CN1255550A - Clone of antisense gene resisting heteroplastic transplantation and in-situ synthetase - Google Patents
Clone of antisense gene resisting heteroplastic transplantation and in-situ synthetase Download PDFInfo
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Abstract
An antisense gene clone with anti-heterorepulsion action and the method for transforming and screening the antisense gene clone in the cells of mammal are disclosed. The DNA of Meishan pig is extracted. The reverse joining carrier of heteroantigen site synthetase gene fragment is amplified, cloned and coded by polymerase chain reaction to obtain transcribed antisense gene plasmid p CN84, which can be used to animal cells fertilized ovum or organa to obtain stably transformed cell, or transgenic organa or animal, or the transformed cell is used for nuclear ovum transplant to clone animal. Obtained cells or organa are short of main heterotransplant antigen, can avoid heteroimmune recognition, binding and challenge, and effectively resist acute repulsion reaction.
Description
The present invention relates to a kind of new xenotransplantation antigen site synthetic enzyme antisense gene technique field, specifically, be the big segmental clone of antisense gene of exon of pig heterologous antigen site, a kind of plum mountain synthetic enzyme (α (1,3) galactoside base translocase) and the method for the expression in mammalian cell thereof.
Human organ or cell debilitating disease in whole latter stage can obtain curing by organ or Transplanted cells.Be used for transplanted organ or cell and must reduce its immunogenicity and immunoreactivity as far as possible.
The eighties, though the natural xenogenesis organ without the biotechnology transformation has been started the generation organ transplantation beginning, at first successful Application and clinical heart transplantation in the world.But because the generation of the unusual violent rejection that is difficult to accept, in the transplantation medicine of clinical long-term surviving, xenotransplant is at present replaced by allotransplantation eventually.Yet the human organ is difficult to obtain, and nearly edge heterogenous animal organ is difficult to obtain more.The organ or the cell that obtain anti-hyperacute rejection with genetic engineering modified easily numerous edge heterogenous animal far away of easily supporting have broad prospect of application clinically.
Since 1994, existing scholar obtains can be used for the recombinant human Complement Regulatory Protein DAF gene transgenic animal of medical transplanting, it is said and to obtain anti-row's effect (G.A.Langford, et al:Production of pigs transgenic for human decay accelerating factor.Transplantation Proceedings 1994,26,3,1400-1401).But, have not yet to see the report that clinical application obtains the achievement in research of promising result and deliver.From Biological Principles, heterologous antigen is not eliminated in the organ of this biotechnology transgenic animal.This mode is only to the anticomplement immunological effect, and many other immunological rejection effects beyond complement kills and wounds can't be eliminated.The xenotransplantation antigen that xenotransplant descendant's immunoglobulin (Ig) and donor organ cell surface are constantly expressed discern and react after started the super acute reaction system in the many aspects of body, for example system etc. is engulfed in blood coagulation system, kinin system, inflammatory mediator system and immunity, cause violent hyperacute rejection to take place, make graft failure.Heterologous antigen is not eliminated, and takes precautions against incomplete to immunity identification and immune response unavoidably.
Transplant of immunity identification and the immune response of back acceptor for eliminating, can effectively intervene heterologous antigen with biological engineering method and express the xenogenesis organ.The invention provides a kind of DNA product and method and can effectively reach this purpose.
Find between Chinese-Mei mountain pig semi-lactosi on the xenogenesis blood group epi-position be the anti-pig IgM of people institute at major antigen decision family molecule (open good equality: xenogenesis is repelled immunoreactive restraining effect with flow cytometer observation semi-lactosi.1996 the 6th volumes of laser medicine, 2 phases: 55-58.) the basis on, the contriver has invented the expression that a kind of method and product are used to eliminate the main transplantation antigen of donor organ, thereby obtains more effective anti-hyperacute rejection medical transplanting animal and cell.
Heterologous antigen site synthetic enzyme (α (1,3) the galactoside base translocase) gene clone of genetically engineered animal at present and mainly follow following method at mammalian cell or expression in vivo:
1, segmental the obtaining of coding heterologous antigen site synthetase dna: mainly contain two kinds of methods:
Planting is to extract hemocyte RNA, builds base through synthetic cDNA library of reverse transcription or extraction DNA
Because of the group library, with special derivated screening and cloning; Another kind is through PCR with special primer
Obtain behind this gene connection carrier again from RNA or DNA cloning.2, structure inverted defined gene: commonly used variously can have the constructive expression to do usefulness in mammalian cell
Promotor and enhanser oppositely place the goal gene coding region segmental 3 ' end, the choosing
The poly adenosine signal of transcribing nucleic acid with effective protection places 5 ' of goal gene to hold.3, the structure of transgenosis cell strain: recombinant plasmid with calcium phosphate, liposome, retrovirus,
Method such as electroporation or microinjection imports mammalian cell and (comprises in zygote or the body
Or in the isolated organ).
Plasmid of the present invention can be used for the antigenic expression of main culture transferring that multiple mammiferous cells such as pig, sheep, ox, monkey, mouse are used to eliminate donorcells and organ.Heterologous antigen is expressed defective after cell or zygote or the organ transgenosis.
