CN105861444B - The cell line and its application of one plant of anti-B subgroup avian leucosis virus - Google Patents
The cell line and its application of one plant of anti-B subgroup avian leucosis virus Download PDFInfo
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Abstract
The present invention provides the cell line and its application of one plant of anti-B subgroup avian leucosis virus (ALV-B).ALV-B env gene is inserted into expression vector, the transfer vector of building is expressed in chicken embryo fibroblasts system DF-1, the chicken embryo fibroblasts system DF-1/B for having Zeocin resistance, stablizing expression ALV-B env gene is obtained by Zeocin drug screening, which is CCTCC No.C201609.Detection and viral infection resisting analysis are carried out to DF-1/B cell line, it is found that the ALV-B env albumen expressed in DF-1 cell line has superinfection resistance to ALV-B virus, can resist titre is 104TCID50Virus infection duplication amount, DF-1/B cell line of the invention can be used for the antidiastole of different subgroup avian leucosis virus infection, and diagnostic value is significant, has a extensive future.
Description
Technical field
The present invention relates to field of immunology, and in particular, to the cell line of one plant of anti-B subgroup avian leucosis virus and its answers
With.
Background technique
Avian leukosis virus (ALV) belongs to Retroviridae fowl α type retrovirus group.Avian leukosis virus and sarcoma
Virus is closely related, and is often commonly referred to as avian leukosis/sarcoma virus.According to cell infection range, disturbance spectrum and envelope antigen feature,
Avian leukosis/sarcoma virus group can be further discriminated between as 10 subgroups such as A to J, wherein A, B, C, D, E, J subgroup see chicken
Deng.A, B, J subgroup are common exogenous virus, and C, D subgroup are the exogenous virus seldom reported, wherein E subgroup is endogenous
Venereal disease poison.Avian leukosis virus envelope protein (the virus glycosylation albumen of env gene coding) is the major surfaces of the viroid
Albumen, it determines the host range of virus infection, can induce generation neutralizing antibody.The albumen and target cell surface specific receptor
Identification combines, and is cell entry cell, necessary to duplication.After infecting specific subgroup ALV, cell surface virus receptor energy quilt
The virus glycosylation protein blocking for the env gene coding that the virus generates, shows powerful superinfection resistance, can limit corresponding
The subinfection again of virus.
Avian leukosis endangers China's aviculture serious at present, and a variety of diseases such as myelocytome type and angiomatous type occur
Phenotype is managed, is also easy to mutually obscure with tumor diseases such as reticuloendotheliosis disease, Marek's diseases in clinical manifestation.
The disease is mainly vertical transmission, and controlling this disease effective measures is to reduce the infection rate of breeder flock and establish without the white blood of exogenous fowl
The breeder flock of sick virus infection, main means are to eliminate band poison sun in time by periodically carrying out exogenous ALV detection to chicken group
Property chicken, with purify and eliminate chicken group in ALV.In exogenous ALV, with J subgroup, A subgroup and B subgroup avian leucosis virus pair
Chicken have it is apparent pathogenic, it is also big to the harm of avian production.
Both at home and abroad for the detection and identification method of avian leukosis virus mainly have cell culture, ELISA, indirectly be immunized it is glimmering
Phototesting, PCR etc., wherein ELISA is clinically most widely used, its advantage is that the method operates relative ease, there is the inspection of commercialization
Test agent box can use, and shortcoming is to cannot distinguish between endogenous and exogenous ALV；Indirect immunofluorescence assay is more straight
It sees, but generally requires to use specific antibody (monoclonal antibody).The PCR primer and its work system of different ALV subgroups can be distinguished
It has had been reported that.
In the separation of exogenous avian leukosis virus, currently used cell has chicken embryo fibroblasts (CEF) and source
It is the fibroblast DF-1 of chicken in 0.The U.S. laboratory ADOL is based on DF1 cell line, establishes J substock lymphoid leuoosis-resistant disease
The DF-1/J cell line of poison.Although there are some specific chicken strains in the U.S. laboratory ADOL, the primary cell generated can divide
Specific exogenous avian leukosis virus growth is not limited, but the rarity of these Strains of Chickens limits the performance of its efficiency.Mesh
The relevant report of the preceding cell line that can specifically limit B subgroup avian leucosis virus not yet.
