral
Test and the application of Virus Type.
Cell state: threadiness, fusiformis, adherent growth.(as shown in Figure 2)
Env gene in PCR detection DF-1/B: the DNA of extracting DF-1/B, according to aforementioned expansion
Increase the primer of env gene, system and response procedures amplification env gene, result as it is shown on figure 3,
Env gene amplification fragment size is 1845bp.
Indirect immunofluorescence (IFA) detection env expression conditions: confluent monolayers DF-1/B
In 24 orifice plates of cell, after cultivating 5d, bottom cell PBS washes 3 times, uses 4% poly
Formaldehyde fixes 20min.Wash 3 times with PBS again, add the ALV-B monoclonal antibody of 1:100 dilution,
4 DEG C of overnight incubation.PBS washs 3 times, adds that the sheep anti-mouse igg-FITC that 1:200 dilutes is glimmering
Photoactivated antibody, after 37 DEG C of effects 1h, PBS wash 3 times, adds 1 drop volume mark 50% glycerol and covers
Cover cell, observation experiment result under fluorescence microscope.Set up a DMEM negative control.
Result as shown in Figure 4, can be observed endochylema and the surface of DF-1/B cell under fluorescence microscope
Specific fluorescence, DF-1 cell is had not to have green fluorescence.Illustrate that external source env fragment is at DF-1/B
Middle successful expression.
The expression of Western-blot detection env gene: DF-1/B and comparison DF-1 cultivates 5d
After carry total protein of cell, carry out electrophoresis with Bio-Rad vertical electrophoresis apparatus, concentrating glue voltage is 80V,
Separation gel electrophoretic voltage is 100V, and electrophoretic buffer is 1 × Tris-glycine running buffer, treats
Bromophenol blue indicator stops electrophoresis after reaching bottom separation gel.After cutting glue transferring film, NC film is used
1 × TBS washs 3 times, each 5min.NC film is placed in the TBS of the defatted milk powder containing 5%
In, room temperature is closed 2h, 1 × TBST and is washed 3 times, each 5min.Add albumen and Actin is mono-
Anti-, hatch 12h for 4 DEG C.Antibody diluted concentration is env (1:500), Actin (1:1000).One
Resist after hatching, film TBST is washed 3 times, each 5min, NC film is placed in 1:10000
During the mountain sheep anti-mouse igg two of HRP labelling resists, hatching 1h for 37 DEG C, 1 × TBST washs 3 times,
5min every time.In darkroom, on NC film, add ECL immunochemiluminescence liquid hatch 5min,
Wrapping NC film with preservative film to be placed on photographic film, development, fixing, after drying, picture is swept
Retouch, preserve.Result is as it is shown in figure 5, there is env protein expression in DF-1/B, and in DF-1
No.External source env fragment successful expression in DF-1/B is described.
The anti-B subgroup avian leucosis virus chick embryo fibroblast name that present invention screening obtains
For DF-1/B, on January 21st, 2016 China typical culture collection center (address:
Wuhan, China Wuhan University, China typical culture collection center, is called for short CCTCC, postcode
430072) preservation, deposit number CCTCC No.C201609.
