CN101857873A - Hemangioma pathotype subgroup J avian leukosis virus gene and construction of infectious clone thereof - Google Patents
Hemangioma pathotype subgroup J avian leukosis virus gene and construction of infectious clone thereof Download PDFInfo
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- CN101857873A CN101857873A CN201010140195A CN201010140195A CN101857873A CN 101857873 A CN101857873 A CN 101857873A CN 201010140195 A CN201010140195 A CN 201010140195A CN 201010140195 A CN201010140195 A CN 201010140195A CN 101857873 A CN101857873 A CN 101857873A
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Abstract
The invention discloses a hemangioma pathotype subgroup J avian leukosis virus gene and the construction of infectious clone thereof. A full-length genome sequence of a subgroup J avian leukosis virus of the invention is shown as SEQ ID NO:1. Then, the hemangioma pathotype subgroup J avian leukosis virus gene shown as SEQ ID NO:1 is subjected to amplification by adopting a specific primer; and pBlueskript SK (I) is used as a vector and chick embryo fibroblast is used as a host cell to construct the infectious clone of the hemangioma pathotype subgroup J avian leukosis virus gene so as to lay the foundation for subsequent research on relation among pathogenic mechanism, a gene structure and functions, virus reproduction, genetic variation rules and the like of the hemangioma pathotype subgroup J avian leukosis virus ALV-J.
Description
Technical field
The present invention relates to biological technical field, be specifically related to the research of ALV-J virus, relate in particular to a kind of hemangioma pathotype subgroup J avian leukosis virus gene and Construction of Infectious Molecular Clone thereof.
Background technology
Avian leukosis virus (ALV) is exactly the RNA polymerase shortage check and correction mechanism of virus as a distinguishing feature of retrovirus, no matter whether immune pressure exists, all can cause heritable variation in the process of going down to posterity, therefore utilizing the infections clone of the single origin of reverse genetic technique construction is very important for research virus.
J subgroup avian leucosis virus (ALV-J) is eighties of last century emerging a kind of exogenous avian leukosis virus at the beginning of the nineties.Different ALV-J can cause multiple different clinical manifestation type, as the myelomatosis modification, and angiomatosis modification etc.But up to the present, also have only the Britain ALV-J prototype-strain HPRS-103 of myelomatosis modification and domestic NX0101 strain to set up infective molecule cloning, still do not have the report of angiomatosis modification ALV-J cloning virus.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of hemangioma pathotype subgroup J avian leukosis virus gene is provided.
Another object of the present invention is to provide the infections clone of above-mentioned hemangioma pathotype subgroup J avian leukosis virus gene.
Another object of the present invention is to provide above-mentioned Construction of Infectious Molecular Clone method.
Above-mentioned purpose of the present invention is achieved by following scheme:
Inventor's separation from the blue brown laying hen in angiomatosis modification avian leukosis sea obtains the new angiomatosis modification J subgroup avian leucosis virus strain (called after SCAU-HN06) of a strain, and obtained the full-length gene group sequence of this virus strain by molecule clone technology, by this gene order is analyzed, find that this genome sequence is different from existing J subgroup fowl leukemia virus gene, therefore the inventor has obtained a new hemangioma pathotype subgroup J avian leukosis virus gene, and its nucleotide sequence is shown in SEQ ID NO:1.
The inventor is on the basis of hemangioma pathotype subgroup J avian leukosis virus gene shown in the above-mentioned SEQ ID NO:1, make up the infections clone that obtains angiomatosis modification J subgroup avian leucosis virus again, this infections clone comprises hemangioma pathotype subgroup J avian leukosis virus gene shown in the SEQ ID NO:1.
The construction process of above-mentioned J subgroup avian leucosis infections cloneization virus rSCAU-HN06, be to adopt Auele Specific Primer that hemangioma pathotype subgroup J avian leukosis virus gene shown in the SEQ ID NO:1 is increased, and employing pBlueskript SK (-) is a carrier, intestinal bacteria are recipient bacterium, structure obtains comprising the plasmid of hemangioma pathotype subgroup J avian leukosis virus gene shown in the complete S EQ ID NO:1, with the chick embryo fibroblast is recipient cell, obtains required infections clone; Its nucleotide sequence of described Auele Specific Primer is shown in SEQ ID NO:2～7.
In the above-mentioned construction process, used Auele Specific Primer is that design gets the inventor according to hemangioma pathotype subgroup J avian leukosis virus gene shown in the SEQ IDNO:1.
