CN107058246A - Single viral purification process in avian leukosis virus mixed infection - Google Patents

Single viral purification process in avian leukosis virus mixed infection Download PDF

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Publication number
CN107058246A
CN107058246A CN201710350008.4A CN201710350008A CN107058246A CN 107058246 A CN107058246 A CN 107058246A CN 201710350008 A CN201710350008 A CN 201710350008A CN 107058246 A CN107058246 A CN 107058246A
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alv
avian leukosis
mixed infection
leukosis virus
purification process
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王培坤
林璐璐
杨永立
李海娟
磨美兰
韦天超
黄腾
韦平
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Guangxi University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/11011—Alpharetrovirus, e.g. avian leucosis virus
    • C12N2740/11051—Methods of production or purification of viral material

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Abstract

The invention discloses single viral purification process in a kind of leukemia virus mixed infection, by the TCID for determining hybrid infection viruses50End dilution is carried out, using the cells of poison disease vaccination DF 1 after dilution, supernatant identifying it with cell culture and with Classification Identification primer and IFA is collected, so as to obtain the avian leukosis virus of single hypotype.The method is simple to experiment condition requirement, is of very high actual application value.The results show, the present invention can be purified successfully to the ALV of different subtype mixed infection, can be that avian leukosis virus molecular biological characteristics and its pathogenic research provide technical support.

