CN102277451A - Universal nest-polymerase chain reaction (PCR) primer group for use in amplification of gp90 gene in main classic strain of equine infectious anemia virus (EIAV) - Google Patents
Universal nest-polymerase chain reaction (PCR) primer group for use in amplification of gp90 gene in main classic strain of equine infectious anemia virus (EIAV) Download PDFInfo
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Abstract
The invention discloses a universal nest-PCR primer group for use in amplification of a gp90 gene in a main classic strain of equine infectious anemia virus and relates to a method for detecting equine infectious anemia virus by using the primer group to perform the nest-PCR amplification of a sample to be tested and the use of the primer group in the preparation of biological reagents for detecting or diagnosing equine infectious anemia virus. In the invention, the amplification primers are designed on the basis of the contrast among full-length genetic sequences of the main classic strains EIAVwyo, EIAVArgen and EIAVLN40 of EIAV and conserved region. The results of experiments indicate the nest-PCR primers designed by the invention can be used for amplification of the gp90 gene in main representative strains of EIAV and also have amplification specificity for on-site separated strains. The nest-PCR primers established through researches can be used in clinic molecular epidemiologic researches on pandemic strains of EIAV and are effective universal primers for amplification of the gp90 gene.
Description
Technical field
The present invention relates to the detection primer of equine infectious anemia virus, relate in particular to general nest-PCR primer sets based on the main classical strains gp90 of the detection equine infectious anemia virus gene of nest-PCR design, and detection method and application, belong to the detection range of equine infectious anemia virus.
Background technology
Equine infectious anemia virus (Equine infectious anemia virus, EIAV) belong to Retroviridae lentivirus member, be transmissible disease-equine infectious anemia (Equine infectious anemia, pathogenic agent EIA) of serious harm equus.
The Gp90 gene is the virogene of coding EIAV envelope protein Gp90, the about 1.4kb of total length.Envelope protein Gp90 interacts as part and host cell receptor ELR, helps virus to enter host cell.Because the ThermoScript II fidelity of slow virus is poor, makes slow virus can produce the high frequency transgenation when duplicating, cause virus antigen to continue drift.No matter be between different EIAV strains, also or by between the different quasispecies of evolving out in the same strain body, the frequency of undergoing mutation in the viral protein is the highest is Gp90 albumen.And the Gp90 protein mutation, be that the viral escape host immune monitors, form and continue the major cause of evolving that amplification in vitro EIAVgp90 gene order is also carried out sequential analysis to it, to carrying out researchs such as EIAV molecular epidemiology, antigenic variation analysis and immunologic escape mechanism, all significant.
Since the influence that sudden change of the height of gp90 gene and interzone are evolved, the genome difference that has higher level between different EIAV strains.The EIAV of EIAV America representative strains EIAVwyo and China for example
LN40Genome difference is up to 30%.This height variation characteristics, make with a certain representative strains genetic background design be used to increase the primer of gp90 gene, when being used for the gene amplification of other strains, may and be not suitable for.Therefore, if can design and not influenced by the EIAV gene diversity, can be common to the primer that main EIAV represents strain and EIAV terrain epidemic strain gp90 gene amplification, for the EIAV molecule epidemic disease-ology research in time, effectively carry out, improve the efficient of the popular and variation work of monitoring EIAV, very necessary.
Summary of the invention
Pass through the main classical strains EIAV of EIAV at the problems referred to above the present inventor
Wyo, EIAV
Argen, EIAV
LN40Complete genome sequence compare, and based on the conserved regions design amplimer, use the auxiliary design of primers of finishing of molecular biology software Oligo6.0.
One of technical problem solved by the invention provides the general nest-PCR primer sets of the one group of main classical strains gp90 of equine infectious anemia virus gene that is used to increase, it is characterized in that described primer sets by the overcoat primer to interior cover primer to forming, described overcoat primer to sequence shown in SEQ ID No.1 and SEQ ID No.2, described in the cover primer to sequence shown in SEQ ID No.3 and SEQ ID No.4.