The present invention also provides one to align reverse primer, and its dna sequence dna is: with the forward (aL84) and the reverse primer (aL82) of (agTL) upstream and downstream coupling: aL84:5 ' cgg gat ccg atacat tga gca tta ctt gga gga gtt ct; AL82:5 ' GCGTCGACGGCACAAT-TTAAAGTCAGATGTTATTTCTAACC3.
The present invention also provides a kind of heterologous antigen site synthase gene fragment dna molecular, 707bp, the 259th bit base is G, document (the Sandrin-GS that has delivered, McKenzie-IF:Gal alpha (1,3) Gal, the major xenoantigen (s) recognized in pig by human natural antibodies.Immuno Rcv1994 141:169-90.) is T;
The present invention also provides a kind of new peptide fragment and application thereof, comprises as immunogen generation monoclonal antibody or antigen making detection reagent or obtaining the xenotransplantation immunological tolerance as the former transplant recipient that is used for of immunological tolerance.
The present invention also provides a kind of new inverted defined gene, and its total order is classified 2550 pairs of based compositions as;
The invention provides a kind of new inverted defined gene recombinant plasmid, its total order is classified 6375 pairs of based compositions as.This plasmid is used to transform strain of heterologous antigen deficient cells or the transgenic animal that the zygote transformed mammalian cell obtains stable conversion.Or as being used to carry out the nuclear transplantation of cloned animal, and then produce the main heterologous antigen of deduction and exemption is expressed in donorcells or the organ effective anti-hyperacute rejection medical transplanting animal and cell for nuclear.Plasmid of the present invention goes down to posterity stable, suppresses the efficient height.
The expression that the present invention also provides a kind of method and product to be used to eliminate the main transplantation antigen of donor organ, thus more effective anti-hyperacute rejection medical transplanting animal and cell produced.These can help to solve the consistency of clinical xenotransplantation.Be used for the treatment of organ damage or whole last depleted disease.
Use the plasmid that contains antisense heterologous antigen site synthetic enzyme that can in mammalian cell, express and screen among the present invention, transformed mammalian cell or zygote or as being used to carry out the nuclear transplantation of cloned animal for nuclear can produce in donorcells or the organ effective anti-hyperacute rejection medical transplanting animal and cell that the main heterologous antigen of deduction and exemption is expressed.
Implementing technical essential of the present invention is:
1, design and synthetic primer, cross the peripheral blood mononuclear cell DNA of plum mountain pig in from fresh blood, extracting, with above-mentioned primer, DNA, rTth DNA synthetic enzyme through the polymerase chain bill should (PCR) the big fragment of exon (α gal T) of heterologous antigen site synthetic enzyme (α (1,3) galactoside base translocase) of synthetic and amplification plum mountain pig.Enzyme is cut the PCR product, is cloned into the plasmid vector pGEM7Z that cut with enzyme, and cohesive end connects back transformed into escherichia coli JM109 competence bacteria; Through screening and the restriction enzyme evaluation, obtaining recombinant molecule be: pZagTL84.With Sp6 primer guiding sequencing analysis;
2, make up antisense agTL gene pCN84: oppositely construct SV40 polyA signal sequence and Xin Meisu selection gene (expressing the neomycin phosphotransferase gene of G418 resistance) at agTL gene fragment upstream flank, oppositely construct CMV promoter sequence and enhancer sequence and artificial intron sequences at the downstream flank, identify that with restriction endonuclease direction of insertion is correct, pCN84 by name.Wherein antisense agTL gene is correct with T7 primer guiding sequencing analysis.
3, pig inoblast transgenosis and select the clone: with the calcium phosphate precipitation method linear antisense agTL gene of transduceing.Select the positive colony cell with G418.Extract the RNA of positive colony, carry out the efficient of transcribing of RT-PCR reaction detection inverted defined gene with T7 primer and aL82; Heterologous antigen is expressed defective after measuring the pig cell transgenosis.Transgenic cell does not compare group.FCM measure genetically modified pig inoblast surface bonding human serum IgM test, measure the complement-mediated cytotoxic reaction, measure people's hemagglutination reaction and prove that all this plasmid and method can be used for eliminating the expression of the main transplantation antigen of donor organ.
4, sheep, ox, mouse fibroblast cell transgenosis and select the clone: with the 3rd, the calcium phosphate precipitation method linear antisense agTL gene of transduceing.Select the positive colony cell with G418.Heterologous antigen is expressed defective after measuring sheep, ox, mouse cell transgenosis.This homology inverted defined gene of xenogeneic can suppress other this genetic expression of Mammals homology; Can suppress xenotransplantation hyperacute rejection antigen presentation after the big fragment transgenosis of antisense α gal-T.The anti-hyperacute rejection effect of antisense xenotransplantation antigen gene.This provides a good model for heterogenous cell or organ transplantation.