Description

Single viral purification process in avian leukosis virus mixed infection
Technical field
The invention belongs to single virus in viral purification technical field, more particularly to a kind of avian leukosis virus mixed infection Purification process.
Background technology
Avian leukosis virus (avian leukosis virus, ALV) is Retroviridae, leukaemia/sarcoma virus Group.Roloff in 1868 reports one " lymphosarcoma ", and this is first report on avian leukosis.Then, ALV is each There is relevant report on ground, and host range gradually expands, and laying hen and Local Chicken Breeds, clinical tumor performance are traveled to from broiler chicken More substantially, larger economic loss is caused to aviculture.
ALV point is A, B, C, D, E, J, K totally 7 subgroups, and wherein A, B, J subgroup are arrived clinic is easily separated, and K subgroups are recent It was found that new subgroup.ALV can cause immunity of organism to suppress, and reduce the success rate of vaccine immunity, have a strong impact on production performance.In recent years Come, ALV mixed infections, which have, increases trend, there is two subgroups even mixed infection of three subgroups mostly.This is accomplished by fowl The fashion trend and mechanism of genetic change of leukemia virus are studied, and it is the basic with before of research to obtain single subtype virus Carry.In clinic, the situation of the ALV mixed infections of different subgroups occurs in chicken group, therefore the purifying of virus is urgently to be resolved hurrily asks Topic.
The content of the invention
The technical problem to be solved in the present invention is to provide single viral purifying in a kind of avian leukosis virus mixed infection Method, is avian leukosis virus molecular biology in order to obtain single subtype virus from avian leukosis virus mixed infection Feature and its pathogenic research provide technical support.
In order to solve the above technical problems, the present invention uses following technical scheme：It is single in avian leukosis virus mixed infection The purification process of virus, by the TCID for determining hybrid infection viruses50End dilution is carried out, using the poison disease vaccination after dilution DF-1 cells, collect supernatant identifying it with cell culture and with Classification Identification primer and IFA, so as to obtain single The avian leukosis virus of hypotype.
Classification Identification primer includes ALV-A primer pairs, ALV-B primer pairs, ALV-J primer pairs, and they have sequence respectively Table SEQ.ID.No.1 to SEQ.ID.No.6 base sequence.
Single viral purification process in above-mentioned avian leukosis virus mixed infection, according to the following steps operation is carried out：
(1) hybrid infection viruses are obtained and bred for 3 generations in DF-1, the 3rd generation, by being inoculated with DF-1 cells, then passed through detection ALV-p27 antigens simultaneously measure viral TCID according to Reed-Muench methods50；
(2) virus liquid is to determine TCID50End dilution is carried out, dilution restrovirus liquid acts on individual layer with 24 orifice plate multiple holes DF-1 cell 2h, then change liquid and maintain culture to 9 days；
(3) collect cell culture supernatant and numbering is stored in -80 DEG C respectively, cell culture is collected DNA extraction and is used in combination Serotype specific primer is identified；
(4) the corresponding aperture cell conditioned medium inoculation DF-1 cells for being accredited as single subtype viral infection breed IFA points of 3 generations progress Type identification (, the virus for being accredited as single hypotype carries out next step research.)
Step (1) detection ALV-p27 antigens use IDEXX ALV-p27 kits.
The dilution that step (2) dilution is used produces DMEM for Gibco.
Step (2) maintains the nutrient solution that culture is used for the DMEM containing 1%FBS.
The situation that trend for current avian leukosis virus different subtype mixed infection increases, inventor establishes one kind Single viral purification process in leukemia virus mixed infection, by the TCID for determining hybrid infection viruses50Carry out terminal dilute Release, using the poison disease vaccination DF-1 cells after dilution, collect supernatant with cell culture and with Classification Identification primer and IFA It is identified, so as to obtain the avian leukosis virus of single hypotype.The method is simple to experiment condition requirement, with very high Actual application value.The results show, the present invention can be purified successfully to the ALV of different subtype mixed infection, can It is that avian leukosis virus molecular biological characteristics and its pathogenic research provide technical support.
Brief description of the drawings
During Fig. 1 is the result figure identified cell culture PCR after purification using serotype specific primer, figure：M is DL2000 DNA Maker；1 is ALV-A amplifications；2 be ALV-B amplifications；3 be ALV-J amplifications；4 be negative control.
Fig. 2 is the result figure of immunofluorescence method detection, in figure：A is that strain GX14MM6-2M is directed to A in DF-1 cells Hypotype monoclonal antibody IFA results；B is that strain GX15MM6-2-Mix is directed to subtype B monoclonal antibody IFA results in DF-1 cells；C is strain GX15MM6-2-Mix is directed to J hypotype monoclonal antibody IFA results in DF-1 cells；D is negative control result.
Embodiment
1.1 strain
(applicant is real from GX15MM6-2-Mix plants of Chinese pathogenic strain for the avian leukosis virus of mixed infection A, B, J hypotype Test room preservation).
1.2 method
1.2.1 design of primers
Primer reference literature is designed, and ALV serotype specific primer is shown in Table 1, and is synthesized by Shanghai Hua Da bioengineering Co., Ltd.
The primer sequence of table 1
1.2.2 strain GX15MM6-2Mix recovery
DF-1 cells are inoculated with strain GX15MM6-2Mix, 37 DEG C are continued to be incubated 1h, abandon supernatant after 1h, be replaced by containing The DMEM culture mediums of 1% hyclone, in 37 DEG C, 5%CO2Under the conditions of cultivate 9 days；Then collect cell culture and be placed on -80 It is DEG C standby.
1.2.3 strain GX15MM6-2Mix TCID50 is determined
By the mixed infection strain GX15MM6-2Mix of recovery DF-1 breed 3 generations, the 3rd generation by being inoculated with DF-1 cells, It is 10 then to withhold the virus liquid of collection according to dilution factor by the 3rd-1-10-10Doubling dilution, and the row of repeat function 8 are carried out, is received after 9 days Collection supernatant measures virus by detecting ALV-p27 antigens (IDEXX ALV-p27 kits) according to Reed-Muench methods TCID50。
1.2.4 strain GX15MM6-2Mix end dilution
According to the TCID of measure50End dilution multiple is determined, containing 1 virus in making it per 0.1ml virus liquids.Dilution Virus liquid repeat function after (dilution is that Gibco produces DMEM) after 24 porocyte plates, 37 DEG C of absorption 1h in abandoning supernatant, more The DMEM culture mediums containing 1% hyclone are changed to, in 37 DEG C, 5%CO2Under the conditions of cultivate 9 days.Cell conditioned medium is collected, continuously Pass after 3 generations, collect cell culture, the PCR detections that DNA carries out A, B, J hypotype respectively are extracted by kit specification.
ALV-A/ALV-B PCR response procedures are 94 DEG C of pre-degenerations 5 minutes, and 94 DEG C are denatured 45 seconds, and 50 DEG C are annealed 45 seconds, 72 DEG C extend 2 minutes, totally 35 circulations, and last 72 DEG C extend 10 minutes.ALV-J PCR response procedures are 94 DEG C of 5 points of pre-degenerations Clock, 94 DEG C are denatured 45 seconds, and 56 DEG C are annealed 45 seconds, and 72 DEG C extend 2 minutes, totally 35 circulations, and last 72 DEG C extend 10 minutes.1% Agarose gel electrophoresis identifies PCR primer.
1.2.5 Classification Identification
DF-1 cells are inoculated with to the corresponding aperture cell conditioned medium for being accredited as single subtype viral infection and bred for 3 generations, after it is thin to its Born of the same parents' culture extracts DNA, and it enters performing PCR Classification Identification (the same 1.2.4 of PCR programs) with ALV-A/B/J Classification Identifications primer pair. Enter row agarose gel electrophoresis analysis to PCR primer afterwards, A, B, J hypotype mixed infection strain are judged according to electrophoresis result Whether GX15MM6-2-Mix purifies successfully (result is shown in Fig. 1).
As a result show, the ALV-J in strain GX15MM6-2-Mix is purified successfully.
By above-mentioned in 37 DEG C, 5%CO2Under the conditions of culture 7 days after cell hole, washed with PBS 3 times, each 5min；Then Using ethanol: acetone=2: 3 fix cell 5min, PBS washing 3 times, each 5min under the conditions of 4 DEG C.By ALV-A/B/J monoclonal antibodies By 1: 250 dilution, add in test group cell hole different with blank control group, per the μ L of hole 150, be incubated in 37 DEG C of wet box respectively 3h, PBS are washed 3 times, each 5min.The rabbit-anti mouse fluorescence antibody of FITC marks is added, by 1: 300 dilution, per the μ L of hole 150, Continue to be incubated 1h in 37 DEG C of wet box.PBS observes (result is shown in Fig. 2) after washing 3 times under inverted fluorescence microscope.As a result show to resist ALV-A and ALV-B monoclonal antibodies are negative, and anti-ALV-J monoclonal antibodies are positive, infected group DF-1 cell born of the same parents Visible obvious green fluorescence in slurry, and green fluorescence is had no in negative control group.
As a result show, the ALV-J in strain GX15MM6-2-Mix is purified successfully.
SEQUENCE LISTING
<110>Guangxi University
<120>Single viral purification process in avian leukosis virus mixed infection
<130>Single viral purification process in avian leukosis virus mixed infection
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Claims (6)

