CN101696398B - Equine infectious anemia virus pathopoiesia virulent strain ass fetus skin cell adapted strain and application - Google Patents
Equine infectious anemia virus pathopoiesia virulent strain ass fetus skin cell adapted strain and application Download PDFInfo
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- CN101696398B CN101696398B CN2009101803181A CN200910180318A CN101696398B CN 101696398 B CN101696398 B CN 101696398B CN 2009101803181 A CN2009101803181 A CN 2009101803181A CN 200910180318 A CN200910180318 A CN 200910180318A CN 101696398 B CN101696398 B CN 101696398B
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Abstract
The invention discloses an equine infectious anemia virus (EIAV) pathopoiesia virulent strain ass fetus skin (FDD) cell adapted strain and application thereof. In the invention, FDD cell is selected as the infection medium, and six passages of the virulent strain EIVADLV 34 are carried out in the FDD cell, the obtained virulent strain is named as EIAV-DLV34-F6, and the microorganism preservation number is CGMCC No.3231. The virulent strain in the invention can be favorably copied on the FDD cell during culture in vitro, and the pathogenicity as well as gene characteristic is maintained. The establishment of virulent strain provides important research material for relative researches of EIAV and slow virus toxicity and immunogenicity.
Description
Technical field
The present invention relates to equine infectious anemia virus (Equine Infectious Anemia Virus; EIAV) virulent strain; Relate in particular to the donkey embryo skin cell adapted strain and the construction process thereof of the pathogenic virulent strain of equine infectious anemia virus; The invention still further relates to the application of donkey embryo skin cell adapted strain in making up tool virulence infections clone of the pathogenic virulent strain of this equine infectious anemia virus, belong to the equine infectious anemia virus field.
Background technology
The equine infectious anemia less toxic vaccine is with field isolating highly pathogenicity strain (EIAV
LV) process obtains a little less than the people in generation surplus the external donkey monocyte 120 is exquisite.Virulent strain (EIAV
LV) through passage, experiencing virulence and weakening gradually, immunogenicity is the enhanced process gradually.
The equine infectious anemia less toxic vaccine is as the slow virus vaccine of unique successful Application, for slow virus immunoprotection mechanism provides important model.The appraisement system of distinctive virulent strain of China and attenuated vaccine strain more helps to illustrate its immunoprotection mechanism.But,, limited its biological Characteristics Study because the distinctive cultural characters of equine infectious anemia virulent strain (former generation monocyte with whole serum cultivation).Therefore, keeping changing the virulent strain training method under the prerequisite of virulence, to the research of its biological characteristics and immunoprotection, and the improvement of equine infectious anemia vaccine and new generation vaccine structure are all significant.
Summary of the invention
One of the object of the invention is under the prerequisite of the virulence that keeps the pathogenic virulent strain of equine infectious anemia virus; Change its training method; Obtain the donkey embryo skin cell adapted strain of the pathogenic virulent strain of a strain equine infectious anemia virus; This donkey embryo skin cell adapted strain can well duplicate on the donkey embryo skin cell FDD of vitro culture, and keeps its pathogenic and gene characteristics;
Two of the object of the invention is that the cause a disease donkey embryo skin cell adapted strain of virulent strain of above-mentioned equine infectious anemia virus is applied to make up infections clone, for equine infectious anemia virus and slow virus virulence and immunogenicity correlative study provide important research material;
Above-mentioned purpose of the present invention realizes through following technical scheme:
The cause a disease donkey embryo skin cell adapted strain of virulent strain of one strain equine infectious anemia virus, its microbial preservation number are: CGMCC No.3231; Classification name: equine infectious anemia virus; The preservation time is: on August 13rd, 2009; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The equine infectious anemia less toxic vaccine is with field isolating highly pathogenicity strain (EIAV
LV) process obtains a little less than the people in generation surplus the external donkey monocyte 120 is exquisite.Virulent strain (EIAV
LV) through passage, experiencing virulence and weakening gradually, immunogenicity is the enhanced process gradually.Therefore, the present invention is strong according to China, low virulent strain cause weak and generation characteristics, process EIAV has selected to go down to posterity
DLV34Strain.The test of horse body shows that this strain still has higher virulence, can cause horse acute death (Fig. 1).Consider a large amount of cultivations of virus and the operability of immunodetection, the present invention selects the FDD cell as infecting medium, with EIAV
DLV34Strain carries out 6 times at the FDD cell and goes down to posterity, the strain called after EIAV-DLV34-F6 of acquisition.