The invention will be further described below by embodiment:
Embodiment one:
The amplification and the order-checking of plum mountain pig α (1,3) galactosyltransferase (α gal T) part encoding sequence: dna fragmentation clone: (1) is synthetic with Beckman Oligo1000M DNA Syntheesizer and the big fragment of α gal T exon (agTL) upstream and downstream mates forward (aL84) and reverse primer (aL82): aL84:84#:5 ' cg-g gat ccg ata cat tga gca tta ctt ggagga gtt ct 38er; AL82:82#:5 '-GC GTCGAC GGC ACA ATT TAA AGT CAGATG TTA TTT CTAACC3 ' 41er; (2) with RT-PCR method amplification agTL: carry out the PCR reaction with above-mentioned primer: 5ml plum mountain pig anticoagulation lymphocyte classical way [7] dissolves with 0.5mlTE after extracting DNA.Get 2 μ l and add the 100pM primer, and add 2u rTth thermal synthesis enzyme, on PE company 2400 type PCR instrument, react.Reaction parameter: 94 ℃, sex change in 3 minutes; Thermal cycling: 94 ℃, 20 seconds, 56 ℃ 20 seconds, 72 ℃ 50 seconds totally 28 times the circulation.(3) the PCR product cloning to JM109:PCR product and plasmid vector pGEM7Z with BamHI with after SalI is cut into cohesive end by document reclaim, be connected, the conversion limitations restriction endonuclease identifies [8].It is by name to obtain the clone: pZagTL84.(4) to inserting fragment agTL sequencing analysis: with the guiding of Sp6 primer, press terminal cessation method Kit (Perkin Elmer Co) the specification sheets operation of the two deoxidations of fluorescent mark, last AppliedBiosystems 373 DNA Sequencer Sttretch automatic sequencers order-checking.Sequence sees Table 1.
1.pZagTL84agTLDNAgatacattga gcattacttg gaggagttct taatatctgc aaatacatacttcatggttg gccacaaagt catctttaac atcatggtgg atgatatctccaggatgcct ttgatagagc tgggtcctct gcgttccttt aaagtgtttgagatcaagtc cgagaagagg tggcaagaca tcagcagtat gcgcatgaagaccatcgggg agcacatcct ggcccacatc cagcacgagg tggacttcctcttctgcatg gacgtggatc aggtcttcca aaacaacttt ggggtggagaccctgggcca gtcggtggct cagctacagg cctggtggta caaggcacatcctgacgagt tcacctacga gaggcggaag gagtccgcag cctacattccgtttggccag ggggattttt attaccacgc agccattttt gggggaacacccactcaggt tctaaacatc actcaggagt gcttcaaggg aatcctccaggacaaggaaa atgacataga agccgagtgg catgatgaaa gccatctaaacaagtatttc cttctcaaca aacccactaa aatcttatcc ccagaatactgctgggatta tcatataggc atgtctgtgg atattaggat tgtcaagatagcttggcaga aaaaagagta taatttggtt agaaataaca tctgactttaaattgtgcc
709 pairs of bases are come self-template.683-685 is to being stop code.
According to the dna sequence dna of measuring, 230 aminoacid sequences of mountain pig heterologous antigen site synthase gene (α (1,3) galactoside base translocase (α [1,3] galactosyl-transferase)) carboxyl terminal after the rainy season of deriving see Table 2..
Pig heterologous antigen site, table 2. plum mountain synthase gene
(α (1,3) galactoside base translocase
(α[1，3]galactosyltransferase))
230 aminoacid sequences of carboxyl terminal: YIEHYLEEFL ISANTYFMVG HKVIFNIMVD DISRMPLIEL GPLRSFKVFEIKSEKRWQDI SSMRMKTIGE HILAHIQHEV DFLFCMDVDQ VFQNNFGVETLGQSVAQLQA WWYKAHPDEF TYERRKESAA YIPFGQGDFY YMAAIGGTTPTQVLNITQEC FYGILQDYEN DIEAEWHDES HLNKYFLLNK PTKILSPEYCWDYHIGMSVD IRIVKIAWGK KEYNLVRNNI
4, the structure of antisense agTL gene: (1) cuts the pZagTL84 plasmid with BamHI, and Klenow fragment enzyme is mended flat 3 ' end.Cut with Xbal then.The LMP agarose reclaims 721b and inserts fragment.Connect again and enter between the Nhel and EcoRV site of pTargetT carrier.Transform JM109 bacterium and screening and cloning, identify direction of insertion with restriction endonuclease.Obtain plasmid pCN84 by name; Bacterial classification is called JM109+pCN84; (2) antisense agTL gene sequencing is analyzed among the cloned plasmids pCN84: with the guiding of T7 primer.Check order with automatic sequencer as stated above.Its sequence sees Table 3..Plasmid pCN84DNA complete sequence sees Table 4.