The nest-PCR primer sets sequence of determining is as follows: the overcoat primer, and upstream: P-EIAV-gp90-f, 5 ' CA CCA GAG TGT TGT GGA AAG GTG A 3 ' (being SEQ ID No.1), the downstream:
P-EIAV-gp90-r, 5 ' GA CCC CAT GAT TCA TTC CA 3 ' (being SEQ ID No.2).Interior cover primer, upstream: p-EIAV-gp90-f-1,5 ' TG TAA GGT TTG GTG TAT GGG 3 ' (being SEQ ID No.3), downstream: p-EIAV-gp90-r-1,5 ' TG GCA GCT ATT ATA GCA GA 3 ' (being SEQ ID No.4).
Two of technical problem solved by the invention provides a kind of method that equine infectious anemia virus detects that is used for, and it is characterized in that: use above-described primer sets that testing sample is carried out the nest-PCR amplification.
Wherein, in the described nest-PCR amplified reaction, preferred, the right annealing temperature of overcoat primer is 53 ℃, and the right annealing temperature of interior cover primer is 51 ℃; The extension time is respectively 2min and 1min30sec, and two secondary responses respectively carry out 35 circulations respectively.
The general nest-PCR primer sets that three of technical problem solved by the invention provides the described main classical strains gp90 of the equine infectious anemia virus gene that is used for increasing detects or the purposes of the biological reagent of diagnosis equine infectious anemia virus in preparation.
This result of study shows, the nest-PCR primer of the present invention's design can be common to the amplification that EIAV mainly represents strain gp90 gene, and for the amplification of the terrain strain isolated of this research use, points out it to have specific amplification equally.Above result of study proves, the nest-PCR primer that this research is set up is the effective universal primer that can be used for clinical EIAV epidemic isolates molecule epidemic disease-ology research, amplification gp90 gene.
Description of drawings
Fig. 1 detects the experimental result of sample for the different EIAV of the nest-PCR primer amplification that uses the present invention's design.Swimming lane 1-8 is respectively 1, dna molecular amount marker2000; 2, EIAV
Wyo3, EIAV
Argen4, EIAV
LN405, EIAV
FDDV126, EIAV-1; 7, EIAV-2; 8, negative control (the negative horse tissue DNA of EIAV).
Embodiment
The present invention will be further described below by specific embodiment, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
1 experiment material, method
1.1. strain, pathological material of disease, detection sample
Be used to verify that the specific EIAV sample of primer amplification comprises EIAV North America representative strains EIAV
Wyo(Genbank No, AF033820), South America representative strains EIAV
Argen(be recorded in: Geng Qinghua.The foundation of China's contagious equine abortion attenuated vaccine strain and America epidemic isolates differential diagnosis method and the research of marker vaccine.China Ph D dissertation, Beijing, graduate school of the Chinese Academy of Agricultural Sciences, 2006.), representative of China's strain EIAV
LN40(Genbank No, AF327877), Chinese EIAV vaccine low virulent strain EIAV
DLV125(Genbank No, AF327878) and within Chinese territory terrain strain isolated EIAV-1, EIAV-2 (preserve by laboratory, patent applicant place, separated from Heilungkiang with 2005) respectively at 2004.
1.2 design of primers and primer sequence
Based on the main classical strains EIAV of EIAV
Wyo, EIAV
Argen, EIAV
LN40Complete genome sequence comparison, based on the conserved regions design amplimer.Use the auxiliary design of primers of finishing of molecular biology software Oligo6.0.The nest-PCR primer sequence of determining is as follows: overcoat primer, upstream: P-EIAV-gp90-f, 5 ' CA CCA GAG TGT TGT GGA AAG GTG A 3 ', downstream: P-EIAV-gp90-r, 5 ' GA CCC CAT GAT TCA TTC CA 3 '.Interior cover primer, upstream: p-EIAV-gp90-f-1,5 ' TG TAA GGT TTG GTG TAT GGG 3 ', downstream: p-EIAV-gp90-r-1,5 ' TG GCA GCT ATT ATA GCA GA3 '.