Summary of the invention
The purpose of the present invention is to provide the cell lines of one plant of specific anti-B subgroup avian leucosis virus.
Another object of the present invention is to provide the applications of the cell line of above-mentioned anti-B subgroup avian leucosis virus.
The construction method of anti-B subgroup avian leucosis virus cell line of the invention, includes the following steps:
(1) utilize the primer with KpnI and NotI restriction enzyme site from ALV-B (CD08 strain) genomic PCR amplification env piece
Section, with restriction enzyme KpnI and NotI respectively to the env gene of amplification and eukaryon expression plasmid pcDNA3.1/Zeo (+) into
B subgroup avian leucosis virus env genetic fragment is connected to pcDNA3.1/Zeo (+) eukaryon table after purification respectively by row double digestion
Up to plasmid, it is built into recombinant plasmid pcDNA-env-B；
(2) transfer vector pcDNA-env-B passes through Zeocin medicine on the basis of DF-1 chicken embryo fibroblasts system expresses
Object repeatedly screens, and obtains the chicken embryo fibroblasts system DF-1/ for having Zeocin resistance, can stablizing expression ALV-B env albumen
B, i.e., the cell line of anti-B subgroup avian leucosis virus.
The anti-B subgroup avian leucosis virus chicken embryo fibroblasts that the present invention screens are named as DF-1/B, in 2016
On January 21, China typical culture collection center (address: Wuhan, China Wuhan University, in China typical culture collection
The heart, abbreviation CCTCC, postcode 430072) preservation, deposit number CCTCC No.C201609.
The present invention provides the cell lines for the anti-B subgroup avian leucosis virus that deposit number is CCTCC No.C201609 to exist
Prepare the application in B subgroup avian leucosis virus diagnostic reagent.
The present invention provides the cell lines for the anti-B subgroup avian leucosis virus that deposit number is CCTCC No.C201609 to exist
Prepare the application in B subgroup avian leucosis virus antibody test reagent.
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, is CCTCC No.C201609's by deposit number
Cell line is prepared.The present invention provides the anti-B subgroup avian leucosis virus that deposit number is CCTCC No.C201609
Cell line is preparing the application in B subgroup avian leucosis virus biological products.
The biological products are vaccine.
The present invention provides the cell lines for the anti-B subgroup avian leucosis virus that deposit number is CCTCC No.C201609 to exist
Prepare the application in anti-B subgroup avian leucosis transgenic chicken.
The present invention also provides a kind of for detecting the reagent of B subgroup avian leucosis virus, is CCTCC containing deposit number
The cell line of the anti-B subgroup avian leucosis virus of No.C201609.
The cell line of anti-B subgroup avian leucosis virus of the invention can be applied to the diagnosis of B subgroup avian leucosis virus.
The present invention provides a kind of for detecting the specific primer pair of the cell line of anti-B subgroup avian leucosis virus,
Upstream and downstream primer sequence are as follows:
Upstream primer: CGGGGTACCGCCACCATGGAAGCCGTCATAAAGATGAGGCGAGCCCTCTTTTTGC；
Downstream primer: AAGGAAAAAAGCGGCCGCCTATACTGCTCTTTCGGGCTGCTTA.
Target fragment size about 1845bp.
Kit containing above-mentioned specific primer pair belongs to the scope of protection of the present invention.
The present invention is inserted into pcDNA3.1/ from env albumen superinfection resistance, by B subgroup avian leucosis virus env gene
Zeo (+) construction of eukaryotic expression vector is at transfer vector pcDNA-env-B, then by carrier pcDNA-env-B transfection chicken embryo at fibre
Cell line DF-1 is tieed up, is screened by drug Zeocin, the anti-B subgroup fowl for obtaining anti-Zeocin, stablizing expression ALV-B env albumen
The cell line of leukemia virus.The cell line of obtained anti-B subgroup avian leucosis virus can stablize passage, long-term preservation；
It is 10 that titre, which can be resisted,4TCID50Virus infection duplication amount, high specificity, DF-1/B cell line of the invention can be used for B Asia
The diagnosis of group avian leukosis virus.
