CN105861445B - The cell line and its application of one plant of anti-K subgroup avian leucosis virus - Google Patents
The cell line and its application of one plant of anti-K 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-K subgroup avian leucosis virus (ALV-K).ALV-K 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/K for having Zeocin resistance, stablizing expression ALV-K env gene is obtained by Zeocin drug screening, which is CCTCC No.C201610.Detection and viral infection resisting analysis are carried out to DF-1/K cell line, it is found that the ALV-K env albumen expressed in DF-1 cell line has superinfection resistance to ALV-K virus, can resist titre is 104TCID50Virus infection duplication amount, DF-1/K 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-K 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 meat
Tumor virus is closely related, and is often commonly referred to as avian leukosis/sarcoma virus.It is special according to cell infection range, disturbance spectrum and envelope antigen
Sign, past often further discriminate between avian leukosis/sarcoma virus group for 10 subgroups such as A to J, wherein A, B, C, D, E, J subgroup
It is mainly seen in chicken.A, B, J subgroup are common exogenous virus, and C, D subgroup are the exogenous virus seldom reported.Exist in recent years
New discovery K subgroup avian leucosis virus in some Chinese native chicken strains.
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 is serious to the harm of China's aviculture at present, and it is a variety of myelocytome type and angiomatous type occur etc.
Pathological manifestations type is also easy to mutually mix with tumor diseases such as reticuloendotheliosis disease, Marek's diseases in clinical manifestation
Confuse.The disease is mainly vertical transmission, and controlling this disease effective measures is to reduce the infection rate of breeder flock and establish without leukaemia
Breeder flock, main means are to eliminate the positive chicken of band poison, in time by periodically carrying out exogenous ALV detection to chicken group with purification
With the ALV in elimination chicken group.In exogenous ALV, have significantly with J subgroup, A subgroup and B subgroup avian leucosis virus to chicken
It is pathogenic, it is also big to the harm of avian production, and newfound K subgroup also belongs to exogenous avian leukosis virus, it may be always
It is present in China regional strain chicken group, and is all separated to the subgroup in many places in China.
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 been had been reported that, but since K subgroup avian leucosis virus is new discovery subgroup, antidiastole technology is still not perfect, without spy
Specific monoclonal antibodies etc., at present mainly by round pcr, after send the method for sequencing to distinguish subgroup.
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 Establishment of Cell Line 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 not limit K subgroup avian leucosis virus specifically both at home and abroad.
Summary of the invention
The purpose of the present invention is to provide the cell lines of one plant of specific anti-K subgroup avian leucosis virus.
Another object of the present invention is to provide the applications of the cell line of above-mentioned anti-K subgroup avian leucosis virus.
The construction method of anti-K subgroup avian leucosis virus cell line of the invention, includes the following steps:
(1) utilize the primer with BamHI and NotI restriction enzyme site from ALV-K (GDFX0601 strain) genomic PCR amplification
Env segment, with restriction enzyme BamHI and NotI respectively to the env gene of amplification and eukaryon expression plasmid pcDNA3.1/
Zeo (+) carries out double digestion, and K subgroup avian leucosis virus env genetic fragment is connected to pcDNA3.1/Zeo after purification respectively
(+) eukaryon expression plasmid is built into recombinant plasmid pcDNA-env-K；
(2) transfer vector pcDNA-env-K 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-K env albumen
K, i.e., the cell line of anti-K subgroup avian leucosis virus.
The anti-K subgroup avian leucosis virus chicken embryo fibroblasts that the present invention screens, are named as DF-1/K, 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.C201610.
Deposit number of the present invention is that the cell line of CCTCC No.C201610 can be used for preparing ALV-K monoclonal antibody.
Further, the present invention provides a kind of ALV-K monoclonal antibody, is CCTCC No.C201610's by deposit number
Cell line is prepared.
The present invention provides the cell lines for the anti-K subgroup avian leucosis virus that deposit number is CCTCC No.C201610 to exist
Prepare the application in K subgroup avian leucosis virus diagnostic reagent.
The present invention provides the cell lines for the anti-K subgroup avian leucosis virus that deposit number is CCTCC No.C201610 to exist
Prepare the application in K subgroup avian leucosis virus antibody test reagent.
The present invention provides the cell lines for the anti-K subgroup avian leucosis virus that deposit number is CCTCC No.C201610 to exist
Prepare the application in K subgroup avian leucosis virus biological products.
The biological products are vaccine.
The present invention provides the cell lines for the anti-K subgroup avian leucosis virus that deposit number is CCTCC No.C201610 to exist
Prepare the application in anti-K subgroup avian leucosis transgenic chicken.
The present invention also provides a kind of for detecting the reagent of K subgroup avian leucosis virus, is CCTCC containing deposit number
The cell line of the anti-K subgroup avian leucosis virus of No.C201610.
The cell line of anti-K subgroup avian leucosis virus of the invention can be applied to the diagnosis of K subgroup avian leucosis virus.
