CN1382697A - Human testis development specific protein-28 gene coding protein - Google Patents
Human testis development specific protein-28 gene coding protein Download PDFInfo
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- CN1382697A CN1382697A CN01127210A CN01127210A CN1382697A CN 1382697 A CN1382697 A CN 1382697A CN 01127210 A CN01127210 A CN 01127210A CN 01127210 A CN01127210 A CN 01127210A CN 1382697 A CN1382697 A CN 1382697A
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Abstract
A human testis development specific protein gene 28 (NYD-SP28) is disclosed. It has 1798 bp of whole cDNA sequence length, 1455 bp of open reading frame, 484 coded amino acid and AF382188 for its number in genbank. The said NYD-SP28 is used to clone fusion protein, which is then used immunize animal for preparing monoclonal or polyclonal antibody. The said gene clone is also used for preparing the expression chip of testis specific function gene.
Description
One, technical field
The invention belongs to the Human genome technical group field.
Two, summary of the invention
Human testis development specific protein-28 gene of the present invention (testis developmentspecific protein gene 28, NYD-SP28) be that the contriver obtains with the method for gene chip screening in reproductive medicine Jiangsu Province key lab of Nanjing Medical University, at first prepare people's testicular function gene expression chip, by comparing embryo and grownup's testicular function expression of gene, acquisition differential expression clone, prove full-length cDNA through sequencing, be 1798bp; Its open reading frame is 1455bp, its 484 amino acid of encoding.Consult gene pool, this gene is new gene.Because this gene is the adult testis high expression level, the embryo is low to express, therefore the human testis development specific protein-28 gene of finding may with testicualr development, particularly spermatogeny is relevant, can be prepared into fusion rotein with this gene clone, with this protein immunization rabbit, can obtain mono-clonal and polyclonal antibody, this gene also can be used for preparing the testicular function gene expression chip.
Because human testis development specific protein-28 gene still unmanned homologous gene in the Genbank database, the inventor all is initiative for the 26S Proteasome Structure and Function achievement in research of this gene.(National Center for BiotechnologyInformation NCBI) formally accepts the newcomer that this gene is Genbank, and accepting number is AF382188 in American National biotechnology information center.1?gggggggaga?gctcagcctc?actgatagcc?ggggatctct?cctgttcgag?ggctgagatc 