Immunofluorescence and RT enzymic activity detect and show the virulent strain (EIAV of monocyte preferendum
DLV34) having obtained the flexibility of in donkey hide skin cell, duplicating gradually, virus strengthens along with the increase of going down to posterity the infection ability of FDD cell.After going down to posterity for 6 times, obtained 4 * 10
7The infection titer of PFU/ml (Fig. 2).For the change of probing into cell tropism influence to virus virulence; The present invention carries out toxicity test with EIAV-DLV34-F6 involution horse body; Experimental result shows that the horse body occurred equine infectious anemia typical clinical symptom, the time of bringing out of acute onset and virulent strain (EIAV in back 10 days, 24 days in inoculation respectively
LV) more consistent, explain that this strain still keeps the virulence similar with parent's virulent strain.It is thus clear that the change of cell tropism does not exert an influence to virus virulence.
In addition, infections clone is the important means of carrying out known viral gene function research.With this strain is that the framework construction infections clone is an important application of the present invention.The virulent strain genetic background that utilizes the infections clone of the virulence strain that this method obtains to be kept perfectly.SEQ ID NO:1 is the EIAV-DLV34-F6 complete genome sequence; Sequence alignment shows the homology average out to 96.1% (table 2) of this strain and parent's virulent strain; In the accurate strain variety of equine infectious anemia virus scope; The virulent strain EIAV-DLV34-F6 that the present invention sets up lays a good foundation for making up tool virulence infections clone.
Above result confirms that the present invention has successfully made up and can on the donkey embryo skin cell FDD of vitro culture, well duplicate; And keeping its pathogenic and equine infectious anemia virulent strains gene characteristics, the be established as equine infectious anemia virus and slow virus virulence and immunogenicity correlative study of this strain provide important research material.
Description of drawings
Fig. 1 DLV34 strain horse body toxicity test test-results
The infection level of different generations behind Fig. 2 immunofluorescence detection strain DLV34 switching donkey hide skin cell
Fig. 3 EIAV-DLV34-F6 virus titer
Fig. 4 EIAV-DLV34-F6 horse body toxicity test test-results
Fig. 5 infections clone makes up synoptic diagram
Fig. 6 infections clone makes up previous work figure as a result
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
The screening and the evaluation of experimental example 1EIAV-DLV34-F6 strain
This test and Selection the process EIAV that goes down to posterity
DLV34Strain (EIAV
DLV34Be EIAV
LVObtain through the external continuous passage of donkey monocyte).The test of horse body shows that this strain still has higher virulence, can cause horse acute death (Fig. 1).The present invention selects the FDD cell as infecting medium, with EIAV
DLV34Strain carries out 6 times at the FDD cell and goes down to posterity, the strain called after EIAV-DLV34-F6 of acquisition.
The active detection of immunofluorescence and reversed transcriptive enzyme (RT) shows (referring to Fig. 2, table 1), the virulent strain (EIAV of monocyte preferendum
DLV34) obtained gradually at donkey hide skin cells growth activity, the virus infection ability strengthens along with the increase of going down to posterity.After going down to posterity for 6 times, obtained 1.8 * 10
7The infection titer of PFU/ml (Fig. 3).For the change of the probing into cell tropism influence to virus virulence, the present invention carries out toxicity test with EIAV-DLV34-F6 involution horse body.Fig. 4 result shows that the horse body occurred equine infectious anemia typical clinical symptom, the time of bringing out of acute onset and virulent strain (EIAV in back 10 days, 24 days in inoculation respectively
LV) more consistent, explain that this strain still keeps the virulence similar with parent's virulent strain.It is thus clear that the change of cell tropism does not exert an influence to virus virulence.