1.3 the extraction of DNA before the virus
(USA D3396-01) to different virus strain infections cell or tissue, carries out total DNA extraction to using-system DNA extraction test kit for Tissue DNA kit, Omega.The proviral DNA that includes EIAV among total DNA.This DNA extraction thing as the template of nest-PCR, is used for follow-up amplification research.
The acquisition process of the proviral DNA of different strains is specific as follows:
EIAV
WyoStrain: the infections clone strain Infection in Vitro donkey embryo skin cell of deriving that uses the EIAVwyo strain.Connect poison after 6 days, inhale the nutrient solution of abandoning in the Tissue Culture Flask, use cell to scrape and get cell.The cell of results is resuspended with the cold PBS of 200 μ l, and afterwards to above-mentioned cell sample, using-system DNA extraction test kit carries out the cell total DNA extraction.The DNA extraction process is specifically referring to the test kit specification sheets.
EIAV
DLV125Strain: the proviral DNA acquisition methods and the EIAV of this strain
WyoStrain is identical.
Use derive the infections clone strain and the EIAV of EIAVwyo strain
DLV125Strain Infection in Vitro donkey embryo skin cell, the donkey fetus cells is homemade former generation skin cells (Zhao Li equality, the improvement of equine infectious anemia antigen production process, the animal and veterinary scientific and technical information of taking from the donkey fetus in present patent application laboratory, 2004 09 phases, the 37-39 page or leaf.)。
EIAV
ArgenStrain: this strain proviral DNA is taken at the lymphoglandula of this virus strain infection horse.Get the mesenteric lymph nodes of this virus strain infection horse, about 30mg fully shreds with scissors, and it is resuspended to add the cold PBS of 200 μ l.Using-system DNA extraction test kit carries out DNA extraction to this sample afterwards, and detailed process is referring to the test kit specification sheets.
This strain proviral DNA is taken at the present patent application laboratory and uses this strain to carry out the lymphoglandula of artificial challenge horse.
EIAV
LN40, EIAV-1 and EIAV-2 strain proviral DNA acquisition process, with EIAV
ArgenStrain is consistent.
1.4Nest-PCR amplification condition optimization
Annealing temperature is the main PCR reaction parameter of optimizing of this research.Under the prerequisite of quantitative templates as amplification template of different copy numbers, be optimized by the grads PCR instrument.
1.5 the order-checking of amplification gene is identified
Send commercial order-checking company to check order to the pcr amplification product of different strains.Sequencing result carries out the sequence alignment analysis by biological software DNAstar, to determine obtaining whether gene is the EIAVgp90 gene.
2 experimental results
2.1Nest-PCR amplification condition
PCR reaction conditions through optimizing is: comprise in the 25 μ L reaction solutions: 1.2mmol MgCl
2, 0.5mmoldNTP, each 10 μ mol of upstream and downstream primer, 1.25U rTaq enzyme and dna profiling 2 μ L.The final pre-denaturation temperature of determining of nest-PCR is 95 ℃, time 5min; Annealing temperature is overcoat: 53 ℃, interior cover: 51 ℃, the time is 40sec; Elongating temperature is 72 ℃, and the time is respectively 2min and 1min30sec.Two secondary responses respectively carry out 35 circulations respectively, carry out 72 ℃ of 10min reactions at last again.The PCR product carries out electrophoresis and observations under ultraviolet lamp with 1.5% sepharose.
2.2 the pcr amplification qualification result of different strains and detection sample
Use above-mentioned pcr amplification condition, test sample DNA is carried out nest-PCR amplification, electrophoretic analysis picture such as Fig. 1 of the PCR product of acquisition.As shown in Figure 1, the different samples that detect have all obtained the PCR product about 1.4kb through the PCR of this experimental design primer amplification.The classical terrain epidemic strain of representing the domestic some areas of strain and China of EIAV that the primer of this experimental design of preliminary proof is selected for use at this research, all tool versatility.