1. single viral purification process in a kind of avian leukosis virus mixed infection, it is characterised in that by determining mixed infection The TCID of virus50End dilution is carried out, using the poison disease vaccination DF-1 cells after dilution, supernatant and cell culture is collected simultaneously With identifying it for Classification Identification primer and IFA, so as to obtain the avian leukosis virus of single hypotype.
2. single viral purification process in avian leukosis virus mixed infection according to claim 1, it is characterised in that： The Classification Identification primer includes ALV-A primer pairs, ALV-B primer pairs, ALV-J primer pairs, and they have sequence table respectively SEQ.ID.No.1 to SEQ.ID.No.6 base sequence.
3. single viral purification process in avian leukosis virus mixed infection according to claim 2, it is characterised in that Operation is carried out according to the following steps：
(1) obtain hybrid infection viruses DF-1 breed 3 generations, the 3rd generation by being inoculated with DF-1 cells, then by detecting ALV- P27 antigens simultaneously measure viral TCID according to Reed-Muench methods50；
(2) virus liquid is to determine TCID50End dilution is carried out, it is thin that dilution restrovirus liquid acts on individual layer DF-1 with 24 orifice plate multiple holes Born of the same parents 2h, then changes liquid and maintains culture to 9 days；
(3) collect cell culture supernatant and numbering is stored in -80 DEG C respectively, cell culture collects DNA extraction and uses parting Primer is identified；
(4) the corresponding aperture cell conditioned medium inoculation DF-1 cells for being accredited as single subtype viral infection breed 3 generations progress IFA parting mirror It is fixed.
4. single viral purification process in avian leukosis virus mixed infection according to claim 1, it is characterised in that Step (1) the detection ALV-p27 antigens use IDEXX ALV-p27 kits.
5. single viral purification process in avian leukosis virus mixed infection according to claim 1, it is characterised in that Step (2) dilution used that dilutes produces DMEM for Gibco.
6. single viral purification process in avian leukosis virus mixed infection according to claim 1, it is characterised in that Step (2) is described to maintain the nutrient solution that culture is used for the DMEM containing 1%FBS.
CN201710350008.4A 2017-05-17 2017-05-17 Single viral purification process in avian leukosis virus mixed infection Pending CN107058246A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109122550A (en) * 2018-03-22 2019-01-04 四川省畜牧科学研究院 A kind of chicken poultry leukaemia purification method

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101988131A (en) * 2010-11-01 2011-03-23 中国农业科学院哈尔滨兽医研究所 Loop-mediated isothermal amplification (LAMP) reaction primer for detecting subgroup-A avian leukosis virus (ALV-A)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101988131A (en) * 2010-11-01 2011-03-23 中国农业科学院哈尔滨兽医研究所 Loop-mediated isothermal amplification (LAMP) reaction primer for detecting subgroup-A avian leukosis virus (ALV-A)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GAO Q ET AL: "Development and application of a multiplex PCR method for rapid differential detection of subgroup A, B, and J avian leukosis viruses", 《J CLIN MICROBIOL》 *
孟凡峰等: "核酸杂交技术在禽白血病病毒TCID50测定中的应用", 《中国家禽》 *
杨永立等: "三个亚群禽白血病病毒混合感染阉鸡的诊断", 《中国家禽》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109122550A (en) * 2018-03-22 2019-01-04 四川省畜牧科学研究院 A kind of chicken poultry leukaemia purification method
CN109122550B (en) * 2018-03-22 2021-02-23 四川省畜牧科学研究院 Method for purifying leukemia of chickens

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