CN1344742A - 人睾丸发育特异性蛋白－24基因编码蛋白 - Google Patents
人睾丸发育特异性蛋白－24基因编码蛋白 Download PDFInfo
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Abstract
本发明属于人类基因组技术领域,所述的人睾丸发育特异性蛋白－24基因(testis development specific protein gene 24,NYD－SP24)基因全长cDNA序列为2823bp,其开放阅读框序列为2178bp,其编码725个氨基酸,该基因在基因库(Genbank)中的编号为AY032876。本发明利用NYD－SP24基因克隆制备融合蛋白,并用该蛋白免疫动物制备单克隆和多克隆抗体,同时将基因克隆用于睾丸特异功能基因表达芯片的制备。
Description
一、技术领域
本发明属于人类基因组技术领域。
二、发明内容
本发明所述人睾丸发育特异性蛋白-24基因(testis developmentspecific protein gene 24，NYD-SP24)是发明人在南京医科大学生殖医学江苏省重点实验室用基因芯片方法筛选获得，首先制备人睾丸功能基因表达芯片，通过比较胚胎和成年人睾丸功能基因的表达，获得差异表达克隆，经序列测定证明为全长cDNA，为2823bp；其开放阅读框架为2178bp，其编码725个氨基酸。查阅基因库，该基因为新基因。由于该基因是成人睾丸高表达，胚胎睾丸低表达，因此发现的人睾丸发育特异性蛋白-24基因可能与睾丸发育，特别是精子发生有关，用该基因克隆可制备成融合蛋白，用该蛋白免疫家兔，可获得单克隆和多克隆抗体，该基因也可用于制备睾丸功能基因表达芯片。
由于人睾丸发育特异性蛋白-24基因在Genbank数据库中尚无人的同源基因，本发明人对于该基因的结构和功能研究成果全都是开创性的。美国国家生物技术情报中心(National Center for BiotechnologyInformation，NCBI)已正式接受该基因为Genbank的新成员，接受号为AY032876。
1 ggggggggcg cgcctgcgca ttatgctggt ctccatggcg gggctcggag caagacgaga
61 gaaaatgctg ctgtgaagac ccaattaaag ctttcacata aatgaagcta cagctatcat
121 gatttagtgc ccccaaaagg aagaacttca gtggacaaga aaggacattt ctgggggtag
181 tagaatgctt gaggcctgga atttaaacct gagccactat ctgaagctat taaataatag
241 aagaaaatgg aattgaatat gatgtcagca tggaacaatc taatgattca ttaagagtca
301 accataatga cggtgaagag tcaaaaacca gtgctcaagt atttgagcat ctaatctgta
361 tggactccag ggattcttcc tttggacaaa atgattctcc tacagttttg cccatcacta
421 ctcgtgaagc aaataattca ctcatatcac agaatatacc agggcccctg actcagacac
481 agactctttc tgcagagcaa ttccatctag tggaccaaaa tgggcaggct attcaatatg
541 aacttcagtc attgggggaa tccaatgcac aaatgatgat cgttgccagc ccaacagaaa
601 atggacaggt acttcgtgta attccaccta cccagacagg aatggcacaa gtgattatac
661 ctcaggggca acttgtggat gtgaatagtc ctcgggatgt ccctgaagag aaacccagta
721 acagaaactt accaactgta agagtggata ctctagcaga caataccagc aattacattc
781 ttcatcctca aacatccttc ccattgccca aaaagtcagt gaccggaatg ctggaagaac
841 cccttctggg gcctcttcag ccactttctt ctaatacacc tatatgggcc tgccgtctta
901 ggagctgtga gaaaattgga gattcatacc gtggctactg tgtaagtgag actgaattag
961 aaagtgtcct aacatttcac gagcagcaaa cacagagtgt ttgggggacc cgtcagtctc
1021 caagcccagc caagcctgct acacgcttga tgtggaaatc ccagtatgtt ccatatgatg
1081 gaatcccatt tgttaatgca gggagtagag ctgtggtaat ggagtgtcag tatgggccaa
1141 gaagaaaagg tttccagtta aaaaaagtca gtgagcagga aagcaggtct tgtcagctct
1201 acaaagccac ttgtccagct cggatttaca ttaaaaaggt acagaagttt cctgaatata