Table 1
| Unit | DLV34-F1 | Positive control | Negative control |
| OD405/492nm | 1.971 | 3.457 | 0.131 |
Concrete experimental technique is following: inspection strain 500 μ l are inserted donkey embryo skin cell (FDD) cell of cultivating into individual layer.37 ℃ of incubators are cultivated to extract after 8 days and are met poison cell culture supernatant 200 μ l.Simultaneously, set up corresponding negative control (not connecing the poison cell culture supernatant) and positive control (EIAVDLV strain cell culture fluid).Add 100 μ l PEG8000 solution, 4 ℃ are spent the night behind the abundant mixing.Next day, connect reverse transcriptase activity in the poison cell culture supernatant according to Reverse Transcriptase AssayColorimetric Kit specification sheets mensuration.Criterion OD >=negative control+2SD is promptly positive, explains that the strain of examining has the virus particle that reverse transcriptase activity can The expressed.
The virulent strain genetic background that utilizes the infections clone of the virulence strain that this method obtains to be kept perfectly.SEQ ID NO:1 shows the EIAV-DLV34-F6 complete genome sequence; Show through the higher several genes of aberration rate in the genome and the sequence alignment of parent's virulent strain; The homology average out to 96.1% of this strain and parent's virulent strain; Its main several aberration rates are seen table 2 than the homology of high gene, and the genovariation degree is in the accurate strain variety of equine infectious anemia virus scope.
Table 2
The application examples of Test Example 2EIAV-DLV34-F6 strain
With the EIAV-DLV34-F6 strain is that the framework construction infections clone is an important application of the present invention.The virulent strain EIAV-DLV34-F6 that the present invention sets up lays a good foundation for making up tool virulence infections clone.
At first design primer (primer sequence: LTR-L:TGT GGG ATT AAT ATA AGA TTCT; LTR-R:TGT TAG ATC TTG AAA ACA AGA C) amplification LTR 316bp gene; Be cloned into pMD18-T and go up acquisition plasmid pMD18-T-LTR; Utilize carrier two ends Hind III and EcoR I site with this goal gene subclone to (pLG338 can buy (the The American TypeCulture Collection in ATCC by low copy carrier pLG338 (pLG338 is gone up disappearance back, Mlu I site)
Www.atcc.org, commercial disignation: 37130
TM) on obtained plasmid pLG-338-LTR; PCR increases respectively and comprises gag gene 3.5kb fragment (primer sequence: Pltr-L:CTC ATT ATA GTT CCG CTT TTG TGA CG; Ppol-R:GATGTG AAT ATA TCA TGT CTG) and comprise pol-env gene (primer sequence: Pgag-L:CTGGAA TTC GTC GAC AGC AGA GGA GAA CTT ACA G; Pltr-R:CAG ACT CGAGCA GGG ACT CAG ACC GCA GAA TC) and the 7.7kb fragment of part 3 ' LTR and clone respectively in pMD18-T and go up to obtain plasmid pMD18-T-3.5kb and pMD18-T-7.7kb.Hind III enzyme is cut pMD18-T-3.5kb, reclaims the endonuclease bamhi of 2.6kb; Hind III enzyme is cut pMD18-T-7.7kb, reclaims the endonuclease bamhi of 8.1kb; Two fragments are connected, obtain plasmid pMD18-T-8.0kb; Utilize Mlu I that two reorganization plasmids are carried out enzyme and cut the back connection, transformed competence colibacillus intestinal bacteria HB101 obtains to comprise the recombinant plasmid of full gene.After sequencing analysis is identified, transfection donkey embryo skin cell (FDD), blind passage 4-6 generation, detect through immunofluorescence and RT enzymic activity, determine whether to obtain infections clone.
Test-results is seen Fig. 6.Test-results shows that the equine infectious anemia virulence Construction of Infectious Molecular Clone skeleton that it is genetic background that this test has obtained with strain EIAV-DLV34-F6 of the present invention can continue the experimental infection in later stage.