Detailed description of the invention
Fig. 1 is the technology path that the cell line of anti-B subgroup avian leucosis virus constructs
Fig. 2 is normal DF-1 cell state: threadiness, shuttle shape, adherent growth.
Fig. 3 is that PCR detects env genetic results in DF-1/B, is successfully detected in DF-1/B with the primer that the present invention designs
To the env gene of 1845bp, and do not have in DF-1.
Fig. 4 is indirect immunofluorescence (IFA) detection env expression conditions as a result, wherein Fig. 4 A is that DF-1 cell is (negative
Control) redgreen fluorescence, Fig. 4 B is that DF-1/B cell has green fluorescence, illustrates the success in DF-1/B of external source env genetic fragment
Expression.
Fig. 5 is the expression of results that Western-blot detects env gene, has env protein expression in DF-1/B, and in DF-1
No, illustrate external source env segment successful expression in DF-1/B.Actin is internal reference albumen, and env albumen is purpose albumen.
Fig. 6 is anti-B subgroup avian leucosis virus (ALV-B) experimental result, and CD08 can normally be bred on DF-1, but
On DF-1/B, CD08 cannot normally be bred, and only be 10 in virus titer5TCID50When replicate, but virus replication obviously by
Duplication to inhibition, substantially less than on DF-1.As a result illustrate, DF-1/B has the ability of very strong anti-ALV-B infected cell.
Fig. 7 is viral (ALV-J CHN06) experimental result of J substock lymphoid leuoosis-resistant, ALV-J on DF-1/B and DF-1 all
It can normally breed, breeding situation does not have marked difference, illustrates that DF-1/B does not have resistance to ALV-J.
Fig. 8 is that clinical virus divides group's diagnosis example as a result, the S/P value of DF-1/B is substantially less than DF-1, illustrates DF-1/B pairs
The ALV is resistant.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
It is chicken embryo fibroblasts that DF-1 cell line of the invention, which is 0, and CD08 is B subgroup avian leucosis virus, is all from
Ministry of Agriculture's live vaccine of Agricultural University Of South China is relied on to formulate key lab.Restriction enzyme Kpn I used in the present invention
NEB company is purchased from Not I.PMD18-T carrier is purchased from purchased from TaKaRa company, eukaryon expression plasmid pcDNA3.1/Zeo (+)
Invitrogen company.ALV-A/B monoclonal antibody specific, the present of the U.S. laboratory ADOL.The mountain sheep anti mouse of FITC label
The goat anti-mouse IgG antibodies of IgG antibody and HRP label are purchased from Simga company.SQ Tissue tissue DNA extracts kit,
Plastic recovery kit DNA Gel Extraction Kit removes endotoxin plasmid extraction kit Plasmid Miniprep Kit
For OMEGA Products.ALV antigen detection kit (Avian Leukosis Virus Antigen Test Kit) is beauty
State's IDEXX Products.DMEM in high glucose medium powder, 0.25% pancreatin (containing EDTA), fetal calf serum are the production of U.S. GIBCO company
Product；Dimethyl sulfoxide (DMSO) is Sigma Products.POLOdeliverer3000 transfection reagent box is purchased from the sharp match life in Shanghai
Object Technology Co., Ltd..
The foundation of anti-B crowds of avian leukosis virus (ALV-B) the cell line DF-1/B of embodiment 1
The present invention expands the primer of ALV-B env gene by inventor's designed, designed:
Upstream primer: CGGGGTACCGCCACCATGGAAGCCGTCATAAAGATGAGGCGAGCCCTCTTTTTGC
Downstream primer: AAGGAAAAAAGCGGCCGCCTATACTGCTCTTTCGGGCTGCTTA
Reaction system (50 μ L): each 1 μ L of 25 μ L of LA Taq, upstream and downstream primer, ddH2O21 μ L, 2 μ L of template.