1261 gagttcctac agaccccaaa attgacaaga aaattatcag aatggagcag gagaaagctt
1321 ttaacatgct aaagaagaac ttggtagatg ctggtggtgt tcttaggtgg tatgtacagt
1381 tacctacaca gcaagctcat cagtatcatg aattagagac tccctgcctc actttgtcac
1441 cttctccttt tcctgtgtct tctcttgaag aagaggaaac tgcagttaga gatgagaatt
1501 gtgcattacc ctcacgttta catcctcaag tagcacataa gattcaagaa ttagtatcac
1561 agggaataga acaagtgtat gcagtaagga aacagctaag aaaatttgtg gaaagggaac
1621 tgttcaaacc cgatgaggta cctgaaagac ataatttatc tttttttcca actgtaaatg
1681 atataaaaaa tcacatccat gaggtacaga aatccttgag aaatggagat acggtatata
1741 actcagagat tattccagca acgcttcaat ggactacaga cagtgggaat attctcaaag
1801 agaccatgac agttacattt gcagaaggaa attcaccagg agaatcaatt accaccaaag
1861 tggaaacaaa tcagaccagg ggttctttgt ctcctgagcc aacccacttg ctctcctcac
1921 tctcctcatt tcagcccaaa atatttacac aactacaggg tttgcagtta caaccaaggt
1981 acacctctcc tgatgaatca ccagctgtgg tatcagtaaa taaccagccg tcctctagtc
2041 cttcaggact tctggataca ataggaagtg ctgtaatgaa taataattct ctactgcttg
2101 gtcaaagtca tagccttcaa agagatacat gcttaaccca aaacaatagt actgcctcca
2161 ccatgggtaa ccttccagaa ccagatcaaa atctagttgc aatggacgag ctggtagaag
2221 ttggagatgt tgaggataca gggaatctgg aaggaactgt tcatcggatt ctgttgggag
2281 atgtgcagac tattccaata cagattatag acaaccactc agctcttatt gaagaaaatc
2341 cagaaagtac catttctgtg agccaagtta aacaagaacc caaagaacca gcattgtcta
2401 tggaagcaaa aaaaactgtg gactataaga aattatctgc tacataaatt attgaagctt
2461 ttcagaattt acaactctgg tgacttctga tgtcttagaa aggaaggtga atatagtcaa
2521 agcgtggcat tgagataatt ggactgaaga ccagtttgat gagaagcttt tatttaaaac
2581 tgatatattt gttgccagtt ccatattttt accttcctta agagatgttt tactcttctt
2641 attttgtata atttttatgc tctttgatta tctaataagg ccagtatttc aaaagctgtt
2701 ctgaatttat ccacagtaat atagtctgaa cacaaatagt ttacttaact tatccttgga
2761 aatattcaat tttggttatt acaaaacaga aaagaacaac agcaaaaaaa aaaaaaaaaa
2821 aaa
人睾丸发育特异性蛋白-24基因cDNA序列QNIPGPLTQTQTLSAEQFHLVDQNGQAIQYELQSLGESNAQMMIVASPTENGQVLRVIPPTQTGMAQVIIPQGQLVDVNSPRDVPEEKPSNRNLPTVRVDTLADNTSNYILHPQTSFPLPKKSVTGMLEEPLLGPLQPLSSNTPIWACRLRSCEKIGDSYRGYCVSETELESVLTFHEQQTQSVWGTRQSPSPAKPATRLMWKSQYVPYDGIPFVNAGSRAVVMECQYGPRRKGFQLKKVSEQESRSCQLYKATCPARIYIKKVQKFPEYRVPTDPKIDKKIIRMEQEKAFNMLKKNLVDAGGVLRWYVQLPTQQAHQYHELETPcLTLSPSPFPVSSLEEEETAVRDENCALPSRLHPQVAHKIQELVSQGIEQVYAVRKQLRKFVERELFKPDEVPERHNLSFFPTVNDIKNHIHEVQKSLRNGDTVYNSEIIPATLQWTTDSGNILKETMTVTFAEGNSPGESITTKVETNQTRGSLSPEPTHLLSSLSSFQPKIFTQLQGLQLQPRYTSPDESPAVVSVNNQPSSSPSGLLDTIGSAVMNNNSLLLGQSHSLQRDTCLTQNNSTASTMGNLPEPDQNLVAMDELVEVGDVEDTGNLEGTVHRILLGDVQTIPIQIIDNHSALIEENPESTISVSQVKQEPKEPALSMEAKKTVDYKKLSAT