In the above-mentioned construction process, adopt Auele Specific Primer that hemangioma pathotype subgroup J avian leukosis virus gene shown in the SEQ ID NO:1 is increased, hemangioma pathotype subgroup J avian leukosis virus gene shown in the SEQ ID NO:1 is divided into three sections to increase, and clone respectively in the T carrier, make up plasmid called after pHN-A-T, pHN-B-T and pHN-C-T; Described T carrier is selected except the carrier of multiple clone site with no EcoRI and KpnI restriction enzyme site in the external carrier sequence, as pMD18-T simple vector carrier, pMD20-T carrier, pSIMPLE-18EcoR V/BAP carrier or pGEM-7Zf (+/-) carrier or the like.
In the above-mentioned construction process, the contriver is according to EcoRI special in the hemangioma pathotype subgroup J avian leukosis virus gene shown in the SEQ ID NO:1 and KpnI single endonuclease digestion site, simultaneously in conjunction with special restriction enzyme site SalI and NotI on pBlueskript SK (-) carrier, the final EcoRI that selects, KpnI, these four restriction enzyme sites of SalI and NotI, with pHN-A-T, pHN-B-T and pHN-C-T enzyme are cut, connect the recombinant plasmid that the back obtains to contain hemangioma pathotype subgroup J avian leukosis virus gene shown in the SEQ ID NO:1, after this recombinant plasmid carried out sequence verification, with hemangioma pathotype subgroup J avian leukosis virus gene fragment subclone shown in the correct SEQ ID NO:1 in pBlueskript SK (-) carrier, and called after pSK-ALV-HN; Adopt pBlueskript SK (-) carrier mainly to be herein because this carrier is a low copy carrier, pBlueskript SK (-) carrier can also replace with other low copy carrier, as pBlueskriptSK (+) or pWSK29 or the like, all can realize the present invention.
In the above-mentioned construction process, the inventor imports the infections clone that obtains to contain hemangioma pathotype subgroup J avian leukosis virus gene shown in the complete S EQ ID NO:1 in the chick embryo fibroblast with the pSK-ALV-HN plasmid that obtains.
In the above-mentioned construction process, can be the bird cell that any one those skilled in the art used always as the chick embryo fibroblast of host cell, as DF-1 cell, chicken embryo kidney cell etc.
Compared with prior art, the present invention has following beneficial effect:
1. thereby the inventor obtains a new hemangioma pathotype subgroup J avian leukosis virus gene by Separation Research, for the research of ALV-J provides abundant content and research means more;
2. the present invention utilizes the reverse genetic manipulation technique construction to obtain the infections clone of angiomatosis modification type J subgroup fowl leukemia virus gene, this pathogenesis, gene structure and function relationship, virus for the ALV-J of follow-up study angiomatosis modification J subgroup avian leucosis virus duplicate and heritable variation rule etc. is laid a good foundation;
3. the present invention utilizes EcoRI special in the hemangioma pathotype subgroup J avian leukosis virus gene and KpnI single endonuclease digestion site, simultaneously in conjunction with special restriction enzyme site SalI and NotI on pBlueskript SK (-) carrier, structure contains the genomic total length plasmid of infections clone virus provirus, has guaranteed integrity and purity in the plasmid construction process;
4. the present invention adopts low copy plasmid pBlueskript SK (-) for carrier, and the constructed plasmid that contains complete hemangioma pathotype subgroup J avian leukosis virus gene can stably be duplicated in recipient bacterium;
5. the present invention adopts intestinal bacteria as the host bacterium, its transformation efficiency height;
6. to adopt chick embryo fibroblast be host cell in the present invention, easy being easy to get.
Description of drawings
Fig. 1 is embodiment 1 a medium vessels knurl pathotype J subgroup fowl leukemia virus gene amplification electrophorogram;
Wherein, M1 is Marker, and 1 is the A fragment, and 2 is the B fragment, and 3 is the C fragment;
Fig. 2 is the structure schema of plasmid pHN-(A+B)-T;
Fig. 3 is the structure schema of plasmid pMD18-ALV-HN;
Fig. 4 is the structure schema of plasmid pSK-ALV-HN;
Fig. 5 verifies fluorescence microscopy Electronic Speculum figure as a result for infectious virus.
Embodiment
Below in conjunction with specific embodiment the present invention is done description further, but specific embodiment is not done any qualification to the present invention.
