CN1344740A - Human testicular development specific protein-20 gene encoding protein - Google Patents
Human testicular development specific protein-20 gene encoding protein Download PDFInfo
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- CN1344740A CN1344740A CN01127203A CN01127203A CN1344740A CN 1344740 A CN1344740 A CN 1344740A CN 01127203 A CN01127203 A CN 01127203A CN 01127203 A CN01127203 A CN 01127203A CN 1344740 A CN1344740 A CN 1344740A
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Abstract
The present invention belongs to the field of human genome technology. The said human testis development specific protein-20 (NYD-SP20) gene has whole length cDNA sequence of 1365 bp, open reading frame sequence of 1092 bp, encoded 363 amino acids and a Genbank number AF367472. The present invention utilizes NYD-SP20 gene to clone and to prepare fusion protein and utilizes the said protein to immunize animal and to prepare monoclonal and polyclonal antibody while using gene cloning in the preparation of chip expressing testis specific function gene.
Description
One, technical field
The invention belongs to the Human genome technical group field.
Two, summary of the invention
Human testicular development specific protein-20 gene of the present invention (testis developmentspecific protein gene 20, NYD-SP20) 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 1365p; Its open reading frame is 1092bp, its 363 amino acid of encoding.Consult gene pool, this gene is new gene.Because this gene is the adult testis high expression level, embryonic testis is low expresses, therefore the human testicular development specific protein-20 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 testicular development specific protein-20 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 AF367472 in American National biotechnology information center.
1?gggggacggg?gccgcctgga?cgacggttga?cggaggcgag?ggaaactgag?ggcgaaagtc
61?tagtgggaca?atgtcatatt?ataaatttgg?aatgctgaat?agaaaattat?agattttgat
121?attgaaggaa?atgaagcgaa?gcctaagtga?aaattcagct?cgaagtacag?caggctgttt
181?gcctgttccg?ttgttcaatc?agaaaaagag?gaacagacag?ccattaactt?ctaatccact
241?taaagatgat?tcaggtatca?gtaccccttc?tgacaattat?gattttcctc?ctctacctac
301?agattgggcc?tgggaagctg?tgaatccaga?gttggctcct?gtaatgaaaa?cagtggacac
361?cgggcaaata?ccacattcag?cttctcgtcc?tctgagaagt?caagattctg?tctttaactc
421?tattcaatca?aatactggaa?gaagccaggg?tggttggagc?tacagagatg?gtaacaaaaa
481?taccagcttg?aaaacttgga?ataaaaatga?ttttaagcct?caatgtaaac?gaacaaactt
541?agtggcaaat?gatggaaaaa?attcttgtcc?agtgagttcg?ggagctcaac?aacaaaaaca
601?attaagaata?cctgaacctc?ctaacttatc?tcgcaacaaa?gaaaccgagc?tactcagaca
661?aacacattca?tcaaaaatat?ctggctgcac?aatgagaggg?ctagacaaaa?acagtgcact
721?acagacactt?aagcccaatt?ttcaacaaaa?tcaatataag?aaacaaatgt?tggatgatat
781?tccagaagac?aacaccctga?aggaaacctc?attgtatcag?ttacagttta?aggaaaaagc
841?tagttcttta?agaattattt?ctgcagttat?tgaaagcatg?aagtattggc?gtgaacatgc
901?acagaaaact?gtacttcttt?ttgaagtatt?agctgttctt?gattcagctg?ttacacctgg
961?cccatattat?tcgaagactt?ttcttatgag?ggatgggaaa?aatactctgc?cttgtgtctt
1021?ttatgaaatc?gatcgtgaac?ttccgagact?gattagaggc?cgagttcata?gatgtgttgg
1081?caactatgac?cagaaaaaga?acattttcca?atgtgtttct?gtcagaccgg?cgtctgtttc