The present invention provides a kind of for detecting the kit of K subgroup avian leucosis virus, contains K subgroup avian leucosis disease
Malicious monoclonal antibody, the monoclonal antibody is by the anti-K subgroup avian leucosis virus that deposit number is CCTCC No.C201610
Cell line is prepared.
The present invention provides a kind of for detecting the kit of K subgroup avian leucosis virus antibody, contains the white blood of K subgroup fowl
Sick viral monoclonal antibodies, the monoclonal antibody is by the anti-K subgroup avian leucosis disease that deposit number is CCTCC No.C201610
The cell line of poison is prepared.
The present invention provides the specific primer pair of the cell line for detecting anti-K subgroup avian leucosis virus, above and below
Trip primer sequence is respectively as follows:
Upstream primer: CGCGGATCCGCCACCATGGAAGCCGTCATAAAGGCATTTCTGACTGGATAC
Downstream primer: AAGGAAAAAAGCGGCCGCTTACACTGCTCCATTTTCG.
Target fragment size about 1791bp.
Kit containing above-mentioned specific primer pair belongs to the scope of protection of the present invention.
K subgroup avian leucosis virus env gene is inserted by the present invention from ALV env albumen superinfection resistance
Then pcDNA3.1/Zeo (+) construction of eukaryotic expression vector turns carrier pcDNA-env-K at transfer vector pcDNA-env-K
Chicken embryo fibroblasts system DF-1 is contaminated, is screened by drug Zeocin, anti-Zeocin is obtained, stablizes expression ALV-K env albumen
Anti- K subgroup avian leucosis virus cell line.The cell line of obtained anti-K subgroup avian leucosis virus can stablize biography
Generation, long-term preservation；It is 10 that titre, which can be resisted,4TCID50Virus infection duplication amount, high specificity, DF-1/K cell of the invention
System can be used for the diagnosis of K subgroup avian leucosis virus and the development of ALV-K vaccine.
Detailed description of the invention
Fig. 1 is the technology path that the cell line of anti-K 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/K, is successfully detected in DF-1/K with the primer that the present invention designs
To the env gene of 1791bp, 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/K cell has green fluorescence, illustrates the success in DF-1/K 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/K, and in DF-1
No, illustrate external source env segment successful expression in DF-1/K.Actin is internal reference albumen, and env albumen is purpose albumen.
Fig. 6 is anti-K subgroup avian leucosis virus (ALV-K) experimental result, and GDFX0601 strain can be normal numerous on DF-1
It grows, but on DF-1/K, GDFX0601 cannot normally be bred, and only be 10 in virus titer5TCID50When replicate, but disease
Poison duplication is obviously suppressed, the extremely significant duplication lower than on DF-1.As a result illustrate, DF-1/K has very strong anti-ALV-K
The ability of infected cell.
Fig. 7 is viral (ALV-J CHN06) experimental result of J substock lymphoid leuoosis-resistant, ALV-J on DF-1/K and DF-1 all
It can normally breed, breeding situation does not have marked difference, illustrates that DF-1/K 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/K is substantially less than DF-1, illustrates DF-1/K 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 GDFX0601 is K subgroup avian leucosis virus,
CHN06 is ALV-J, is all from the Ministry of Agriculture's live vaccine initiative key lab for relying on College of Veterinary Medicine, South China Agricultural University.This
Invention restriction enzyme BamHI and Not I used is purchased from NEB company.LA Taq is purchased from TaKaRa company.PMD18-T is carried
Body is purchased from Invitrogen company purchased from TaKaRa company, eukaryon expression plasmid pcDNA3.1/Zeo (+).Anti- ALV-K single-factor
Serum is provided by relying on Ministry of Agriculture's live vaccine of College of Veterinary Medicine, South China Agricultural University to formulate key lab.FITC label
The goat anti-mouse IgG antibodies of goat anti-mouse IgG antibodies and HRP label are purchased from Simga company.SQ Tissue tissue DNA extracts
Kit, removes endotoxin plasmid extraction kit Plasmid at plastic recovery kit DNA Gel Extraction Kit
Miniprep Kit is OMEGA Products.ALV antigen detection kit (Avian Leukosis Virus Antigen
Test Kit) it is U.S. IDEXX Products.Cell culture: DMEM in high glucose medium powder, 0.25% pancreatin (contain EDTA),
Fetal calf serum is 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 anti-K crowds of avian leukosis virus (ALV-K) the cell line DF-1/K of embodiment 1
The present invention expands the primer of ALV-K env gene by inventor's designed, designed:
Upstream primer: CGCGGATCCGCCACCATGGAAGCCGTCATAAAGGCATTTCTGACTGGATAC
Downstream primer: AAGGAAAAAAGCGGCCGCTTACACTGCTCCATTTTCG
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-K (GDFX0601) genome with the above-mentioned primer of designed, designed, is recycled according to glue
Kit DNA Gel Extraction Kit specification is recycled；According to NEB company restriction enzyme BamHI, NotI
Specification carries out double digestion to eukaryon expression plasmid pcDNA3.1/Zeo (+) and pMD18-T-env segment respectively；Enzyme is separately recovered
Product is cut, and recovery product is attached according to 4 ligase specification of NEB company's T, construction recombination plasmid pcDNA-env-K；
Transformed competence colibacillus cell DH5 α goes endotoxin plasmid extraction kit specification according to OMEGA company, carries out recombinant plasmid
PcDNA-env-K 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-e nv-K plasmids and 10 μ L rouge
Plastid uses 100 μ L respectively5min is diluted and be incubated for, is then remixed, is incubated for 15min jointly, by 200 μ L matter
Grain-liposome complex is added in the Tissue Culture Dish of ready six hole.5h changes cell culture fluid (DMEM+10% after transfection
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 (DMEM+ of hole
15%FBS+200 μ g/mL zeocin), the cell culture fluid of primary same system was changed every 3-4 days, 10-15d forms unicellular
Colony, 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 25cm later2Carefully
It is cultivated in born of the same parents' bottle, cell culture is (DMEM+10%FBS+200 μ g/mL zeocin) at this time.With drug zeocin to cell
In 30 generation of step sizing, detects intracellular env from nucleic acid and protein level, and carries out Antiviral breeding and virus type
The application of type.