人睾丸发育特异性蛋白-24基因编码蛋白氨基酸序列
本发明的目的是：鉴于人睾丸发育特异性蛋白-24基因是本发明人发现的全新基因，目前人类对此基因的认识和了解仅限于本发明人所做的科学研究工作。由于该基因是与睾丸特异功能相关的基因，而且是胚胎低表达，成人高表达，因此该基因很可能与睾丸发育，特别是精子发生有关，其异常表达可能影响男性的生殖功能，因此该基因的研究可用于：
①通过制备该基因的融合蛋白，用于研究该基因与睾丸功能的关系，还可能成为一种治疗男性睾丸相关疾病的生物药品。
②制备特异性抗体：基因编码蛋白的特异性抗体是研究基因结构和功能的不可缺少的重要工具，人睾丸发育特异性蛋白-24基因编码蛋白的多克隆和单克隆抗体，可用作免疫组织化学染色、ELISA、免疫电镜、免疫共沉淀等多种根据免疫学原理设计的检测方法，可用于该基因编码蛋白在组织细胞中的定位和定量研究，也可用于各种生物样本(如血液、精液、尿液等)中的含量变化研究以及蛋白质-蛋白质相互作用研究等。因此，该抗体有可能用于制备与人睾丸发育特异性蛋白-24基因表达异常相关疾病的诊断试剂盒，并有可能用于制备抗生育药物。
③制备基因诊断芯片：将功能基因用于制备基因诊断芯片是基因开发研究的一个重要方面，在疾病诊断(如男性不育、性功能低下)和药物筛选等方面均具有重要意义。本发明技术方案1.人睾丸发育特异性蛋白-24基因融合蛋白的制备
以人睾丸发育特异性蛋白-24基因开放阅读框的核苷酸序列与质粒pDESTTM15制备成GST-SP2-pDEST15表达质粒，在无NaCl的LB中30℃培养到0.5OD600，经0.3M NaCl诱导，超声粉碎后经GST亲和层析柱纯化，得到纯化的融合蛋白。2.人睾丸发育特异性蛋白-24基因编码蛋白特异性多克隆抗体
取融合蛋白与载体蛋白KLH(Keyhole Limpet Hymocyanin)结合，经透析后再与完全/不完全佐剂(Complete/Incomplete Freund’sAdjuvant)等量混合，免疫新西兰兔(背部皮下注射)，每隔3～4周激发注射一次，共三次，于三个月后取血分离血清。以免疫前动物血清作为对照做ELISA，测定各免疫兔血清中抗体滴度。筛选高滴度血清，进一步用抗原作竞争抑制免疫试验确定抗体的特异性。根据ELISA结果确定其用于免疫组织化学染色的稀释倍数为1∶200～500。3.人睾丸发育特异性蛋白-24基因编码蛋白特异性单克隆抗体
取融合蛋白与载体蛋白KLH(Keyhole Limpet Hymocyanin)结合，经透析后再与完全/不完全佐剂(Complete/Incomplete Freund’sAdjuvant)混合，免疫Balb/C小鼠(腹腔内注射)，隔周一次，连续2-3次，融合前静脉加强一次，用ELISA方法证实抗体的产生后，取脾脏分离脾细胞，然后与骨髓瘤细胞融合产生杂交瘤细胞。阳性克隆经多次亚克隆，直至获得分泌抗人睾丸发育特异性蛋白-24的单克隆杂交瘤细胞株。4.人睾丸发育特异性蛋白-24基因用于制备睾丸功能基因表达芯片
(1)以获取的人睾丸发育特异性蛋白-24基因的核苷酸序列设计引物，扩增全长的人睾丸发育特异性蛋白-24基因序列，用作点膜的样品。
(2)用英国BioRobotics自动点膜仪将标本点在8×12cm尼龙膜上，每一标本点2个点，每点的DNA量约几ng。；阳性对照8个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)。阴性对照加λ噬菌体DNA和P-blue质粒。(3)将含有该基因的芯片用于人睾丸发育特异性蛋白-24基因缺失的诊断和药物筛选33P标记：
抽提待检标本mRNA或DNA，用Random Primer标记法标记待检标本mRNA或DNA，作为杂交的探针。杂交：
将点制好的尼龙膜标记探针杂交后，杂交炉(68℃)内烘干，用磷屏压片。信号扫描和分析：
用记录仪分析杂交信号。根据杂交信号的强弱分析人睾丸发育特异性蛋白-24基因蛋白表达水平或人睾丸发育特异性蛋白-24基因的缺失。
人睾丸发育特异性蛋白-24基因编码蛋白
人睾丸发育特异性蛋白-24基因cDNA序列
1 ggggggggcg cgcctgcgca ttatgctggt ctccatggcg gggctcggag caagacgaga
61 gaaaatgctg ctgtgaagac ccaattaaag ctttcacata aatgaagcta cagctatcat
121 gatttagtgc ccccaaaagg aagaacttca gtggacaaga aaggacattt ctgggggtag
181 tagaatgctt gaggcctgga atttaaacct gagccactat ctgaagctat taaataatag
241 aagaaaatgg aattgaatat gatgtcagca tggaacaatc taatgattca ttaagagtca
301 accataatga cggtgaagag tcaaaaacca gtgctcaagt atttgagcat ctaatctgta
