CN1382697A - 人睾丸发育特异性蛋白－28基因编码蛋白 - Google Patents
人睾丸发育特异性蛋白－28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－28基因(testis development specific protein gene28,NYD－SP28)基因全长cDNA序列为1798bp,其开放阅读框序列为1455bp,其编码484个氨基酸,该基因在基因库(Genbank)中的编号为AF382188。本发明利用NYD－SP28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-28基因(testis developmentspecific protein gene 28，NYD-SP28)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1798bp；其开放阅读框架为1455bp，其编码484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，胚胎低表达，因此发现的人睾丸发育特异性蛋白-28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-28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AF382188。1 gggggggaga gctcagcctc actgatagcc ggggatctct cctgttcgag ggctgagatc61 ttcggtccca caaccgccca caaccagggg cacttctgga agtccccttt ctagggacat121 ttttcttcta agctctgtaa cgcgggccga ggcagaccga ggcttctttc ccggatccaa181 tcccctgccc ccatgcctaa gaaagaaaaa atggccaaga cgcccctgtc cgatgagaag241 cagctgcttc tgtttcagca gaagttgctg gcagaggagg agatggccaa aaagaaggag301 aggctcctca gccagttctt gaaggacaag ctggccaagg aggaacacaa cagtgctctg361 aaccttaata agattaacac acagtggaga actgtccttc gggaagtcaa gaccagagaa421 cttcataagg acattgagat cctcagccaa acatttgaac gagtggtgga ctgtaaggac481 aatgtcatca agtctttagc taaagacctg tccgaagccg aggagcagta cgcccatgcc541 ctgcgcagcc acttgcacaa tgttgaccag ctcttggccc tgcagaggca ccggctcagt601 ctcctggagg aaagttacaa catggagctg gaagccctaa ccaaggagtt tgagacagaa661 aggaagacaa ttattgacca acatgagaaa gagattcact atctgcaaga tatcttcatg721 gccatggagc agaactatat agattctgag tatgaaagca agctggagtt ccagagcatg781 tggaatgatc tcaaagacat gaatttagaa gagaagcact ttctaagact gcacctggag841 aacagagtag aagatctgtg gagaaagttc caggatgtac tcaagaatta cactgatgcc901 acagaggatc gaaagactgc ctttgagacc ctgcaggtga aggatgagaa gagctccaaa961 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
人睾丸发育特异性蛋白-28基因cDNA序列MPKKEKMAKTPLSDEKQLLLFQQKLLAEEEMAKKKERLLSQFLKDKLAKEEHNSALNLNKINTQWRTVLREVKTRELHKDIEILSQTFERVVDCKDNVIKSLAKDLSEAEEQYAHALRSHLHNVDQLLALQRHRLSLLEESYNMELEALTKEFETERKTIIDQHEKEIHYLQDIFMAMEQNYIDSEYESKLEFQSMWNDLKDMNLEEKHFLRLHLENRVEDLWRKFQDVLKNYTDATEDRKTAFETLQVKDEKSSKEIEVQMKKIQKLQDAITISKGKIMIHSRESEDENRYIRNDKELVLVQLRKLKAQRTQARAASQKNLVRLTLESNATLKALRKIVDKGEKILKLAEICRKFETEEEKVLPFYSSVLTPKEQEGIQKNNLEELTEELTKVMVDYIGMENFWKRYNKVKLEQLSLQHRRAQLLDINGKLREMLKQYLDGISVSDEVLSQLNPLFIVNYQSNLLQPLSIRIAHPGDKQHPTT
人睾丸发育特异性蛋白-28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-28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-28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一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-28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-28基因融合蛋白的制备以人睾丸发育特异性蛋白-28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-28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-28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-28的单克隆杂交瘤细胞株。4.人睾丸发育特异性蛋白-28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-28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-28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。；阳性对照为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)。阴性对照为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-28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-28基因蛋白表达水平或人睾丸发育特异性蛋白-28基因的缺失。