KLPI090889_2
SEQUENCE?LISTING
< 110>Harbin Veterinary Medicine Inst., China Academy of Agriculture
< 120>equine infectious anemia virus pathopoiesia virulent strain ass fetus skin cell adapted strain and application
<130>KLPI080678
<160>1
<170>PatentIn?version?3.1
<210>1
<211>8237
<212>DNA
<213>Equine?Infectious?Anemia?Virus
<400>1
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gtataattat?gtatgagtct?aataatgttc?aatacttgtt?atgcaatact?agtaatacta 6420
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ctatgtctta?tatcgctttg?acagaagtca?acaaattgga?tagtgtgcaa?aatcatactt 6780
ttgaagtaga?gaacaatact?atcaatagca?tggagttaat?agaagagcaa?attcatatat 6840
tatatgctat?ggttctccaa?acacatacag?atgttcaatt?gttaaaagaa?cgacaaaaga 6900
ttgaggaaac?atttaatttg?attggatgta?tagaaagatc?acatacattt?tgtcatactg 6960
gacatccctg?gaatgaatca?tggggtcagc?taaatgattc?tacacagtaa?gatgactggg 7020
taaataagat?ggaaaattta?aatcataata?tactaacaac?acttcatact?gctagaaata 7080
atctagaaca?atctatgata?acttttaata?cacctgacag?tatagcacaa?tttggtaaaa 7140
atatttggag?tcatattgca?aattggattc?ctggattagg?agcttccata?attaaatata 7200
tagtgttatt?attacttgta?tatgtgttac?taacctctgc?acctaagatc?ctcagaggcc 7260
tcttgacaac?gatgagtggt?gcaggatcct?ccgccagtcg?ctacctgagg?aaaagatacc 7320
atcacagacg?tgcatcgcga?ggagacatct?gggcccaggt?ccagtatcat?gcgtacctgg 7380
cagacgagac?tcgtggatca?gaggacaagt?ccaacatgcg?gaagctctcc?aggaacaatt 7440
ggaatggcga?atcagaggag?tacaacagac?ggcaaaagaa?ttggaaaagg?ttaataaaga 7500
gatctggaga?gaattacaat?acacacgaag?acaacatggg?gactatgggt?cgtttggtga 7560
ctaccgccgc?cgagaagaag?aacgttgggg?tgaatcctca?ccaagggtcc?ttaaacctgg 7620
agattcaaag?cgaaggagga?aacatctatg?actgttgcat?taaggctcaa?gaaggaactc 7680
ttgctattcc?ttgctgtggc?ttcccactat?ggctgctttg?gggacttata?atcatattag 7740
gacgcttgtt?gggatatggg?cttcggggaa?ttgcaaaaat?cataatgatt?ctggggaagg 7800
gactaaatgt?aataattaca?ggattaagaa?aactatgtga?ttatattggg?aaaatgctaa 7860
atccagctac?atctcatgta?acaatgcctc?aatatgatgt?ttagaaaaac?aaggggggaa 7920
ctgtgggatt?aatataagat?tcttataagt?gaatatgaaa?gttgctgatg?ctctcataac 7980
cttatgtaac?ccaaaagact?agctcatgtt?gctaggcaac?tgaactgtga?taaccttttg 8040
ttcctcatta?tagttccgct?tttgtgacgc?gttaagttcc?tgtttttaca?gtatataagt 8100
gcttgtattc?tgacatttgg?acactcagat?tctgcggtct?gagtcccttc?tctgctgggc 8160
taactctagc?cttggtaata?aatataattc?tctgctaagt?ccctgttctt?agtttgtctt 8220
gttttcaaga?tctaaca 8237
Claims (2)
1. a strain equine infectious anemia virus (Equine Infectious Anemia Virus) the donkey embryo skin cell adapted strain of virulent strain that causes a disease is characterized in that, its microbial preservation number is: CGMCC No.3231.
2. the purposes of the described donkey embryo skin cell adapted strain of claim 1 in making up tool equine infectious anemia virulence infections clone.
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| CN101696398B true CN101696398B (en) | 2012-05-23 |
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