Response procedures: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 55 DEG C of annealing 1min, 72 DEG C of extension 2min, 30 are followed
Ring；Extend 8min after 72 DEG C.
Env segment is amplified from ALV-B (CD08 strain) genome with the above-mentioned primer of designed, designed, is recycled according to glue
Kit DNA Gel Extraction Kit specification is recycled；It is said according to NEB company restriction enzyme KpnI, NotI
Bright book carries out double digestion to eukaryon expression plasmid pcDNA3.1/Zeo (+) and pMD18-T-env segment respectively；Digestion is separately recovered
Product, and recovery product is attached according to 4 ligase specification of NEB company's T, construction recombination plasmid pcDNA-env-B；Turn
Change competent cell DH5 α, goes endotoxin plasmid extraction kit specification according to OMEGA company, carry out recombinant plasmid pcDNA-
Env-B extracting.Plasmid send Invitrogen (Invitrogen) company in Guangzhou to be sequenced.
Correct plasmid is sequenced according to Shanghai Rui Sai Bioisystech Co., Ltd POLOdeliverer3000 transfection reagent box
Specification is transfected.
The DF-1 cell of early-stage preparations is digested, six porocyte culture plates, 10%FBS, 5%CO are spread2, 37 DEG C of cultures.In for 24 hours
It is transfected, the every hole rotaring redyeing system of six porocyte culture plates of Corning company are as follows: 2 μ g pcDNA-env-B plasmids and 10 μ L rouge
Plastid is used respectively5min is diluted and be incubated for, is then remixed, is incubated for 15min jointly, by 200 μ L
Plasmid-liposome complex is added in the Tissue Culture Dish of ready six hole.5h changes cell culture fluid (DMEM+ after transfection
10%FBS), the cell dissociation in a hole of six orifice plates is got off afterwards for 24 hours, spreads 24 orifice plates, every 500 μ L cell culture fluid of hole
(DMEM+15%FBS+200 μ g/mL zeocin), the cell culture fluid of primary same system was changed every 3-4 days, and 10-15d is formed
Single cell colonies, three weeks or so, single cell colonies were grown up, and were digested and were placed in culture in 6 orifice plates, and adherent cover with is transferred to later
25cm2It is cultivated in cell bottle, cell culture is (DMEM+10%FBS+200 μ g/mL zeocin) at this time.With drug zeocin
To 30 generation of cell step sizing, intracellular env is detected from nucleic acid and protein level, and carry out Antiviral breeding and
The application of Virus Type.
Cell state: threadiness, shuttle shape, adherent growth.(as shown in Figure 2)
Env gene in PCR detection DF-1/B: extracting the DNA of DF-1/B, according to the primer of aforementioned amplification env gene, system
And response procedures expand env gene, as a result as shown in figure 3, env gene amplification fragment size is 1845bp.
Indirect immunofluorescence (IFA) detects env expression conditions: in 24 orifice plates of confluent monolayers DF-1/B cell, training
After supporting 5d, bottom cell is washed 3 times with PBS, fixes 20min with 4% paraformaldehyde.It is washed 3 times with PBS again, 1:100 dilution is added
ALV-B monoclonal antibody, 4 DEG C overnight incubation.PBS is washed 3 times, in addition the diluted sheep anti-mouse igg-FITC fluorescence antibody of 1:200,37 DEG C
1h is acted on, after PBS is washed 3 times, 1 drop volume score, 50% glycerol is added to cover cell, the observation experiment result under fluorescence microscope.
Set up a DMEM negative control.As shown in Figure 4, as a result the endochylema and table of DF-1/B cell can be observed under fluorescence microscope
There is specific fluorescence in face, and DF-1 cell does not have green fluorescence.Illustrate external source env segment successful expression in DF-1/B.