61?ttcggtccca?caaccgccca?caaccagggg?cacttctgga?agtccccttt?ctagggacat?121?ttttcttcta?agctctgtaa?cgcgggccga?ggcagaccga?ggcttctttc?ccggatccaa?181?tcccctgccc?ccatgcctaa?gaaagaaaaa?atggccaaga?cgcccctgtc?cgatgagaag?241?cagctgcttc?tgtttcagca?gaagttgctg?gcagaggagg?agatggccaa?aaagaaggag?301?aggctcctca?gccagttctt?gaaggacaag?ctggccaagg?aggaacacaa?cagtgctctg?361?aaccttaata?agattaacac?acagtggaga?actgtccttc?gggaagtcaa?gaccagagaa?421?cttcataagg?acattgagat?cctcagccaa?acatttgaac?gagtggtgga?ctgtaaggac?481?aatgtcatca?agtctttagc?taaagacctg?tccgaagccg?aggagcagta?cgcccatgcc?541?ctgcgcagcc?acttgcacaa?tgttgaccag?ctcttggccc?tgcagaggca?ccggctcagt?601?ctcctggagg?aaagttacaa?catggagctg?gaagccctaa?ccaaggagtt?tgagacagaa?661?aggaagacaa?ttattgacca?acatgagaaa?gagattcact?atctgcaaga?tatcttcatg?721?gccatggagc?agaactatat?agattctgag?tatgaaagca?agctggagtt?ccagagcatg?781?tggaatgatc?tcaaagacat?gaatttagaa?gagaagcact?ttctaagact?gcacctggag?841?aacagagtag?aagatctgtg?gagaaagttc?caggatgtac?tcaagaatta?cactgatgcc?901?acagaggatc?gaaagactgc?ctttgagacc?ctgcaggtga?aggatgagaa?gagctccaaa?961?gagattgaag?tacagatgaa?aaaaatacag?aaactacagg?atgccataac?tatttcaaaa1021?ggcaagatca?tgatacacag?ccgtgagagt?gaagatgaga?accggtatat?ccgtaatgac1081?aaggaattgg?tccttgtaca?actgcgaaaa?cttaaggccc?aaagaactca?ggcccgggca1141?gcatcccaga?agaacttagt?cagactcacc?ctggaaagta?atgccaccct?caaggccctg
1201?agaaagattg?ttgataaggg?tgaaaagatc?cttaaacttg?ctgaaatatg?taggaaattt
1261?gaaaccgaag?aagaaaaagt?gctgcctttt?tattcatcag?tattgactcc?taaggagcag
1321?gaggggattc?agaagaataa?tctagaagag?cttactgagg?agctcaccaa?ggtgatggtg
1381?gactacatag?gaatggagaa?tttctggaaa?aggtacaaca?aagtgaaact?ggagcaactg
1441?agcctccaac?atagacgagc?ccagctgcta?gatatcaatg?ggaagctgcg?ggagatgctg
1501?aagcagtact?tggatggcat?ctcagtgagt?gacgaagtgc?tgagccagct?caacccactc
1561?tttatagtca?actatcaaag?caacttactc?cagcccttgt?ccatacgtat?agcccatcca
1621?ggtgataaac?aacatccaac?cacttaaaat?ataattgaag?cagcccagat?gatcttccac
1681?aacctgtgat?ctaaggaaaa?aaatctttca?actcctagag?attttttttt?ttttttgaga
1741?aatgtaggga?tgttgctatt?aaaaatttcc?atggagtttg?ggaaaaaaaa?aaaaaaaa
Human testis development specific protein-28 gene cDNA sequence MPKKEKMAKTPLSDEKQLLLFQQKLLAEEEMAKKKERLLSQFLKDKLAKEEHNSAL NLNKINTQWRTVLREVKTRELHKDIEILSQTFERVVDCKDNVIKSLAKDLSEAEEQ YAHALRSHLHNVDQLLALQRHRLSLLEESYNMELEALTKEFETERKTIIDQHEKEI HYLQDIFMAMEQNYIDSEYESKLEFQSMWNDLKDMNLEEKHFLRLHLENRVEDLWR KFQDVLKNYTDATEDRKTAFETLQVKDEKSSKEIEVQMKKIQKLQDAITISKGKIM IHSRESEDENRYIRNDKELVLVQLRKLKAQRTQARAASQKNLVRLTLESNATLKAL RKIVDKGEKILKLAEICRKFETEEEKVLPFYSSVLTPKEQEGIQKNNLEELTEELT KVMVDYIGMENFWKRYNKVKLEQLSLQHRRAQLLDINGKLREMLKQYLDGISVSDE VLSQLNPLFIVNYQSNLLQPLSIRIAHPGDKQHPTT
Human testis development specific protein-28 gene proteins encoded aminoacid sequence
The objective of the invention is: in view of human testis development specific protein-28 gene is the brand-new gene that the inventor finds, human at present understanding and the understanding to this gene only limits to the scientific research that the inventor does.Because this gene is the gene relevant with the testis specific function, and be the low expression of embryo, adult's high expression level, therefore this gene probably with testicualr development, particularly spermatogeny is relevant, and its unconventionality expression may influence the male sex's reproductive function, so the research of this gene can be used for:
1. by this gene Fusion albumen of preparation, be used to study the relation of this gene and testicular function, also may become a kind of biologics for the treatment of the male testical relative disease.