Antisense coding agTL gene among the table 3. plasmid pCN84 inserts the fragment dna sequence dna
gctagactcg?acggcacaat?ttaaagtcag?atgttatttc?taaccaaatt
atactctttt?ttctgccaag?ctatcttgac?aatcctaata?tccacagaca
tgcctatatg?ataatcccag?cagtattctg?gggataagat?tttagtgggt
ttgttgagaa?ggaaatactt?gtttagatgg?ctttcatcat?gccactcggc
ttctatgtca?ttttccttgt?cctggaggat?tcccttgaag?cactcctgag
tgatgtttag?aacctgagtg?ggtgttcccc?caaaaatggc?tgcgtggtaa
taaaaatccc?cctggccaaa?cggaatgtag?gctgcggact?ccttccgcct
ctcgtaggtg?aactcgtcag?gatgtgcctt?gtaccaccag?gcctgtagct
gagccaccga?ctggcccagg?gtctccaccc?caaagttgtt?ttggaagacc
tgatccacgt?ccatgcagaa?gaggaagtcc?acctcgtgct?ggatgtgggc
caggatgtgc?tccccgatgg?tcttcatgcg?catcatgctg?atgtcttgcc
acctcttctc?ggacttgatc?tcaaacactt?taaaggaacg?cagaggaccc
agctctatca?aaggcatcct?ggagatatca?tccaccatga?tgtaaaagat
gactttgtgg?ccaaccatga?agtatgtatt?tgcagatatt?aagaactcct
ccaagtaatg?ctcaatgtat?cggatcatct?ttcccggggg?taccgtcgac
tgcggccgcg?aattccaagc?ttga
774 pairs of bases.Table 4. contains the plasmid pCN84 full DNA sequence of antisense heterologous antigen site synthetic enzyme: totally 6375 base pairs.tcaatattgg?ccattagcca?tattattcat?tggttatata?gcataaatca
100
*atattggcta?ttggccattg?catacgttgt?atctatatca?taatatgtacatttatattg?gctcatgtcc?aatatgaccg?ccatgttggc?attgattatt
200gactagttat?taatagtaat?caattacggg?gtcattagtt?catagcccatatatggaggt?ccgcgttaca?taacttcgg?taaatggccc?gcctggctga
300ccgcccaacg?acccccgccc?attgacgtca?ataatgacgt?atgttcccatagtaacgcca?atagggactt?tccattgacg?tcaatgggtg?gagtatttac
400ggtaaactgc?ccacttggca?gtacatcaag?tgtatcatat?gccaagtccgccccctattg?acgtcaatga?cggtaaatgg?cccgcctggc?attatgccca
500gtacatgacc?ttacgggact?ttcctacttg?gcagtacatc?tacgtattagtcatcgctat?taccatggtg?atgcggtttt?ggcagtacac?caatgggcgt
600ggatagcggt?ttgactcacg?gggatttcca?agtctccacc?ccattgacgtcaatgggagt?ttgttttggc?accaaaatca?acgggacttt?ccaaaatgtc
700gtaacaactg?cgatcgcccg?ccccgttgac?gcaaatgggc?ggtaggcgtgtacggtggga?ggtctatata?agcagagctc?gtttagtgaa?ccgtcagatc
800actagaagct?ttattgcggt?agtttatcac?agttaaattg?ctaacgcagtcagtgcttct?gacacaacag?tctcgaactt?aagctgcagt?gactctctta
900aggtagcctt?gcagaagttg?gtcgtgaggc?actgggcagg?taagtatcaaggttacaaga?caggtttaag?gagaccaata?gaaactgggc?ttgtcgagac
1000agagaagact?cttgcgtttc?tgataggcac?ctattggtct?tactgacatccactttgcct?ttctctccac?aggtgtccac?tcccagttca?attacagctc
1100ttaaaaattg?gatctccatt?cgccattcag?gctgcgcaac?tgctgggaaggacgatcaga?gcgggcctct?tcgctattac?gccagctggc?gaaagggacg
1200tggcaagcaa?ggcgattaag?ttgagttacg?ccaggatttt?cccagtcacgacgttgtaaa?acgacggcca?gagaattata?atacgactca?ctatagggcg
1300aattcggatc?cttgctagac?tcgacggcac?aatttaaagt?cagatgttatttctaaccaa?attatactct?tttttctgcc?aagctatctt?gacaatccta
1400atatccacag?acatgcctat?atgataatcc?cagcagtatt?ctggggataagattttagtg?ggtttgttga?gaaggaaata?cttgtttaga?tggctttcat
1500catgccactc?ggcttctatg?tcattttcct?tgtcctggag?gattcccttgaagcactcct?gagtgatgtt?tagaacctga?gtgggtgttc?ccccaaaaat
1600ggctgcgtgg?taataaaaat?ccccctggcc?aaacggaatg?taggctgcggactccttccg?cctctcgtag?gtgaactcgt?caggatgtgc?cttgtaccac
1700caggcctgta?gctgagccac?cgactggccc?agggtctcca?ccccaaagttgttttggaag?acctgatcca?cgtccatgca?gaagaggaag?tccacctcgt
1800gctggatgtg?ggccaggatg?tgctccccga?tggtcttcat?gcgcatcatgctgatgtctt?gccacctctt?ctcggacttg?atctcaaaca?ctttaaagga
1900acgcagagga?cccagctcta?tcaaaggcat?cctggagata?tcatccaccatgatgtaaaa?gatgactttg?tggccaacca?tgaagtatgt?atttgcagat
2000attaagaact?cctccaagta?atgctcaatg?tatcggatca?tctttcccgggggtaccgtc?gactgcggcc?gcgaattcca?agcttgagta?ttctatcgtg
2100tcacctaaat?aacttggcgt?aatcatggtc?atatctgttt?cctgtgtgaaattgttatcc?gctcacaatt?ccacacaaca?tacgagccgg?aagcataaag
2200tgtaaagcct?ggggtgccta?atgagtgagc?taactcacat?taattgcgttgcgcgatgct?tccattttgt?gagggttaat?gcttcgagaa?gacatgataa
2300gatacattga?tgagtttgga?caaaccacaa?caagaatgca?gtgaaaaaaatgctttattt?gtgaaatttg?tgatgctatt?gctttatttg?taaccattat