The acquisition of embodiment 1 hemangioma pathotype subgroup J avian leukosis virus gene
Inventor's separation from the blue brown laying hen in angiomatosis modification avian leukosis sea obtains angiomatosis modification J subgroup avian leucosis virus SCAU-HN06 strain, and this virus strain is existing is preserved by Agricultural University Of South China's veterinary college.
The inventor to angiomatosis modification J subgroup avian leucosis virus SCAU-HN06 strain carry out molecular biology research, at first with reference to the disclosed correlated series of GenBank, after comparative analysis, utilize Primer Premier 5.0 professional primer-design software design degenerate primers, divide A, B, three sections of C to increase to its gene, concrete primer is as shown in table 1, and the amplification electrophoresis result as shown in Figure 1.
With three fragments of above-mentioned A, B and C cut respectively that glue reclaims, the T carrier is connected, those skilled in the art's such as transformed competence colibacillus cell and positive colony screening routine operation, the positive colony that finishing screen is chosen is delivered order-checking, three fragments that order-checking obtains are spliced by corresponding software again, the final hemangioma pathotype subgroup J avian leukosis virus gene that obtains, its nucleotide sequence is shown in SEQ ID NO:1.
Table 1 primer sequence
In the above-mentioned table 1, F represents upstream primer, and R represents downstream primer
The design of embodiment 2 Auele Specific Primers
The hemangioma pathotype subgroup J avian leukosis virus gene design Auele Specific Primer that present embodiment obtains according to embodiment 1, its nucleotide sequence are shown in SEQ ID NO:2～7, and it is synthetic to deliver biotech firm.
1. experiment material
Chick embryo fibroblast and ALV-J subgroup specific monoclonal antibody JE9 take from Yangzhou University's veterinary college; The FITC-sheep anti-mouse igg is a ROCLAND company product; The T4DNA ligase enzyme is a Promega company product; Prime STAR
TMHS DNA Polymerase, pMD18-Tsimple vector etc. are TaKaRa company product; PBlueskriptII (-) is a Generay company product; Restriction enzyme Sal I, Not I, Kpn I, EcoR I are Fermentas company product; ALV-Ag ELISA test kit is an IDEXX company product.
2. Construction of Infectious Molecular Clone method
The hemangioma pathotype subgroup J avian leukosis virus gene (its nucleotide sequence is shown in SEQ ID NO:1) that obtains with embodiment 1 is as template, utilize six special primers of embodiment 2 synthetic to divide three sections to increase to this gene order, and clone carrier respectively in pMD18-T simplevector, make up and obtain three plasmids (called after pHN-A-T, pHN-B-T and pHN-C-T respectively).
As shown in Figure 1, plasmid pHN-A-T, pHN-B-T respectively with SalI and the digestion of EcoRI double digestion, are reclaimed the purpose fragment, after connection and transforming, the single bacterium colony of picking is identified the clone, with recombinant plasmid called after pHN-(the A+B)-T that obtains.
As shown in Figure 2, pHN-(A+B)-T and pHN-C-T are used KpnI and the two enzymic digestions of NotI respectively, reclaim the purpose fragment, after connection and transforming, the single bacterium colony of picking is identified the clone, with the recombinant plasmid called after pMD18-ALV-HN that obtains at last, and sequence verification.
As shown in Figure 3, the fragment subclone is to pBlueskript SK (-) carrier, and called after pSK-ALV-HN for the hemangioma pathotype subgroup J avian leukosis virus gene that the sequence verification result is correct (its nucleotide sequence is shown in SEQ ID NO:1).
Get pSK-ALV-HN plasmid transfection chick embryo fibroblast, get supernatant liquor after 6 days, inoculate another blank chick embryo fibroblast, keep harvested cell after 6 days, continue 4 generations of blind passage, obtain required angiomatosis modification ALV-J infections clone virus.
The steps such as double digestion, connection and conversion that present embodiment is related all adopt the routine operation of this area.
Utilize IDEXX ALV-Ag ELISA test kit to transfection and feel right host cell culture supernatant and detect, transfection and infected cell culture supernatant ELISA detected result (shown in the table 2) are all positive.
Table 2ELISA detected result
| PSK-ALV-HN-Δ E transfection amount (μ g) | The transfection supernatant | Infect the |
| 2 | ??1.339 | ??1.070 |
| 0 | ??0.045 | ??0.044 |
With the cell that infects with methyl alcohol fixing after, through the PBS washing, drip specificity ALV-J monoclonal antibody JE9, hatch 30min for 37 ℃,, add the goat anti-mouse igg fluorescent-labeled antibody then with the PBS washing, hatch 30min for 37 ℃, and, under fluorescent microscope, observe at last through the PBS washing, cells infected can be observed tangible green fluorescence, and is typical tenuigenin dyeing, and negative control is reactionless, as shown in Figure 5, among Fig. 5, the chick embryo fibroblast of A for infecting, B is the barren chick embryo fibroblast.