361 tggactccag ggattcttcc tttggacaaa atgattctcc tacagttttg cccatcacta
421 ctcgtgaagc aaataattca ctcatatcac agaatatacc agggcccctg actcagacac
481 agactctttc tgcagagcaa ttccatctag tggaccaaaa tgggcaggct attcaatatg
541 aacttcagtc attgggggaa tccaatgcac aaatgatgat cgttgccagc ccaacagaaa
601 atggacaggt acttcgtgta attccaccta cccagacagg aatggcacaa gtgattatac
661 ctcaggggca acttgtggat gtgaatagtc ctcgggatgt ccctgaagag aaacccagta
721 acagaaactt accaactgta agagtggata ctctagcaga caataccagc aattacattc
781 ttcatcctca aacatccttc ccattgccca aaaagtcagt gaccggaatg ctggaagaac
841 cccttctggg gcctcttcag ccactttctt ctaatacacc tatatgggcc tgccgtctta
901 ggagctgtga gaaaattgga gattcatacc gtggctactg tgtaagtgag actgaattag
961 aaagtgtcct aacatttcac gagcagcaaa cacagagtgt ttgggggacc cgtcagtctc
1021 caagcccagc caagcctgct acacgcttga tgtggaaatc ccagtatgtt ccatatgatg
1081 gaatcccatt tgttaatgca gggagtagag ctgtggtaat ggagtgtcag tatgggccaa
1141 gaagaaaagg tttccagtta aaaaaagtca gtgagcagga aagcaggtct tgtcagctct
1201 acaaagccac ttgtccagct cggatttaca ttaaaaaggt acagaagttt cctgaatata
1261 gagttcctac agaccccaaa attgacaaga aaattatcag aatggagcag gagaaagctt
1321 ttaacatgct aaagaagaac ttggtagatg ctggtggtgt tcttaggtgg tatgtacagt
1381 tacctacaca gcaagctcat cagtatcatg aattagagac tccctgcctc actttgtcac
1441 cttctccttt tcctgtgtct tctcttgaag aagaggaaac tgcagttaga gatgagaatt
1501 gtgcattacc ctcacgttta catcctcaag tagcacataa gattcaagaa ttagtatcac
1561 agggaataga acaagtgtat gcagtaagga aacagctaag aaaatttgtg gaaagggaac
1621 tgttcaaacc cgatgaggta cctgaaagac ataatttatc tttttttcca actgtaaatg
1681 atataaaaaa tcacatccat gaggtacaga aatccttgag aaatggagat acggtatata
1741 actcagagat tattccagca acgcttcaat ggactacaga cagtgggaat attctcaaag
1801 agaccatgac agttacattt gcagaaggaa attcaccagg agaatcaatt accaccaaag
1861 tggaaacaaa tcagaccagg ggttctttgt ctcctgagcc aacccacttg ctctcctcac
1921 tctcctcatt tcagcccaaa atatttacac aactacaggg tttgcagtta caaccaaggt
1981 acacctctcc tgatgaatca ccagctgtgg tatcagtaaa taaccagccg tcctctagtc
2041 cttcaggact tctggataca ataggaagtg ctgtaatgaa taataattct ctactgcttg
2101 gtcaaagtca tagccttcaa agagatacat gcttaaccca aaacaatagt actgcctcca
2161 ccatgggtaa ccttccagaa ccagatcaaa atctagttgc aatggacgag ctggtagaag
2221 ttggagatgt tgaggataca gggaatctgg aaggaactgt tcatcggatt ctgttgggag
2281 atgtgcagac tattccaata cagattatag acaaccactc agctcttatt gaagaaaatc
2341 cagaaagtac catttctgtg agccaagtta aacaagaacc caaagaacca gcattgtcta