1141?tgaacaaaaa?actttccagg?catttgtcaa?aattgcagat?gttgagatgc?agtattatat
1201?taatgtgatg?aatgaaactt?aagtagtgat?aaaaggaagt?ttagcataaa?ttatagtagt
1261?tttctgttat?tgcttaattt?accatctcca?tagttttata?gctactattg?tatttcactt
1321?gttgaattaa?agtatttgaa?ttcttttaaa?aaaaaaaaaa?aaaaa
Human testicular development specific protein-20 gene cDNA sequence MKRSLSENSARSTAGCLPVPLFNQKKRNRQPLTSNPLKDDSGISTPSDNYDFPPLP TDWAWEAVNPELAPVMKTVDTGQIPHSASRPLRSQDSVFNSIQSNTGRSQGGWSYR DGNKNTSLKTWNKNDFKPQCKRTNLVANDGKNSCPVSSGAQQQKQLRIPEPPNLSR NKETELLRQTHSSKISGCTMRGLDKNSALQTLKPNFQQNQYKKQMLDDIPEDNTLK ETSLYQLQFKEKASSLRIISAVIESMKYWREHAQKTYLLFEVLAVLDSAVTPGPYY SKTFLMRDGKNTLPCVFYEIDRELPRLIRGRVHRCVGNYDQKKNIFQCVSVRPASV SEQKTFQAFVKIADVEMQYYINVMNET
Human testicular development specific protein-20 gene proteins encoded aminoacid sequence
The objective of the invention is: in view of human testicular development specific protein-20 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 testicular development specific protein-20 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-protein interaction research etc.Therefore, this antibody might be used to prepare the diagnostic kit with human testicular development specific protein-20 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 testicular development specific protein-20 gene fusion roteins
Nucleotide sequence and plasmid pDEST with the human testicular development specific protein-20 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 testicular development specific protein-20 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 testicular development specific protein-20 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 testicular development specific protein-20 of secretion.4. human testicular development specific protein-20 gene is used to prepare the nucleotide sequence design primer of testicular function gene expression chip (1) with the human testicular development specific protein-20 gene that obtains, the human testicular development specific protein-20 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. 8 housekeeping genes of positive control (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 testicular development specific protein-20 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 testicular development specific protein-20 gene protein expression level of hybridization signal or the disappearance of human testicular development specific protein-20 gene.
The human testicular development specific protein-20 gene proteins encoded
Human testicular development specific protein-20 gene cDNA sequence
1?gggggacggg?gccgcctgga?cgacggttga?cggaggcgag?ggaaactgag?ggcgaaagtc
61?tagtgggaca?atgtcatatt?ataaatttgg?aatgctgaat?agaaaattat?agattttgat
121?attgaaggaa?atgaagcgaa?gcctaagtga?aaattcagct?cgaagtacag?caggctgttt
181?gcctgttccg?ttgttcaatc?agaaaaagag?gaacagacag?ccattaactt?ctaatccact
241?taaagatgat?tcaggtatca?gtaccccttc?tgacaattat?gattttcctc?ctctacctac
301?agattgggcc?tgggaagctg?tgaatccaga?gttggctcct?gtaatgaaaa?cagtggacac
361?cgggcaaata?ccacattcag?cttctcgtcc?tctgagaagt?caagattctg?tctttaactc
421?tattcaatca?aatactggaa?gaagccaggg?tggttggagc?tacagagatg?gtaacaaaaa
481?taccagcttg?aaaacttgga?ataaaaatga?ttttaagcct?caatgtaaac?gaacaaactt