2. prepare specific antibody: the specific antibody of gene coded protein is the indispensable important tool of research gene structure and function, the polyclone of human testis development specific protein-28 gene proteins encoded and monoclonal antibody, can be used as immunohistochemical staining, ELISA, immuno-electron microscope, multiple detection method such as co-immunoprecipitation according to the immunology principle design, can be used for location and the quantitative examination of this gene coded protein in histocyte, also can be used for various biological specimens (as blood, seminal fluid, urine etc.) content research in and protein one protein interaction research etc.Therefore, this antibody might be used to prepare the diagnostic kit with human testis development specific protein-28 gene abnormal expression relative disease, and might be used to prepare antifertility drug.
3. prepare gene diagnosis chip: functional gene is used to prepare the importance that gene diagnosis chip is the gene development research, all significant at aspects such as medical diagnosis on disease (as male sterility, sexual disorder) and drug screenings.The preparation of technical solution of the present invention 1. human testis development specific protein-28 gene fusion roteins is with the nucleotide sequence and the plasmid pDEST of human testis development specific protein-28 gene open reading frame
TM15 are prepared into the GST-SP2-pDEST15 expression plasmid, cultivate 0.5OD600 for 30 ℃ in the LB of no NaCl, induce through 0.3M NaCl, and ultrasonication obtains the fusion rotein of purifying after GST affinity chromatography column purification.2. human testis development specific protein-28 gene coded protein specific polyclonal antibody
Getting fusion rotein combines with carrier proteins KLH (Keyhole Limpet Hymocyanin), through the dialysis after again with fully/Freund (Complete/Incomplete Freund ' sAdjuvant) balanced mix, immunity new zealand rabbit (back subcutaneous injection), every 3～4 all booster injections once, totally three times, after three months, get blood system from serum.Be ELISA in contrast with animal serum before the immunity, measure antibody titers in each immunize rabbit serum.Screen high titre serum, further do competition and suppress the specificity that immunity test is determined antibody with antigen.Determine that according to ELISA result its extension rate that is used for immunohistochemical staining is 1: 200～500.3. human testis development specific protein-28 gene coded protein specific monoclonal antibody
Getting fusion rotein combines with carrier proteins KLH (Keyhole Limpet Hymocyanin), after dialysis again with fully/Freund (Complete/Incomplete Freund ' sAdjuvant) mixes, immunity Balb/C mouse (intraperitoneal injection), every other week once, continuous 2-3 time, vein is strengthened once before merging, after ELISA method confirmation production of antibodies, get the spleen separating Morr. cell, merge the generation hybridoma with the myeloma cell then.Positive colony is through subclone repeatedly, until the monoclonal hybridoma strain that obtains the anti-human testis development specific protein-28 of secretion.4. human testis development specific protein-28 gene is used to prepare the testicular function gene expression chip
(1) with the nucleotide sequence design primer of the human testis development specific protein-28 gene that obtains, the human testis development specific protein-28 gene sequence of amplification total length is as the sample of some film.
(2) with Britain BioRobotics put automatically the film instrument with specimens point on 8 * 12cm nylon membrane, 2 points of each specimens point, several approximately ng of every DNA amount. Positive control is 8 housekeeping genes (a.ribosomal protein S9; B.Actin gamma; C.glyceraldehyde-3-phosphate dehydrogenase; D.hypoxanthinephosphoribosyltransferase 1; E.H.sapiens mRNA for 23KD highly basicprotein; F.ubiqnitin C; G.Phospholipase A2; H.ubiquitin carboxy I-ferminal esterase L I).Negative control is lambda bacteriophage dna and P-blue plasmid.(3) chip that will contain this gene is used for the diagnosis and the drug screening of human testis development specific protein-28 gene disappearance
33The P mark:
Extracting sample mRNA to be checked or DNA are with Random Primer labelling method mark sample mRNA to be checked or DNA, as the probe of hybridization.Hybridization:
After the nylon membrane label probe hybridization that point is made, oven dry is shielded compressing tablet with phosphorus in the hybrid heater (68 ℃).Signal scanning and analysis:
Analyze hybridization signal with registering instrument.According to the strong and weak analyst's testis development specific protein-28 gene protein expression level of hybridization signal or the disappearance of human testis development specific protein-28 gene.