人睾丸发育特异性蛋白-28基因编码蛋白
人睾丸发育特异性蛋白-28基因cDNA序列1 gggggggaga gctcagcctc actgatagcc ggggatctct cctgttcgag ggctgagatc61 ttcggtccca caaccgccca caaccagggg cacttctgga agtccccttt ctagggacat121 ttttcttcta agctctgtaa cgcgggccga ggcagaccga ggcttctttc ccggatccaa181 tcccctgccc ccatgcctaa gaaagaaaaa atggccaaga cgcccctgtc cgatgagaag241 cagctgcttc tgtttcagca gaagttgctg gcagaggagg agatggccaa aaagaaggag301 aggctcctca gccagttctt gaaggacaag ctggccaagg aggaacacaa cagtgctctg361 aaccttaata agattaacac acagtggaga actgtccttc gggaagtcaa gaccagagaa421 cttcataagg acattgagat cctcagccaa acatttgaac gagtggtgga ctgtaaggac481 aatgtcatca agtctttagc taaagacctg tccgaagccg aggagcagta cgcccatgcc541 ctgcgcagcc acttgcacaa tgttgaccag ctcttggccc tgcagaggca ccggctcagt601 ctcctggagg aaagttacaa catggagctg gaagccctaa ccaaggagtt tgagacagaa661 aggaagacaa ttattgacca acatgagaaa gagattcact atctgcaaga tatcttcatg721 gccatggagc agaactatat agattctgag tatgaaagca agctggagtt ccagagcatg781 tggaatgatc tcaaagacat gaatttagaa gagaagcact ttctaagact gcacctggag841 aacagagtag aagatctgtg gagaaagttc caggatgtac tcaagaatta cactgatgcc901 acagaggatc gaaagactgc ctttgagacc ctgcaggtga aggatgagaa gagctccaaa961 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人睾丸发育特异性蛋白-28基因编码蛋白氨基酸序列MPKKEKMAKTPLSDEKQLLLFQQKLLAEEEMAKKKERLLSQFLKDKLAKEEHNSALNLNKINTQWRTVLREVKTRELHKDIEILSQTFERVVDCKDNVIKSLAKDLSEAEEQYAHALRSHLHNVDQLLALQRHRLSLLEESYNMELEALTKEFETERKTIIDQHEKEIHYLQDIFMAMEQNYIDSEYESKLEFQSMWNDLKDMNLEEKHFLRLHLENRVEDLWRKFQDVLKNYTDATEDRKTAFETLQVKDEKSSKEIEVQMKKIQKLQDAITISKGKIMIHSRESEDENRYIRNDKELVLVQLRKLKAQRTQARAASQKNLVRLTLESNATLKALRKIVDKGEKILKLAEICRKFETEEEKVLPFYSSVLTPKEQEGIQKNNLEELTEELTKVMVDYIGMENFWKRYNKVKLEQLSLQHRRAQLLDINGKLREMLKQYLDGISVSDEVLSQLNPLFIVNYQSNLLQPLSIRIAHPGDKQHPTT
Claims (5)
1.一种睾丸功能特异基因(NYD-SP28)克隆制备的融合蛋白，其特征是NYD-SP28基因全部开放阅读框氨基酸，序列为：MPKKEKMAKTPLSDEKQLLLFQQKLLAEEEMAKKKERLLSQFLKDKLAKEEHNSALNLNKINTQWRTVLREVKTRELHKDIEILSQTFERVVDCKDNVIKSLAKDLSEAEEQYAHALRSHLHNVDQLLALQRHRLSLLEESYNMELEALTKEFETERKTIIDQHEKEIHYLQDIFMAMEQNYIDSEYESKLEFQSMWNDLKDMNLEEKHFLRLHLENRVEDLWRKFQDVLKNYTDATEDRKTAFETLQVKDEKSSKEIEVQMKKIQKLQDAITISKGKIMIHSRESEDENRYIRNDKELVLVQLRKLKAQRTQARAASQKNLVRLTLESNATLKALRKIVDKGEKILKLAEICRKFETEEEKVLPFYSSVLTPKEQEGIQKNNLEELTEELTKVMVDYIGMENFWKRYNKVKLEQLSLQHRRAQLLDINGKLREMLKQYLDGISVSDEVLSQLNPLFIVNYQSNLLQPLSIRIAHPGDKQHPTT
2.根据权利要求1所述针对NYD-SP28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-SP28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-SP28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-SP28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-SP28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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| CN112695033A (zh) * | 2020-12-29 | 2021-04-23 | 华南师范大学 | 基于美洲大蠊雄性附腺生殖相关基因SP28设计的siRNA及其制备方法与应用 |
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| CN112695033A (zh) * | 2020-12-29 | 2021-04-23 | 华南师范大学 | 基于美洲大蠊雄性附腺生殖相关基因SP28设计的siRNA及其制备方法与应用 |
| CN112695033B (zh) * | 2020-12-29 | 2021-08-24 | 华南师范大学 | 基于美洲大蠊雄性附腺生殖相关基因SP28设计的siRNA及其制备方法与应用 |
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