The above results proves and successfully obtains the venereal infection poison strain.
A kind of hemangioma pathotype subgroup J avian leukosis virus gene and Construction of Infectious Molecular Clone sequence table thereof
SEQUENCE?LISTING
＜110〉Agricultural University Of South China
＜120〉a kind of hemangioma pathotype subgroup J avian leukosis virus gene and Construction of Infectious Molecular Clone thereof
<130>
<160>13
<170>PatentIn?version?3.5
<210>1
<211>7642
<212>DNA
＜213〉sea blue brown laying hen (HY-LINEVARIETYBROWN)
<400>1
tgtagtctta?tgcaataccc?ttatgtaacg?atgaaacagc?aatatgccct?ataaggagaa?????60
agaaggcact?gtacacgttg?attggtggaa?gtaaggtggt?acggtcatgg?tgtgatcgtg????120
ccttattagg?aaggcaacgg?acgggtcaga?catggattgg?atgatcccct?tagttccgcg????180
ttgcagagaa?attgtattta?agtgcctagc?ttgatacaat?aaacgccatt?ttacctccca????240
ccacattggt?gtgcgcctgg?gttgatggcc?ggaccgtcgg?ttccctgacg?attacgaaca????300
cctgcatgaa?gcggaaggct?tcatttggtg?accccgacgt?gatcgttagg?gaatagtggt????360
cggccaccgg?cggcgtggcg?atcctgccct?catccgtccc?gcttaacggg?gagcgggcga????420
tgaccctagt?agagggggct?gcggcttagg?agggcagaag?ctgagtggcg?tcggggggag????480
ctctactgca?gggagcccac?ataccctacc?gagcactcag?agggtcgttg?gaagacgagg????540
aggaagcccg?acgactgagt?ggtccgcacc?aggcgcggtt?ccggttgctc?tgcgtgattc????600
tggtcgccct?gtggatcaag?catggaagcc?gtcataaagg?tgatttcgtc?cgcgtgtaaa????660
acctattgcg?ggaaaacctc?tccttctaag?aaggaaatag?gggccatgtt?gtccctctta????720
caagatgaag?ggttgcttat?gtccccctca?gacttatatt?ccccggggtc?ctgggatccc????780
attaccgcgg?cgctctccca?gcgggctatg?gtgcttggga?aatcgggaga?gttaaaaacc????840
tggggattga?ttttgggggc?attgaaggcg?gctcgagagg?aacaggctac?atctgagcaa????900
gcaaagtttt?ggttcgggct?agggggaggg?ggggtctctc?ccccaggtcc?ggagtgtatc????960
gaaaaaccag?caacggagcg?gcggatcggc?aaaggggagg?aagcgagaga?gacaactgcg???1020
cagcgagatg?cgaagatggc?gccggaggaa?gcggccacac?ctaaaaccgt?tggcacatcc???1080
tgctatcatt?gcgggacagc?tattggctgt?aattgcgcca?cagcctcggc?tcctcctcct???1140
ccttatgtgg?ggagtggttt?gtatccttcc?ctggcggggg?tgggagagca?gcagggccag???1200
gggggtgaca?cacctcgggg?agcggaacag?ccaagggcgg?agccagggca?cgcgggcccg???1260
gcccctgggc?cggccctgac?tgactgggca?agggtaaggg?aggagcttgc?gagtactggt???1320
ccgcccgtgg?tggccatgcc?tgtagtgatt?aagacagagg?gacccgcctg?gacccctctg???1380
gagccaaaat?tgatcacaag?actggccgat?acggtcagga?ccaagggctt?acgatccccg???1440
atcactatgg?cagaagtgga?agcgcttatg?tcctccccgc?tgctgccgca?tgacgtcacg???1500
aatctaatga?gggttatttt?aggacctgcc?ccatacgcct?tatggatgga?cgcttgggga???1560
gtccaactac?agacggttat?agcggcagcc?actcgcgacc?cccgacaccc?agcgaatggt???1620
caagggcgag?gggaacggac?taatttggat?cgcttaaagg?gcctagctga?tgggatggtg???1680
ggcaacccgc?agggtcaggc?cgcattattg?agaccggggg?aattggttgc?tatcacagca???1740
tcggctctcc?aggcgtttag?agaggtcgct?cggctggcgg?aacctgcagg?cccatgggcg????1800
gatatcacac?agggaccatc?tgagtccttt?gttgatttcg?ctaatcgtct?tataaaggcg????1860
gttgaagggt?cagatcttcc?atcttccgcg?cgggctccgg?tgatcattga?ctgctttagg????1920