2400aagctgcaat?aaacaagtta?acaacaacaa?ttgcattcat?tttatgtttcaggttcaggg?ggagatgtgg?gaggtttttt?aaagcaagta?aaacctctac
2500aaatgtggta?aaatccgata?aggatcgatt?ccggagcctg?aatggcgaatggacgcgccc?tgtagcggcg?cattaagcgc?ggcgggtgtg?gtggttacgc
2600gcacgtgacc?gctacacttg?ccagcgccct?agcgcccgct?cctttcgctttcttcccttc?ctttctcgcc?acgttcgccg?gctttccccg?tcaagctcta
2700aatcgggggc?tccctttagg?gttccgattt?agtgctttac?ggcacctcgaccccaaaaa ?cttgattagg?gtgatggttc?acgtagtggg?ccatcgccct
2800gatagacggt?ttttcgccct?ttgacgttgg?agtccacgtt?ctttaatagtggactcttgt?tccaaactgg?aacaacactc?aaccctatct?cggtctattc
2900ttttgattta?aagggatttt?gccgatttcg?gccttattgg?ttaaaaaatgagctgattta?acaaaaattt?aacgcgaatt?ttaacaaaat?attaacgctt
3000acaatttcgc?ctgtgtacct?tctgaggcgg?aaagaaccag?ctgtggaatgtgtgtcagtt?agggtgtgga?aagtccccag?gctccccagc?aggcagaagt
3100atgcaaagca?tgcatctcaa?ttagtcagca?accaggtgtg?gaaagtccccaggctcccca?gcaggcagaa?gtatgcaaag?catgcatctc?aattagtcag
3200caaccatagt?cccgccccta?actccgccca?tcccgcccct?aactccgcccagttccgccc?attctccgcc?ccatggctga?ctaatttttt?ttatttatgc
3300agaggccgag?gccgcctcgg?cctctgagag?attccagaag?tagtgaggaggcttttttgg?aggcctaggc?ttttgcaaaa?agcttgattc?ttctgacaca
3400acagtctcga?acttaaggct?agagccacca?tgattgaaca?agatggattgcacgcaggtt?ctaaggcgcc?ttgggtggag?aggctattcg?gctatgactg
3500ggcacaacag?acaatcggct?gctctgatgc?cgccgtgttc?cggctgtcagcgcaggggcg?cccggttctt?tttgtcaaga?ccgacctgtc?cggtgccctg
3600aatgaactgc?aggacgaggc?agcgcggcta?tcgtggctgg?ccacgacgggcgttccttgc?gcagctgtgc?tcgacgttgt?cactgaagcg?ggaagggact
3700ggctgctatt?gggcgaagtg?ccggggcagg?atctcctgtc?atctcaccttgctcctgccg?agaaagtatc?catcatggct?gatgcaatgc?ggcggctgca
3800tacgcttgat?ccggctacct?gcccattcga?ccaccaagcg?aaacatcgcatcgagcgagc?acgtactcgg?atggaagccg?gtcttgtcga?tcaggatgat
3900ctggacgaag?agcatcaggg?gctcgcgcca?gccgaactgt?tcgccaggctcaaggcgcgc?atgcccgacg?gcgaggatct?cgtcgtgacc?catggcgatg
4000cctgcttgcc?gaatatcatg?gtggaaaatg?gccgcttttc?tggattcatcgactgtggcc?ggctgggtgt?ggcggaccgc?tatcaggaca?tagcgttgga
4100tacccgtgat?attgctgaag?agcttggcgg?cgaatgggct?gaccgcttcctcgtgcttta?cggtatcgcc?gctcccgatt?cgcagcgcat?cgccttctat
4200cgccttcttg?acgagttctt?ctgagcggga?ctctggggtt?cgaaatgaccgaccaagcga?cgcccaacct?gccatcacga?tggccgcaat?aaaatatctt
4300tattttcatt?acatctgtgt?gttggttttt?tgtgtgaaga?tccgcgtatggtgcactctc?agtacaatct?gctctgatgc?cgcatagtta?agccagcccc
4400gacacccgcc?aacacccgct?gacgcgccct?gacgggcttg?tctgctcccggcatccgctt?acagacaagc?tgtgaccgtc?tccgggagct?gcatgtgtca
4500gaggttttca?ccgtcatcac?cgaaacgcgc?gagacgaaag?ggcctcgtgatacgcctatt?tttataggtt?aatgtcatga?taataatggt?ttcttagacg
4600tcaggtggca?cttttcgggg?aaatgtgcgc?ggaaccccta?tttgtttatttttctaaata?cattcaaata?tgtatccgct?catgagacaa?taaccctgat
4700aaatgcttca?ataatattga?aaaaggaaga?gtatgagtat?tcaacatttccgtgtcgccc?ttattccctt?ttttgcggca?ttttgccttc?ctgtttttgc
4800tcacccagaa?acgctggtga?aagtaaaaga?tgctgaagat?cagttgggtgcacgagtggg?ttacatcgaa?ctggatctca?acagcggtaa?gatccttgag
4900agttttcgcc?ccgaagaacg?ttttccaatg?atgagcactt?ttaaagttctgctatgtggc?gcggtattat?cccgtattga?cgccgggcaa?gagcaactcg
5000gtcgccgcat?acactattct?cagaatgact?tggttgagta?ctcaccagtcacagaaaagc?atcttacgga?tggcatgaca?gtaagagaat?tatgcagtgc
5100tgccataacc?atgagtgata?acactgcggc?caacttactt?ctgacaacgatcggaggacc?gaaggagcta?accgcttttt?tgcacaacat?gggggatcat
5200gtaactcgcc?ttgatcgttg?ggaaccggag?ctgaatgaat?ccataccaaacgacgagcgt?gacaccacga?tgcctgtagc?aatggcaaca?acgttgcgca
5300aactattaac?tggcgaacta?cttactctag?cttcccggca?acaattaatagactggatgg?aggcggataa?gttgcaggac?cacttctgcg?ctcggccctt
5400tccggctggc?tggtttattg?ctgataaatc?tggagccggt?gagcgtgggtctcgcggtat?cattgcagca?ctggggccag?atggtaagcc?ctcccgtatc
5500gtagttatct?acacgacggg?gagtcaggca?actatggatg?aacgaaatagacagatcgct?gagataggtg?cctcactgat?taagcattgg?taactgtcag
5600accaagttta?ctcatatata?ctttagattg?atttaaaact?tcatttttaatttaaaagga?tctaggtgaa?gatccttttt?gataatctca?tgaccaaaat