Cell state: threadiness, shuttle shape, adherent growth.(as shown in Figure 2)
Env gene in PCR detection DF-1/K: extracting the DNA of DF-1/K, 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 1791bp.
Indirect immunofluorescence (IFA) detects env expression conditions: in 24 orifice plates of confluent monolayers DF-1/K 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
4 DEG C of ALV-K mono-specific antiserum overnight incubation.PBS is washed 3 times, in addition the diluted sheep anti-mouse igg-FITC fluorescence of 1:200 is anti-
Body, 37 DEG C of effect 1h, after PBS is washed 3 times, adds 1 drop volume score, 50% glycerol to cover cell, real under the microscope in fluorescence microscopy
Test result.Set up a DMEM negative control.As shown in Figure 4 A and 4 B shown in FIG., as a result DF-1/K can be observed under fluorescence microscope
There is specific fluorescence on the endochylema of cell and surface, and DF-1 cell does not have green fluorescence.Illustrate the success in DF-1/K of external source env segment
Expression.
The expression of Western-blot detection env gene: total protein of cell is mentioned after DF-1/K 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/K, and do not have in DF-1.Illustrate external source env piece
Section successful expression in DF-1/K.
The anti-K subgroup avian leucosis virus chicken embryo fibroblasts that the present invention screens, are named as DF-1/K, 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.C201610.
The biological activity of anti-K crowds of avian leukosis virus (ALV-K) the cell line DF-1/K of embodiment 2 is tested
Antiviral breeding: respectively with DMEM by ALV-K strain GDFX0601 from 100To 105Carry out doubling dilution.Disappear respectively
Change DF-1/K cell and DF-1 cell, be respectively inoculated in one piece of 24 porocyte culture plates, every hole is inoculated with 1mL cell suspension, cell
Concentration is 1.7 × 105cells/mL。
The venom of each viral dilution gradient of GDFX0601 is inoculated in respectively in two plate cell suspensions, every hole is inoculated with 100 μ L
Venom, each viral dilution gradient do three repetitions, and do the positive control and DMEM negative control of a known strain, and 5%
CO2, 10%FBS, cultivate in 37 DEG C of incubators.Second day after venom to be seeded, after cell covers with single layer, it is changed to the thin of 1%FBS
Born of the same parents' maintaining liquid is cultivated.Continuous culture 6d, during which continues to monitor the growing state of cell, and in collection cell culture in the 6th day
Cell conditioned medium is collected by centrifugation in object, freeze thawing afterwards three times.The cell supernatant of collection is carried out with the ELISA kit of the same batch
The detection of p27 antigen ELISA.As a result as shown in fig. 6, GDFX0601 can normally be bred on DF-1, but on DF-1/K,
GDFX0601 cannot normally be bred, and only be 10 in virus titer5TCID50When replicate, but virus replication is obviously pressed down
System, the serious duplication lower than on DF-1.As a result illustrate, DF-1/K has the ability of very strong anti-ALV-K infected cell.
With same method by CHN06 plants of inoculation DF-1/K cells of ALV-J and DF-1 cell.As a result such as Fig. 7 is shown,
ALV-J can normally be bred on DF-1/K and DF-1, and breeding situation does not have marked difference, illustrate that DF-1/K 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/
K, 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/K is significantly lower than DF-1, says
Bright DF-1/K is resistant to the ALV, tentatively judges the ALV for ALV-K.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-K.
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-K subgroup avian leucosis virus, is chicken embryo fibroblasts, entitled DF-1/K, preservation is compiled
Number be CCTCC No.C201610.
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| 我国地方品种鸡分离到的一个禽白血病病毒新亚群的鉴定;王鑫 等;《病毒学报》;20121231;第28卷(第6期);摘要、第609页右栏第1段-第611页右栏第2段、第613页左栏第1段 * |
| 抗J亚群禽白血病病毒的鸡胚成纤维细胞系建立;叶建强 等;《病毒学报》;20051120;第21卷(第06期);第456页右栏第1段-第459页右栏第2段 * |
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