cagaagtcac?agccagatat?tcagcagctt?atacgggcag?caccctccac?gctgaccacc????1980
ccaggagaga?taatcaaata?tgtgctagac?aggcagaaga?ctgcccctct?tacggatcaa????2040
ggcatagccg?cggccatgtc?gtctgctatt?cagcccttag?ttatggcagt?agtcaataga????2100
gagagggatg?gacaaactgg?gtcgggtggt?cgtgcccgag?ggctctgcta?cacttgtgga????2160
tccccgggac?attatcaggc?gcagtgcccg?aaaaaacgaa?agtcagggaa?cagccgtgag????2220
cgatgtcagc?tgtgtgacgg?gatgggacac?aacgctaaac?aatgcagaag?gcgggatggc????2280
aaccagggcc?aacgcccagg?aagaggtctc?tcttcggggc?cgtggcccgg?ccctgaccag????2340
cctgccgtct?cgttagcgat?gacaatggaa?cataaagatc?gccccttggt?tagggtcatt????2400
ctgactaaca?ctgggagtca?tccagtcaaa?cagcgttcgg?tgtatatcac?cgcgctgttg????2460
gactctggag?cggacatcac?tattatttcg?gaggaggatt?ggcctactga?ttggccggtg????2520
gtggacaccg?cgaacccaca?gatccatggc?ataggagggg?gaattcccat?gcgaaaatct????2580
cgtgacatga?tagaggtggg?ggttattaac?cgagacgggt?ctttggagcg?acccctgctc????2640
ctcttccccg?cagtagctat?ggttagagga?agcatcctag?gaagggattg?tctgcagggc????2700
ctagggctcc?gcttgacaaa?tttataggga?gggccactgt?tcttactgct?gcgctacatc????2760
tggctattcc?gctcaaatgg?aagccagacc?acacgcccgt?gtggattgac?cagtggcccc????2820
ttcctgaggg?taaacttgta?gcgctaacgc?aattagtgga?aaaagaatta?cagttaggac????2880
atatagaacc?ttcacttagt?tgttggaaca?cacctgtctt?tgtgatccgg?aaggcttccg????2940
ggtcttatcg?cttattgcat?gacttgcgcg?ctgttaacgc?caagcttgtt?ccttttgggg????3000
ctgtccaaca?gggggcgcca?gttctctccg?cgctcccgcg?tggctggccc?ctgatggttc????3060
tagacctcaa?ggattgcttc?ttttctattc?ctcttgcgga?acaagatcgc?gaagcttttg????3120
catttacgct?cccctctgtg?aataaccagg?cccccgctcg?aagattccaa?tggaaggtct????3180
tgccccaagg?gatgacctgt?tctcccacta?tctgtcagtt?ggtagtgggt?caggtacttg????3240
agcccttgcg?actcaagcac?ccatctctgc?gcatgttgca?ttatatggat?gatcttttgc????3300
tagccgcctc?aagtcatgat?gggttggaag?tggcagggga?ggaggttatt?agtacattgg????3360
aaagagctgg?gttcaccatt?tcgcctgata?aggtccagag?ggagcccgga?gtacaatatc????3420
ttgggtacaa?gttaggcagt?acgtatgtag?cacccgtagg?cttggtagca?gaacccagga????3480
tagccacctt?gtgggatgtt?caaaagctgg?tggggtcact?tcagtggctt?cgcccagcgt????3540
taggaatccc?gccacgactg?atgggcccct?tttatgagca?gttacgaggg?tcagatccta????3600
acgaggcgag?ggagtggaat?ctagacatga?aaatggcctg?gagagagatc?gtacagctta????3660
gcaccactgc?tgccttggaa?cgatgggacc?ctgccctgcc?tctggaagga?gcggtcgcta????3720
gatgtgaaca?gggggctata?ggggtcctgg?gacagggact?gtccacacac?ccaaggccat????3780
gtttgtggtt?attctccacc?caacccacca?aggcgtttac?tgcttggtta?gaagtgctca????3840
cccttttgat?tactaagcta?cgtgcttcgg?cagtgcgaac?ctttggcaag?gaggttgaca????3900
tcctcctgtt?gcctgcatgc?tttcgggagg?accttccgct?cccggagggg?atcctgttag????3960
cccttagggg?gtttgcagga?aaaatcagga?gtagtgacac?gccatctatt?tttgacattg????4020
cgcgtccact?gcatgtttct?ctgaaagtga?gggttaccga?ccaccctgta?ccgggaccca????4080
ctgtctttac?cgacgcctcc?tcaagcaccc?ataagggggt?ggtagtctgg?agggagggcc????4140
caaggtggga?gataaaagaa?atagctgatt?tgggggcaag?cgtacaacaa?ctggaagcac????4200
gcgctgtggc?catggcgctt?ctgctgtggc?cgacaacgcc?cactaatgtg?gtgacggact????4260