2401 tggaagcaaa aaaaactgtg gactataaga aattatctgc tacataaatt attgaagctt
2461 ttcagaattt acaactctgg tgacttctga tgtcttagaa aggaaggtga atatagtcaa
2521 agcgtggcat tgagataatt ggactgaaga ccagtttgat gagaagcttt tatttaaaac
2581 tgatatattt gttgccagtt ccatattttt accttcctta agagatgttt tactcttctt
2641 attttgtata atttttatgc tctttgatta tctaataagg ccagtatttc aaaagctgtt
2701 ctgaatttat ccacagtaat atagtctgaa cacaaatagt ttacttaact tatccttgga
2761 aatattcaat tttggttatt acaaaacaga aaagaacaac agcaaaaaaa aaaaaaaaaa
2821 aaa
人睾丸发育特异性蛋白-24基因编码蛋白氨基酸序列QNIPGPLTQTQTLSAEQFHLVDQNGQAIQYELQSLGESNAQMMIVASPTENGQVLRVIPPTQTGMAQVIIPQGQLVDVNSPRDVPEEKPSNRNLPTVRVDTLADNTSNYILHPQTSFPLPKKSVTGMLEEPLLGPLQPLSSNTPIWACRLRSCEKIGDSYRGYCVSETELESVLTFHEQQTQSVWGTRQSPSPAKPATRLMWKSQYVPYDGIPFVNAGSRAVVMECQYGPRRKGFQLKKVSEQESRSCQLYKATCPARIYIKKVQKFPEYRVPTDPKIDKKIIRMEQEKAFNMLKKNLVDAGGVLRWYVQLPTQQAHQYHELETPCLTLSPSPFPVSSLEEEETAVRDENCALPSRLHPQVAHKIQELVSQGIEQVYAVRKQLRKFVERELFKPDEVPERHNLSFFPTVNDIKNHIHEVQKSLRNGDTVYNSEIIPATLQWTTDSGNILKETMTVTFAEGNSPGESITTKVETNQTRGSLSPEPTHLLSSLSSFQPKIFTQLQGLQLQPRYTSPDESPAVVSVNNQPSSSPSGLLDTIGSAVMNNNSLLLGQSHSLQRDTCLTQNNSTASTMGNLPEPDQNLVAMDELVEVGDVEDTGNLEGTVHRILLGDVQTIPIQIIDNHSALIEENPESTISVSQVKQEPKEPALSMEAKKTVDYKKLSAT
Claims (5)
1.一种睾丸功能特异基因(NYD-SP24)克隆制备的融合蛋白，其特征是NYD-SP24基因全部开放阅读框氨基酸，序列为：QNIPGPLTQTQTLSAEQFHLVDQNGQAIQYELQSLGESNAQMMIVASPTENGQVLRVIPPTQTGMAQVIIPQGQLVDVNSPRDVPEEKPSNRNLPTVRVDTLADNTSNYILHPQTSFPLPKKSVTGMLEEPLLGPLQPLSSNTPIWACRLRSCEKIGDSYRGYCVSETELESVLTFHEQQTQSVWGTRQSPSPAKPATRLMWKSQYVPYDGIPFVNAGSRAVVMECQYGPRRKGFQLKKVSEQESRSCQLYKATCPARIYIKKVQKFPEYRVPTDPKIDKKIIRMEQEKAFNMLKKNLVDAGGVLRWYVQLPTQQAHQYHELETPCLTLSPSPFPVSSLEEEETAVRDENCALPSRLHPQVAHKIQELVSQGIEQVYAVRKQLRKFVERELFKPDEVPERHNLSFFPTVNDIKNHIHEVQKSLRNGDTVYNSEIIPATLQWTTDSGNILKETMTVTFAEGNSPGESITTKVETNQTRGSLSPEPTHLLSSLSSFQPKIFTQLQGLQLQPRYTSPDESPAVVSVNNQPSSSPSGLLDTIGSAVMNNNSLLLGQSHSLQRDTCLTQNNSTASTMGNLPEPDQNLVAMDELVEVGDVEDTGNLEGTVHRILLGDVQTIPIQIIDNHSALIEENPESTISVSQVKQEPKEPALSMEAKKTVDYKKLSAT
2.根据权利要求1所述针对NYD-SP24基因开放阅读框氨基酸序列制备的多克隆抗体，其特征是融合蛋白与完全和不完全佐剂混合后，免疫新西兰兔制备多克隆抗体。
3.根据权利要求1所述针对NYD-SP24基因开放读框氨基酸序列制备的单克隆抗体，其特征是融合蛋白与完全和不完全佐剂混合，免疫Balb/C小鼠，分离脾脏B淋巴细胞并和骨髓瘤细胞融合产生杂交瘤细胞，进一步用亚克隆方法、ELISA和抗体分型方法筛选分泌IgG抗体的单克隆，制备单克隆抗体。
4.根据权利要求1所述针对将NYD-SP24基因开放读框氨基酸序列用于制备睾丸功能基因表达芯片，其特征是PCR扩增NYD-SP24基因cDNA，点于芯片用于芯片制备。
5.根据权利要求1所述，针对NYD-SP24基因开放读框氨基酸序列用于制备融合蛋白，其特征是融合蛋白可作为制备治疗相关疾病的药物。
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