The human testis development specific protein-28 gene proteins encoded
-28cDNA 1 gggggggaga gctcagcctc actgatagcc ggggatctct cctgttcgag ggctgagatc 61 ttcggtccca caaccgccca caaccagggg cacttctgga agtccccttt ctagggacat 121 ttttcttcta agctctgtaa cgcgggccga ggcagaccga ggcttctttc ccggatccaa 181 tcccctgccc ccatgcctaa gaaagaaaaa atggccaaga cgcccctgtc cgatgagaag 241 cagctgcttc tgtttcagca gaagttgctg gcagaggagg agatggccaa aaagaaggag 301 aggctcctca gccagttctt gaaggacaag ctggccaagg aggaacacaa cagtgctctg 361 aaccttaata agattaacac acagtggaga actgtccttc gggaagtcaa gaccagagaa 421 cttcataagg acattgagat cctcagccaa acatttgaac gagtggtgga ctgtaaggac 481 aatgtcatca agtctttagc taaagacctg tccgaagccg aggagcagta cgcccatgcc 541 ctgcgcagcc acttgcacaa tgttgaccag ctcttggccc tgcagaggca ccggctcagt 601 ctcctggagg aaagttacaa catggagctg gaagccctaa ccaaggagtt tgagacagaa 661 aggaagacaa ttattgacca acatgagaaa gagattcact atctgcaaga tatcttcatg 721 gccatggagc agaactatat agattctgag tatgaaagca agctggagtt ccagagcatg 781 tggaatgatc tcaaagacat gaatttagaa gagaagcact ttctaagact gcacctggag 841 aacagagtag aagatctgtg gagaaagttc caggatgtac tcaagaatta cactgatgcc 901 acagaggatc gaaagactgc ctttgagacc ctgcaggtga aggatgagaa gagctccaaa 961 gagattgaag tacagatgaa aaaaatacag aaactacagg atgccataac tatttcaaaa1021 ggcaagatca tgatacacag ccgtgagagt gaagatgaga accggtatat ccgtaatgac1081 aaggaattgg tccttgtaca actgcgaaaa cttaaggccc aaagaactca ggcccgggca1141 gcatcccaga agaacttagt cagactcacc ctggaaagta atgccaccct caaggccctg1201 agaaagattg ttgataaggg tgaaaagatc cttaaacttg ctgaaatatg taggaaattt1261 gaaaccgaag aagaaaaagt gctgcctttt tattcatcag tattgactcc taaggagcag1321 gaggggattc agaagaataa tctagaagag cttactgagg agctcaccaa ggtgatggtg1381 gactacatag gaatggagaa tttctggaaa aggtacaaca aagtgaaact ggagcaactg1441 agcctccaac atagacgagc ccagctgcta gatatcaatg ggaagctgcg ggagatgctg1501 aagcagtact tggatggcat ctcagtgagt gacgaagtgc tgagccagct caacccactc1561 tttatagtca actatcaaag caacttactc cagcccttgt ccatacgtat agcccatcca1621 ggtgataaac aacatccaac cacttaaaat ataattgaag cagcccagat gatcttccac1681 aacctgtgat ctaaggaaaa aaatctttca actcctagag attttttttt ttttttgaga1741 aatgtaggga tgttgctatt aaaaatttcc atggagtttg ggaaaaaaaa aaaaaaaa-28MPKKEKMAKTPLSDEKQLLLFQQKLLAEEEMAKKKERLLSQFLKDKLAKEEHNSALNLNKINTQWRTVLREVKTRELHKDIEILSQTFERVVDCKDNVIKSLAKDLSEAEEQYAHALRSHLHNVDQLLALQRHRLSLLEESYNMELEALTKEFETERKTIIDQHEKEIHYLQDIFMAMEQNYIDSEYESKLEFQSMWNDLKDMNLEEKHFLRLHLENRVEDLWRKFQDVLKNYTDATEDRKTAFETLQVKDEKSSKEIEVQMKKIQKLQDAITISKGKIMIHSRESEDENRYIRNDKELVLVQLRKLKAQRTQARAASQKNLVRLTLESNATLKALRKIVDKGEKILKLAEICRKFETEEEKVLPFYSSVLTPKEQEGIQKNNLEELTEELTKVMVDYIGMENFWKRYNKVKLEQLSLQHRRAQLLDINGKLREMLKQYLDGISVSDEVLSQLNPLFIVNYQSNLLQPLSIRIAHPGDKQHPTT
Claims (5)