2.3 sequencing and comparison result
The above-mentioned different pcr amplification products that detect sample are checked order, and the sequence and the reference sequences of acquisition compare.Analytical results shows that above-mentioned sequence is the EIAVgp90 gene, proves that on behalf of detection sample standard deviations such as strain and terrain epidemic strain, the nest-PCR primer of this experimental design to different EIAV have versatility.
3 experiment conclusion
This result of study shows that the nest-PCR primer of design can be common to the amplification that EIAV mainly represents strain gp90 gene, and the amplification of the terrain strain isolated that uses for this research points out it to have specific amplification equally.Above result of study proves, the nest-PCR primer that this research is set up is the effective universal primer that can be used for clinical EIAV epidemic isolates molecule epidemic disease-ology research, amplification gp90 gene.
The above only is the preferred embodiments of the present invention, only is illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and can carry out many changes to it in the spirit and scope that claim of the present invention limited, revise, even the equivalence change, but all will fall within the scope of protection of the present invention.
Claims (4)
1. the general nest-PCR primer sets of amplification equine infectious anemia virus main classical strains gp90 gene, it is characterized in that described primer sets by the overcoat primer to interior cover primer to forming, described overcoat primer to sequence shown in SEQ ID No.1 and SEQ ID No.2, described in the cover primer to sequence shown in SEQ ID No.3 and SEQ ID No.4.
2. one kind is used for the method that equine infectious anemia virus detects, and it is characterized in that: use the described primer sets of claim 1 that testing sample is carried out the nest-PCR amplification.
3. in accordance with the method for claim 2, it is characterized in that in the described nest-PCR amplified reaction that the right annealing temperature of overcoat primer is 53 ℃, the right annealing temperature of interior cover primer is 51 ℃; The extension time is respectively 2min and 1min30sec, and two secondary responses respectively carry out 35 circulations respectively.
The general nest-PCR primer sets of the main classical strains gp90 of the described amplification equine infectious anemia virus of claim 1 gene preparation detect or the biological reagent of diagnosis equine infectious anemia virus in purposes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501273A (en) * | 2020-07-27 | 2021-03-16 | 暨南大学 | Nested PCR primer and kit for amplifying horse antibody and application thereof |
| CN112521462A (en) * | 2020-12-14 | 2021-03-19 | 杭州亿米诺生物科技有限公司 | Equine infectious anemia virus p26-gp90 recombinant protein, and preparation method and application thereof |
-
2011
- 2011-08-17 CN CN2011102356712A patent/CN102277451A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| JIAN MA等: "In vivo evolution of the gp90 gene and consistently low plasma viral load during transient immune suppression demonstrate the safety of an attenuated equine infectious anemia virus (EIAV) vaccine", 《ARCHIVES OF VIROLOGY》 * |
| 马建: "EIAV疫苗株gp90基因的多克隆构成和体内进化与免疫保护的相关性", 《中国博士学位论文全文数据库 农业科技辑》 * |
| 马建: "免疫抑制对EIAV弱毒疫苗免疫马体内疫苗弱毒株载量的影响", 《生物化学与生物物理进展》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501273A (en) * | 2020-07-27 | 2021-03-16 | 暨南大学 | Nested PCR primer and kit for amplifying horse antibody and application thereof |
| CN112501273B (en) * | 2020-07-27 | 2021-06-29 | 暨南大学 | Nested PCR primer and kit for amplifying horse antibody and application thereof |
| CN112521462A (en) * | 2020-12-14 | 2021-03-19 | 杭州亿米诺生物科技有限公司 | Equine infectious anemia virus p26-gp90 recombinant protein, and preparation method and application thereof |
| CN112521462B (en) * | 2020-12-14 | 2023-09-19 | 杭州爱谨生物科技有限公司 | Horse infectious anemia virus p26-gp90 recombinant protein and preparation method and application thereof |
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Application publication date: 20111214 |