The biology of embodiment 2 anti-B group avian leukosis virus (ALV-B) cell line DF-1/B lives
Property experiment
Antiviral breeding: respectively with DMEM by ALV-B strain CD08 from 100To 105
Carry out doubling dilution.Digestion DF-1/B cell and DF-1 cell, be respectively inoculated in one piece 24 respectively
In porocyte culture plate, every hole inoculation 1mL cell suspension, cell concentration is
1.7×105cells/mL。
The venom of each for CD08 viral dilution gradient is inoculated in two plate cell suspension respectively, often
100 μ L venom are inoculated in hole, and each viral dilution gradient does three repetitions, and does a known sun
The comparison of property strain and DMEM negative control, 5%CO2, 10%FBS, in 37 DEG C of incubators
Cultivate.After venom to be seeded second day, after cell covers with monolayer, it is changed to the cell dimension of 1%FBS
Hold liquid to cultivate.Cultivating 6d continuously, period continues to monitor the growing state of cell, and
Within 6 days, collect centrifugal collecting cell supernatant after cell culture, freeze thawing three times.On the cell collected
The clear liquid ELISA kit of same batch carries out p27 antigen ELISA detection.Result is such as
Shown in Fig. 6, CD08 normally can breed on DF-1, but on DF-1/B, CD08 can not
Normal breeding, is only 10 at virus titer5TCID50Time occur replicating, but virus replication is obvious
It is suppressed, is substantially less than the duplication on DF-1.Result illustrates, DF-1/B has the strongest
The ability of anti-ALV-B infected cell.
By same method by thin to ALV-J strain CHN06 inoculation DF-1/B cell and DF-1
Born of the same parents.Result such as Fig. 7 shows, ALV-J can normally breed on DF-1/B and DF-1, numerous
The situation of growing does not has marked difference, illustrates that DF-1/B does not has resistance to ALV-J.
Clinical virus is hived off Differential Diagnosis example: as shown in Figure 8, by the ALV that is clinically separated with
Time inoculation DF-1 and DF-1/B, after cultivating 7 days, centrifugal collecting cell supernatant after freeze thawing three times,
ELISA detects, and the S/P value of DF-1/B is significantly lower than DF-1, illustrates that DF-1/B is to this ALV
There is resistance, tentatively judge that this ALV is ALV-B.The DF-1 cell of this ALV is inoculated in extracting
DNA, expands gp85, send order-checking, it was demonstrated that this strain is really ALV-B after recovery.
Although, the most with a general description of the specific embodiments the present invention has been made in detail
Most description, but on the basis of the present invention, it can be made some modifications or improvements, this is to this
It is apparent from for skilled person.Therefore, on the basis without departing from spirit of the present invention
Upper these modifications or improvements, belong to the scope of protection of present invention.
Claims (10)
1. the cell line of a strain anti-B subgroup avian leucosis virus, it is chick embryo fibroblast,
Entitled DF-1/B, its deposit number is CCTCC No.C201609.
2. the cell line of the anti-B subgroup avian leucosis virus described in claim 1 is sub-at preparation B
Application in group's avian leukosis virus diagnostic reagent.
3. the cell line of the anti-B subgroup avian leucosis virus described in claim 1 is sub-at preparation B
Application in group's avian leukosis virus antibody test reagent.
4. the cell line of the anti-B subgroup avian leucosis virus described in claim 1 is sub-at preparation B
Application in group's avian leukosis virus monoclonal antibody.
5. a B subgroup avian leucosis virus monoclonal antibody, it is characterised in that compiled by preservation
Number it is prepared into for the cell line of anti-K subgroup avian leucosis virus of CCTCC No.C201609
Arrive.
6. the cell line of the anti-B subgroup avian leucosis virus described in claim 1 is sub-at preparation B
Application in group's avian leukosis virus biological product.
7. as claim 6 application, it is characterised in that described biological product are vaccine.
8. the cell line of the anti-B subgroup avian leucosis virus described in claim 1 is preparing anti-B
Application in subgroup avian leucosis transgenic chicken.
9. the reagent being used for detecting B subgroup avian leucosis virus, it is characterised in that contain
Have the right the cell line of anti-B subgroup avian leucosis virus described in requirement 1.
10. the specific primer being used for detecting the cell line of anti-B subgroup avian leucosis virus
Right, it is characterised in that its upstream and downstream primer sequence is:
Forward primer: CGGGGTACCGCCACCATGGAAGCCGTCATAAAGA
TGAGGCGAGCCCTCTTTTTGC；
Downstream primer: AAGGAAAAAAGCGGCCGCCTATACTGCTCTTTCGGG
CTGCTTA。
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