1. a testicular function specific gene (NYD-SP28) is cloned the fusion of preparation; It is characterized in that the whole ORFs amino acid of NYD-SP28 gene, sequence is: MPKKEKMAKTPLSDEKQLLLFQQKLLAEEEMAKKKERLLSQFLKDKLAKEEHNSAL NLNKINTQWRTVLREVKTRELHKDIEILSQTFERVVDCKDNVIKSLAKDLSEAEEQ YAHALRSHLHNVDQLLALQRHRLSLLEESYNMELEALTKEFETERKTIIDQHEKEI HYLQDIFMAMEQNYIDSEYESKLEFQSMWNDLKDMNLEEKHFLRLHLENRVEDLWR KFQDVLKNYTDATEDRKTAFETLQVKDEKSSKEIEVQMKKIQKLQDAITISKGKIM IHSRESEDENRYIRNDKELVLVQLRKLKAQRTQARAASQKNLVRLTLESNATLKAL RKIVDKGEKILKLAEICRKFETEEEKVLPFYSSVLTPKEQEGIQKNNLEELTEELT KVMVDYIGMENFWKRYNKVKLEQLSLQHRRAQLLDINGKLREMLKQYLDGISVSDE VLSQLNPLFIVNYQSNLLQPLSIRIAHPGDKQHPTT
2. according to the described polyclonal antibody at the preparation of NYD-SP28 gene open reading frame aminoacid sequence of claim 1, after it is characterized in that fusion rotein and complete and Freund mixing, immune new zealand rabbit prepares polyclonal antibody.
3. according to the described monoclonal antibody of claim 1 at the preparation of NYD-SP28 gene open reading frame aminoacid sequence, it is characterized in that fusion rotein mixes with complete and Freund, immunity Balb/C mouse, the separating spleen bone-marrow-derived lymphocyte and and the myeloma cell merge the generation hybridoma, further, prepare monoclonal antibody with the mono-clonal of subclone method, ELISA and antibody typing method screening IgG secretion antibody.
4. it is characterized in that pcr amplification NYD-SP28 gene cDNA according to claim 1 is described at NYD-SP28 gene open reading frame aminoacid sequence is used to prepare the testicular function gene expression chip, put in chip and be used for chip preparation.
5. according to claim 1, be used to prepare fusion rotein at NYD-SP28 gene open reading frame aminoacid sequence, it is characterized in that fusion rotein can be used as the medicine of preparation treatment relative disease.
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| CN01127210A CN1382697A (en) | 2001-09-13 | 2001-09-13 | Human testis development specific protein-28 gene coding protein |
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| CN01127210A CN1382697A (en) | 2001-09-13 | 2001-09-13 | Human testis development specific protein-28 gene coding protein |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112695033A (en) * | 2020-12-29 | 2021-04-23 | 华南师范大学 | siRNA designed based on periplaneta americana male periglandular reproduction related gene SP28 and preparation method and application thereof |
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2001
- 2001-09-13 CN CN01127210A patent/CN1382697A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112695033A (en) * | 2020-12-29 | 2021-04-23 | 华南师范大学 | siRNA designed based on periplaneta americana male periglandular reproduction related gene SP28 and preparation method and application thereof |
| CN112695033B (en) * | 2020-12-29 | 2021-08-24 | 华南师范大学 | siRNA designed based on periplaneta americana male periglandular reproduction related gene SP28 and preparation method and application thereof |
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