ctgcgttcgt?tgcgaaaatg?ttacttaaga?tgggacagga?gggagtcccg?tccacggcgg????4320
cggcctttat?tttagaggat?gcgttaagcc?aaaggtcagc?catggccgcc?gttctccacg????4380
tgcggagtca?ttctgaagtg?ccagggtttt?tcacagaagg?aaatgatgtg?gcagatagcc????4440
aagccacctt?tcaagcgtat?cccttgagag?aggctaaaga?tcttcatacc?gctctccata????4500
ttggaccccg?cgcgctatcc?aaagcgtgta?atatatctat?gcagcaggct?agggaggttg????4560
ttcagacctg?cccgcattgt?aattcagccc?ctgcgttgga?ggccggggta?aaccctaggg????4620
gtttgggacc?cctacagata?tggcagacag?actttacgct?tgagcctaga?atggcccccc????4680
gttcctggct?tgctgttact?gtggataccg?cctcatcagc?gatagtcgta?actcagcatg????4740
gccgtgtcac?atcggttgct?gcacaacatc?attgggccac?ggctatcgcc?gttctgggaa????4800
ggccaaaggc?cataaagaca?gataacgggt?cctgcttcac?gtctaaatcc?acgcgagagt????4860
ggcttgcgag?atgggggata?gcacacacca?ccgggattcc?gggtaattcc?cagggtcaag????4920
ctatggtaga?gcgggccaac?cggctcctga?aggataagat?ccgcgtgctt?gcggaggggg????4980
acggctttat?gaaaagaatc?cccaccggca?aacaggggga?actactagcc?aaggctatgt????5040
atgccctcaa?ccactttgag?cgtggtgaaa?acacaaaaac?accgatacaa?aaacactgga????5100
gacctaccgt?tcttacagaa?ggacccccgg?ttaaaatacg?aatagagaca?ggggagtggg????5160
aaaaaggatg?gaacgtgctg?gtctggggac?gaggttatgc?cgctgtgaaa?aacagggaca????5220
ctgataaggt?tatttgggta?ccctctcgaa?aagttaaacc?ggacatcacc?caaaaggatg????5280
aggtgactaa?gaaagatgag?gcgagccctc?tctttgcagg?catttctgac?tgggcaccct????5340
ggaaaggtga?gcaagaagga?ctctaagaag?aagccgccag?caacaagcaa?gaaagacccg????5400
gagaagacac?ccttgctgcc?gtcgagaggt?tatttcttct?ttcaaatgat?acttgtgtgc????5460
gtggttatta?tttctgttgt?cccaggggtg?aagggagttc?atctgttgca?acaaccagga????5520
aacgtgtggg?tcacctgggc?aaataagacg?ggccaaacag?atttttgcct?tagtctgcag????5580
tcagcgacct?cgccattccg?cacctgcttg?ataggcattc?cacagtatcc?tctgagcacc????5640
tttgagggat?atgtcactaa?tgttactgcc?tgccaaaacg?acaccgattt?agccagccaa????5700
acagcatgct?tgatacagac?tctaaatacg?accctccctt?gggaccccca?agaattagat????5760
attttagggt?cccagatgat?caagaacgga?acaacacgta?cgtgtgttac?ctttggttcg????5820
gtgtgctata?aagagaacaa?tggcagtaga?gtctgccaca?tttttgatgg?gaactttaat????5880
gggactggtg?gggcggaagc?agaactgcgt?gacttcataa?caaaatggaa?aggtgatgac????5940
catcttataa?ggccctatgt?caaccaatca?tggacgatgg?taagtccaat?aaacacagag????6000
agcttttcaa?taagtcgtag?atattgtgga?ttcaccagta?acgagactcg?ttactataga????6060
ggggaccttt?ctaattggtg?taattcaaaa?gggggtgaat?ggtcagcggg?gtacagcaac????6120
gggacaaaat?gttccagcaa?cacgacaggt?tgtggtggta?attgcacgac?ggaatggaat????6180
tattatgcat?atgggtttac?ctttgggaaa?cagccagagg?tgttgtggaa?caatgggact????6240
gctaaggcac?tccctccagg?tattttcttg?atttgtgggg?acagagcttg?gcaaggcatc????6300
ccgagtaatg?ccttgggagg?gccctgttat?ctaggacaat?tgactatgct?ctctcctaac????6360
tttaccacct?ggatgacgta?tgggccgaac?attacgggtc?accgccgtag?caggcgctcg????6420
gtgagtcatc?tctcgtctga?ctgcagtgac?gaggtacagc?tatggagtgc?gacagcccgg????6480
atatttgctt?ctttctttgc?tcctggtgta?gctgcagcac?aggccttaag?ggaggttgaa????6540
cgcttggcat?gttggtcggt?taagcaagcg?aatttaacat?cattaatatt?gaatgcgatg????6600