The expression of Western-blot detection env gene: total protein of cell is mentioned after DF-1/B and control DF-1 culture 5d, is used
Bio-Rad vertical electrophoresis apparatus carries out electrophoresis, concentrate glue voltage 80V, and separation gel electrophoretic voltage is 100V, electrophoretic buffer 1
× Tris- glycine running buffer stops electrophoresis after bromophenol blue indicator reaches separation gel bottom.It, will after cutting glue transferring film
NC film is washed 3 times with 1 × TBS, each 5min.NC film is placed in the TBS containing 5% skimmed milk power, room temperature close 2h, 1 ×
TBST is washed 3 times, each 5min.Add albumen and Actin monoclonal antibody, 4 DEG C of incubation 12h.Antibody diluted concentration is env (1:500),
Actin(1:1000).After primary antibody is incubated for, film is washed 3 times, each 5min with TBST, NC film is placed in 1:10000HRP mark
In the mountain sheep anti-mouse igg secondary antibody of note, 37 DEG C of incubation 1h, 1 × TBST are washed 3 times, each 5min.In darkroom, add on NC film
Enter ECL immunochemiluminescence liquid and be incubated for 5min, wrap NC film with preservative film and be placed on photographic film, develops, fixing, after drying
Picture scanning saves.As a result as shown in figure 5, there is env protein expression in DF-1/B, and do not have in DF-1.Illustrate external source env piece
Section successful expression in DF-1/B.
The anti-B subgroup avian leucosis virus chicken embryo fibroblasts that the present invention screens are named as DF-1/B, in 2016
On January 21, China typical culture collection center (address: Wuhan, China Wuhan University, in China typical culture collection
The heart, abbreviation CCTCC, postcode 430072) preservation, deposit number CCTCC No.C201609.
The biological activity of anti-B crowds of avian leukosis virus (ALV-B) the cell line DF-1/B of embodiment 2 is tested
Antiviral breeding: respectively with DMEM by ALV-B strain CD08 from 100To 105Carry out doubling dilution.DF- is digested respectively
1/B cell and DF-1 cell are respectively inoculated in one piece of 24 porocyte culture plates, and every hole is inoculated with 1mL cell suspension, and cell concentration is
1.7×105cells/mL。
The venom of each viral dilution gradient of CD08 is inoculated in respectively in two plate cell suspensions, every hole is inoculated with 100 μ L venom,
Each viral dilution gradient does three repetitions, and is the control of positive strain and DMEM negative control, 5%CO known to one2、
It is cultivated in 10%FBS, 37 DEG C of incubators.Second day after venom to be seeded, after cell covers with single layer, it is changed to the cell dimension of 1%FBS
Liquid is held to be cultivated.During which continuous culture 6d continues to monitor the growing state of cell, and in the 6th day collection cell culture, freeze
Melt and cell conditioned medium is collected by centrifugation afterwards three times.The cell supernatant of collection carries out p27 antigen with the ELISA kit of the same batch
ELISA detection.As a result as shown in fig. 6, CD08 can normally be bred on DF-1, but on DF-1/B, CD08 cannot normally be bred,
It is only 10 in virus titer5TCID50When replicate, but virus replication is obviously suppressed, substantially less than on DF-1
Duplication.As a result illustrate, DF-1/B has the ability of very strong anti-ALV-B infected cell.
ALV-J strain CHN06 is inoculated with DF-1/B cell and DF-1 cell with same method.As a result such as Fig. 7 is shown,
ALV-J can normally be bred on DF-1/B and DF-1, and breeding situation does not have marked difference, illustrate that DF-1/B does not have ALV-J
Resistance.
Clinical virus divides group's antidiastole example: as shown in figure 8, by the ALV being clinically separated while being inoculated with DF-1 and DF-1/
B, after culture 7 days, cell conditioned medium is collected by centrifugation in freeze thawing afterwards three times, and the S/P value of ELISA detection, DF-1/B is significantly lower than DF-1, says
Bright DF-1/B is resistant to the ALV, tentatively judges the ALV for ALV-B.Extracting is inoculated with the DF-1 cell DNA of the ALV, amplification
Gp85 send sequencing after recycling, it was demonstrated that the strain is really ALV-B.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (1)
1. the cell line of one plant of anti-B subgroup avian leucosis virus, is chicken embryo fibroblasts, entitled DF-1/B, preservation is compiled
Number be CCTCC No.C201609.
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