5700cccttaacgt?gagttttcgt?tccactgagc?gtcagacccc?gtagaaaagatcaaaggatc?ttcttgagat?cctttttttc?tgcgcgtaat?ctgctgcttg
5800caaacaaaaa?aaccaccgct?accagcggtg?gtttgtttgc?cggatcaagagctaccaact?ctttttccga?aggtaactgg?cttcagcaga?gcgcagatac
5900caaatactgt?ccttctagtg?tagccgtagt?taggccacca?cttcaagaactctgtagcac?cgcctacata?cctcgctctg?ctaatcctgt?taccagtggc
6000tgctgccagt?ggcgataagt?cgtgtcttac?cgggttggac?tcaagacgatagttaccgga?taaggcgcag?cggtcgggct?gaacgggggg?ttcgtgcaca
6100cagcccagct?tggagcgaac?gacctacacc?gaactgagat?acctacagcgtgagctatga?gaaagcgcca?cgcttcccga?agggagaaag?gcggacaggt
6200atccggtaag?cggcagggtc?ggaacaggag?agcgcacgag?ggagcttccagggggaaacg?cctggtatct?ttatagtcct?gtcgggtttc?gccacctctg
6300acttgagcgt?cgatttttgt?gatgctcgtc?aggggggcgg?agcctatggaaaaacgccag?caacgcggcc?tttttacggt?tcctggcctt?ttgcttgcct
6375
*tttgctcaca?tggctcgaca?gatct
5, plum mountain pig inoblast transgenosis and selection clone:
With calcium phosphate precipitation method transduction wire antisense agTL gene plasmid: the pCN84 of purifying adds 1 ° of Scal restriction endonuclease by 1 μ g/ μ l, cuts in 1 times of damping fluid of 37 ℃, and purifying is stand-by.
The pig inoblast is cultivated: with plum mountain pig anesthesia aseptic subcutis 1 * 0.5 * 0.3cm that cuts in back.Be incubated in the RPMI1640 perfect medium after shredding washing, wherein contain 20% calf serum, be incubated at 5%CO
2, 37 ℃ of environment.Go down to posterity 3
-Be used for gene transfer after 5 generations.Add with the calcium phosphate liquid 0.5ml of instant preparation and to go in the pig inoblast ware of nutrient solution, static 20-30 minute, add the 5ml nutrient solution again and cultivate to survey after 48-60 hour and transcribe efficient.Changed a not good liquor in three days with selecting nutrient solution (500 μ g/ml G418) to cultivate later on.Select after 10 days and change culturing bottle over to after single positive colony changes the amplification of 24 orifice plates over to.6, the stable of inverted defined gene transcribed and heterologous antigen expression defective after the mensuration cell transgenosis:
Extract the RNA of positive colony, carry out the efficient of transcribing of RT-PCR reaction detection inverted defined gene with T7 primer and aL82: get 20 μ lRNA and add the 100pM primer, and add 200MMLV reverse transcription synthetic enzyme, on PE company 2400 type PCR instrument, carry out reverse transcription reaction.Reaction parameter: 42 ℃, 15 minutes, 56 ℃ 15 minutes.Get reverse transcription reaction liquid 2 μ l and make template, the same application of sample carries out the PCR reaction to 100 μ l: thermal cycling: 94 ℃ of sex change of every circulation are 20 seconds after 94 ℃ of abundant sex change in 3 fens, anneal 15 seconds for 55 ℃, and 72 ℃ are extended totally 28 circulations in 30 seconds.Positive colony RT-PCR reacting positive.FCM measures the human serum lgM test of genetically modified pig inoblast surface bonding: the transgenic cell that tryptic digestion scatters is adjusted to 10 with PBS washing three times
6/ ml concentration, the people AB inactivated serum that adds equivalent, 37 ℃ were reacted 1 hour, and the 4 ℃ of incubations of anti-people IgM monoclonal antibody (DAKO company, titre 1: 100) that add the FITC mark then wash three times with PBS after 30 minutes again, constant volume is to 600 μ l, with flow cytometer (FACStar Plus) detect (488nm, 150mW), with not transgenic cell contrast, the result shows transgenic cell fluorescent mark rate to blank similar, and transgenic cell fluorescent mark rate height not.Measure the expression that this plasmid of proof and method can be used for eliminating the main transplantation antigen of donor organ.