ctggaggaca?tgaacagcat?ccggcacgcg?gtgttgcaga?atcgagcagc?catcgatttc????6660
ttactcctgg?cgcagggaca?cgggtgtcaa?gacgtagaag?ggatgtgttg?cttcaatctc????6720
agcgatcaca?gtgagtccat?tcacaaggcg?ctccaagcca?tgaaggaaca?tacagagaag????6780
atacgggtgg?aagacgatcc?cataggggat?tggtttacgc?gcacgtttgg?taattttgga????6840
gggtggctcg?cgaaaggtgt?taagacgcta?ctgtttgcat?tgcttgtcat?agtctgtcta????6900
ttagctatca?ttccatgtat?aatcaagtgc?tttcaggatt?gtctatcgag?aacaatgtat????6960
cagtttatgg?atgaacgcat?aagatatcat?agaattaggg?agcagctgta?ggttccgaac????7020
gcgaagtgac?gggaggctgg?taggggtatg?aaacttgcga?atcgggctgt?aacggggcaa????7080
ggcttgactg?aggggactgt?agcatgtata?ggcgctgagc?ggggcttcgg?ttgtacgcgg????7140
ataggaatcc?cctcaggaca?attctgcttg?aaatgtgatg?acaccttcct?gctttgccct????7200
tagactattc?aagttgcctc?tgtggattag?gactggagac?agctcggatg?gtctgatggc????7260
caaatagagc?aagctagata?ggtagctgcg?aaatacgctt?ttgcataggg?agggggaaat????7320
gtagtcttat?gcaataccct?tatgtaacga?tgaaacagca?atatgcccta?taaggagaaa????7380
gaaggcactg?tacacgttga?ttggtggaag?taaggtggta?cggtcatggt?gtgatcgtgc????7440
cttattagga?aggcgacgga?cgggtcagac?atggattgga?tgatcccctt?agttccgcgt????7500
tgcagagaaa?ttgtatttaa?gtgcctagct?tgatacaata?aacgccattt?tacctcccac????7560
cacattggtg?tgcgcctggg?ttgatggccg?gaccgtcgat?tccctgacga?ttacgaacac????7620
ctgcatgaag?cggaaggctt?ca?????????????????????????????????????????????7642
<210>2
<211>33
<212>DNA
＜213〉artificial sequence
<400>2
gcgcggtcga?ctgtagtctt?atgcaatacc?ctt?????????????????????????????????33
<210>3
<211>35
<212>DNA
＜213〉artificial sequence
<400>3
gttaagcggc?cgcacctcta?tcatgtcacg?agatt???????????????????????????????35
<210>4
<211>33
<212>DNA
＜213〉artificial sequence
<400>4
gagcggtcga?cactattatt?tcggaggagg?att?????????????????????????????????33
<210>5
<211>35
<212>DNA
＜213〉artificial sequence
<400>5
gagaggcggc?cgctcatctt?tcttagtcac?ctcat???????????????????????35
<210>6
<211>24
<212>DNA
＜213〉artificial sequence
<400>6
ctggggacga?ggttatgccg?ctgt???????????????????????????????????24
<210>7
<211>37
<212>DNA
＜213〉artificial sequence
<400>7
tatatgcggc?cgctgaagcc?ttccgcttca?tgcaggt?????????????????????37
<210>8
<211>22
<212>DNA
＜213〉artificial sequence
<400>8
tgtagtctta?trcaatacyc?tt?????????????????????????????????????22
<210>9
<211>20
<212>DNA
＜213〉artificial sequence
<400>9
ctgttcacat?ctagcgaccg????????????????????????????????????????20
<210>10
<211>20
<212>DNA
＜213〉artificial sequence
<400>10
cgtatgtagc?acccgtaggt????????????????????????????????????????20
<210>11
<211>24
<212>DNA
＜213〉artificial sequence
<400>11
ggtaataacc?acgcacacaa?gtat???????????????????????????????????24
<210>12
<211>22
<212>DNA
＜213〉artificial sequence
<400>12
agcgatagtc?gtaactcagc?at?????????????????????????????????????22
<210>13
<211>30
<212>DNA
＜213〉artificial sequence
<400>13
gaattctgaa?gccttccgct?tcatkcaggt?????????????????????????????30
Claims (5)
1. hemangioma pathotype subgroup J avian leukosis virus gene, its nucleotide sequence is shown in SEQ ID NO:1.