541?agtggcaaat?gatggaaaaa?attcttgtcc?agtgagttcg?ggagctcaac?aacaaaaaca
601?attaagaata?cctgaacctc?ctaacttatc?tcgcaacaaa?gaaaccgagc?tactcagaca
661?aacacattca?tcaaaaatat?ctggctgcac?aatgagaggg?ctagacaaaa?acagtgcact
721?acagacactt?aagcccaatt?ttcaacaaaa?tcaatataag?aaacaaatgt?tggatgatat
781?tccagaagac?aacaccctga?aggaaacctc?attgtatcag?ttacagttta?aggaaaaagc
841?tagttcttta?agaattattt?ctgcagttat?tgaaagcatg?aagtattggc?gtgaacatgc
901?acagaaaact?gtacttcttt?ttgaagtatt?agctgttctt?gattcagctg?ttacacctgg
961?cccatattat?tcgaagactt?ttcttatgag?ggatgggaaa?aatactctgc?cttgtgtctt
1021?ttatgaaatc?gatcgtgaac?ttccgagact?gattagaggc?cgagttcata?gatgtgttgg
1081?caactatgac?cagaaaaaga?acattttcca?atgtgtttct?gtcagaccgg?cgtctgtttc
1141?tgaacaaaaa?actttccagg?catttgtcaa?aattgcagat?gttgagatgc?agtattatat
1201?taatgtgatg?aatgaaactt?aagtagtgat?aaaaggaagt?ttagcataaa?ttatagtagt
1261?tttctgttat?tgcttaattt?accatctcca?tagttttata?gctactattg?tatttcactt
1321?gttgaattaa?agtatttgaa?ttcttttaaa?aaaaaaaaaa?aaaaa
Human testicular development specific protein-20 gene encoding proteins amino acid sequence MKRSLSENSARSTAGCLPVPLFNQKKRNRQPLTSNPLKDDSGISTPSDNYDFPPLP TDWAWEAVNPELAPVMKTVDTGQIPHSASRPLRSQDSVFNSIQSNTGRSQGGWSYR DGNKNTSLKTWNKNDFKPQCKRTNLVANDGKNSCPVSSGAQQQKQLRIPEPPNLSR NKETELLRQTHSSKISGCTMRGLDKNSALQTLKPNFQQNQYKKQMLDDIPEDNTLK ETSLYQLQFKEKASSLRIISAVIESMKYWREHAQKTVLLFEVLAVLDSAVTPGPYY SKTFLMRDGKNTLPCVFYEIDRELPRLIRGRVHRCVGNYDQKKNIFQCVSVRPASV SEQKTFQAFVKIADVEMQYYINVMNET
Claims (5)
1. the testicular function specific gene NYD-SP20) fusion of clone's preparation; It is characterized in that the whole ORFs amino acid of NYD-SP20 gene, sequence is: MKRSLSENSARSTAGCLPVPLFNQKKRNRQPLTSNPLKDDSGISTPSDNYDFPPLP TDWAWEAVNPELAPVMKTVDTGQIPHSASRPLRSQDSVFNSIQSNTGRSQGGWSYR DGNKNTSLKTWNKNDFKPQCKRTNLVANDGKNSCPVSSGAQQQKQLRIPEPPNLSR NKETELLRQTHSSKISGCTMRGLDKNSALQTLKPNFQQNQYKKQMLDDIPEDNTLK ETSLYQLQFKEKASSLRIISAVIESMKYWREHAQKTVLLFEVLAVLDSAVTPGPYY SKTFLMRDGKNTLPCVFYEIDRELPRLIRGRVHRCVGNYDQKKNIFQCVSVRPASV SEQKTFQAFVKIADVEMQYYINVMNET
2. according to the described polyclonal antibody at the preparation of NYD-SP20 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-SP20 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-SP20 gene cDNA according to claim 1 is described at NYD-SP20 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-SP20 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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| CN01127203A CN1344740A (en) | 2001-09-13 | 2001-09-13 | Human testicular development specific protein-20 gene encoding protein |
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| CN01127203A CN1344740A (en) | 2001-09-13 | 2001-09-13 | Human testicular development specific protein-20 gene encoding protein |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9731970B2 (en) | 2012-04-16 | 2017-08-15 | Seerstone Llc | Methods and systems for thermal energy recovery from production of solid carbon materials by reducing carbon oxides |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9731970B2 (en) | 2012-04-16 | 2017-08-15 | Seerstone Llc | Methods and systems for thermal energy recovery from production of solid carbon materials by reducing carbon oxides |
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