Caption: Fig. 1. the structural representation pCN84 plasmid structure C MVe/p cmv enhancer 1-659 of plasmid pCN84
The plain resistant gene 2965-4286AMP-r of the new enzyme of CMV promoter 669-750 introne 890-1022T7 promoter 1227-1251 reverse agIT plum mountain pig agTL gene antisense code area 1276-2034SV40pA polyadenylic acid tailing signal fragment 2140-2460NEO-r ampicillin resistance gene 4683-5543ScaI 4989
Claims (7)
1, one align reverse primer, it is characterized by: (1) dna sequence dna: with the forward (aL84) and the reverse primer (aL82) of (agTL) upstream and downstream coupling: aL84:84#:5 ' cgg gat ccg ata cat tga gca tta ctt gga gga gtt ctaL82:82#:5 ' GCGTCGACGGCACAATTTAAAGTCAGATGTTATTTCTAACC
2、DNA ( agTL ) ,： ( 1 ) ：gatacattga gcattacttg gaggagttct taatatctgc aaatacatacttcatggttg gccacaaagt catctttaac atcatggtgg atgatatctccaggatgcct ttgatagagc tgggtcctct gcgttccttt aaagtgtttgagatcaagtc cgagaagagg tggcaagaca tcagcagtat gcgcatgaagaccatcgggg agcacatcct ggcccacatc cagcacgagg tggacttcctcttctgcatg gacgtggatc aggtcttcca aaacaacttt ggggtggagaccctgggcca gtcggtggct cagctacagg cctggtggta caaggcacatcctgacgagt tcacctacga gaggcggaag gagtccgcag cctacattccgtttggccag ggggattttt attaccacgc agccattttt gggggaacacccactcaggt tctaaacatc actcaggagt gcttcaaggg aatcctccaggacaaggaaa atgacataga agccgagtgg catgatgaaa gccatctaaacaagtatttc cttctcaaca aacccactaa aatcttatcc ccagaatactgctgggatta tcatataggc atgtctgtgg atattaggat tgtcaagatagcttggcaga aaaaagagta taatttggtt agaaataaca tctgactttaaattgtgcc709。 The 260th bit base is G, and the document of having delivered is T; Corresponding amino-acid residue is that Met is a methionine (Met), and the document of having delivered is that Ile is an Isoleucine.
3, a kind of new inverted defined gene is characterized by: (1) total order is classified as: tcaatattgg ccattagcca tattattcat tggttatata gcataaatca
100atattggcta?ttggccattg?catacgttgt?atctatatca?taatatgtacatttatattg?gctcatgtcc?aatatgaccg?ccatgttggc?attgattatt
200gactagttat?taatagtaat?caattacggg?gtcattagtt?catagcccatatatggaggt?ccgcgttaca?taacttcgg?taaatggccc?gcctggctga
300ccgcccaacg?acccccgccc?attgacgtca?ataatgacgt?atgttcccatagtaacgcca?atagggactt?tccattgacg?tcaatgggtg?gagtatttac
400ggtaaactgc?ccacttggca?gtacatcaag?tgtatcatat?gccaagtccgccccctattg?acgtcaatga?cggtaaatgg?cccgcctggc?attatgccca
500gtacatgacc?ttacgggact?ttcctacttg?gcagtacatc?tacgtattagtcatcgctat?taccatggtg?atgcggtttt?ggcagtacac?caatgggcgt
600ggatagcggt?ttgactcacg?gggatttcca?agtctccacc?ccattgacgtcaatgggagt?ttgttttggc?accaaaatca?acgggacttt?ccaaaatgtc
700gtaacaactg?cgatcgcccg?ccccgttgac?gcaaatgggc?ggtaggcgtgtacggtggga?ggtctatata?agcagagctc?gtttagtgaa?ccgtcagatc
800actagaagct?ttattgcggt?agtttatcac?agttaaattg?ctaacgcagtcagtgcttct?gacacaacag?tctcgaactt?aagctgcagt?gactctctta
900aggtagcctt?gcagaagttg?gtcgtgaggc?actgggcagg?taagtatcaaggttacaaga?caggtttaag?gagaccaata?gaaactgggc?ttgtcgagac
1000agagaagact?cttgcgtttc?tgataggcac?ctattggtct?tactgacatccactttgcct?ttctctccac?aggtgtccac?tcccagttca?attacagctc
1100ttaaaaattg?gatctccatt?cgccattcag?gctgcgcaac?tgctgggaaggacgatcaga?gcgggcctct?tcgctattac?gccagctggc?gaaagggacg
1200tggcaagcaa?ggcgattaag?ttgagttacg?ccaggatttt?cccagtcacgacgttgtaaa?acgacggcca?gagaattata?atacgactca?ctatagggcg
1300aattcggatc?cttgctagac?tcgacggcac?aatttaaagt?cagatgttatttctaaccaa?attatactct?tttttctgcc?aawctatctt?gacaatccta
1400atatccacag?acatgcctat?atgataatcc?cagcagtatt?ctggggataagattttagtg?ggtttgttga?gaaggaaata?cttgtttaga?tggctttcat
1500catgccactc?ggcttctatg?tcattttcct?tgtcctggag?gattcccttgaagcactcct?gagtgatgtt?tagaacctga?gtgggtgttc?ccccaaaaat