2. an angiomatosis modification J is subgroup avian leucosis virus infective cloned, it is characterized in that this infections clone contains the described hemangioma pathotype subgroup J avian leukosis virus gene of claim 1.
3. the subgroup avian leucosis virus infective cloned construction process of the described angiomatosis modification of claim 2 J, it is characterized in that this method is to adopt Auele Specific Primer that hemangioma pathotype subgroup J avian leukosis virus gene shown in the SEQ ID NO:1 is increased, and employing pBlueskript SK (-) is a carrier, chick embryo fibroblast is a host cell, structure obtains required infections clone, and its nucleotide sequence of described Auele Specific Primer is shown in SEQ ID NO:2～7.
4. according to the described construction process of claim 3, it is characterized in that described construction process specifically comprises the steps:
(1) adopt nucleotide sequence Auele Specific Primer shown in SEQ ID NO:2～7 to become three sections to increase the provirus full-length gene component of J subgroup avian leucosis virus SCAU-HN06 strain, and clone respectively in the T carrier, make up plasmid called after pHN-A-T, pHN-B-T and pHN-C-T;
(2) adopt EcoRI, KpnI, SalI and these four restriction enzyme sites of NotI, with pHN-A-T, pHN-B-T and pHN-C-T through double digestion, be connected and conversion after obtain to contain the recombinant plasmid of J subgroup fowl leukemia virus gene shown in the SEQ ID NO:1;
(3) step (2) gained recombinant plasmid and pBlueskript SK (-) carrier are carried out SalI and NotI double digestion respectively, connect the conversion back and obtain recombinant plasmid pSK-ALV-HN;
(4) will prepare required infections clone in the recombinant plasmid pSK-ALV-HN importing chick embryo fibroblast.
5. according to the described construction process of claim 3, it is characterized in that in the described step (2), earlier plasmid called after pHN-A-T and pHN-B-T are carried out EcoRI and SalI double digestion respectively, be connected and conversion after obtain plasmid pHN-(A+B)-T, then plasmid pHN-(A+B)-T and pHN-C-T are carried out KpnI and NotI double digestion respectively, be connected and conversion after obtain containing the recombinant plasmid of J subgroup fowl leukemia virus gene shown in the SEQID NO:1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110384800A (en) * | 2019-07-19 | 2019-10-29 | 广东省实验动物监测所 | Application of the LncRNA XLOC_075168 in the drug that preparation promotes angiogenesis |
| CN111575288A (en) * | 2020-05-16 | 2020-08-25 | 扬州大学 | ch-MYC-AS1 for inhibiting chicken c-MYC gene expression and J subgroup avian leukosis virus proliferation and application thereof |
-
2010
- 2010-03-30 CN CN201010140195A patent/CN101857873A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110384800A (en) * | 2019-07-19 | 2019-10-29 | 广东省实验动物监测所 | Application of the LncRNA XLOC_075168 in the drug that preparation promotes angiogenesis |
| CN110384800B (en) * | 2019-07-19 | 2021-06-08 | 广东省实验动物监测所 | Application of LncRNA XLOC _075168 in preparation of medicine for promoting angiogenesis |
| CN111575288A (en) * | 2020-05-16 | 2020-08-25 | 扬州大学 | ch-MYC-AS1 for inhibiting chicken c-MYC gene expression and J subgroup avian leukosis virus proliferation and application thereof |
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