1600ggctgcgtgg?taataaaaat?ccccctggcc?aaacggaatg?taggctgcggactccttccg?cctctcgtag?gtgaactcgt?caggatgtgc?cttgtaccac
1700caggcctgta?gctgagccac?cgactggccc?agggtctcca?ccccaaagttgttttggaag?acctgatcca?cgtccatgca?gaagaggaag?tccacctcgt
1800gctggatgtg?ggccaggatg?tgctccccga?tggtcttcat?gcgcatcatgctgatgtctt?gccacctctt?ctcggacttg?atctcaaaca?ctttaaagga
1900acgcagagga?cccagctcta?tcaaaggcat?cctggagata?tcatccaccatgatgtaaaa?gatgactttg?tggccaacca?tgaagtatgt?atttgcagat
2000attaagaact?cctccaagta?atgctcaatg?tatcggatca?tctttcccgggggtaccgtc?gactgcggcc?gcgaattcca?agcttgagta?ttctatcgtg
2100tcacctaaat?aacttggcgt?aatcatggtc?atatctgttt?cctgtgtgaaattgttatcc?gctcacaatt?ccacacaaca?tacgagccgg?aagcataaag
2200tgtaaagcct?ggggtgccta?atgagtgagc?taactcacat?taattgcgttgcgcgatgct?tccattttgt?gagggttaat?gcttcgagaa?gacatgataa
2300gatacattga?tgagtttgga?caaaccacaa?caagaatgca?gtgaaaaaaatgctttattt?gtgaaatttg?tgatgctatt?gctttatttg?taaccattat
2400aagctgcaat?aaacaagtta?acaacaacaa?ttgcattcat?tttatgtttcaggttcaggg?ggagatgtgg?gaggtttttt?aaagcaagta?aaacctctac
2500aaatgtggta?aaatccgata?aggatcgatt?ccggagcctg?aatggcgaat
By 2550 pairs of based compositions.
4, a kind of new inverted defined gene recombinant plasmid is characterized by: (1) this plasmid is by 6370 based compositions of table 1.; (2) the 1st～2550 base in this plasmid is the dna molecular in the right 3: (3) this plasmid is used for transformed mammalian zygote or cell, obtains stable heterologous antigen
The cell strain of defective or transgenic animal, or transformant is as being used to clone for nuclear
The nuclear transplantation of animal, and then produce donorcells or organ.This cell or organ or animal
Lacking main heterologous antigen in the body expresses.Be used for clinically preventing effectively that hyperacute rejection is anti-
Should.Plasmid of the present invention goes down to posterity stable, in mammalian cell the transcriptional level height,
Heterologous antigen site synthase gene (α (1,3) semi-lactosi to mammalian cell
Glycosides base translocase (α [1,3] galactosyltransferase)) suppresses the efficient height
Characteristics.
5, use method of the present invention to prepare antisense heterologous antigen site synthase gene;
6, use method of the present invention to prepare the thin of antisense heterologous antigen site synthase gene production
Born of the same parents, organ (all are mammiferous for the bag Chinese juniper) and corresponding product.
7, the 2nd corresponding peptide fragment of the described gene of power and application thereof comprise as immunity anti-
Be subjected to former etc.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98122057 CN1255550A (en) | 1998-11-30 | 1998-11-30 | Clone of antisense gene resisting heteroplastic transplantation and in-situ synthetase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98122057 CN1255550A (en) | 1998-11-30 | 1998-11-30 | Clone of antisense gene resisting heteroplastic transplantation and in-situ synthetase |
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| CN1255550A true CN1255550A (en) | 2000-06-07 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018512876A (en) * | 2015-04-22 | 2018-05-24 | ミナ セラピューティクス リミテッド | saRNA compositions and methods of use |
-
1998
- 1998-11-30 CN CN 98122057 patent/CN1255550A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018512876A (en) * | 2015-04-22 | 2018-05-24 | ミナ セラピューティクス リミテッド | saRNA compositions and methods of use |
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